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Phosphorothioated Primers Lead to Loop-Mediated Isothermal Amplification at Low Temperatures

  • Sheng Cai
    Sheng Cai
    Laboratory of Pharmaceutical Analysis and Drug Metabolism, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, Zhejiang University, Hangzhou, Zhejiang 310058, China
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  • , 
  • Cheulhee Jung
    Cheulhee Jung
    Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 02841, Republic of Korea
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    Orcidhttp://orcid.org/0000-0001-7392-5862
  • , 
  • Sanchita Bhadra
    Sanchita Bhadra
    Institute for Cellular and Molecular Biology, Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas 78712, United States
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  • , and 
  • Andrew D. Ellington*
    Andrew D. Ellington
    Institute for Cellular and Molecular Biology, Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas 78712, United States
    *E-mail: [email protected]
    More by Andrew D. Ellington
Cite this: Anal. Chem. 2018, 90, 14, 8290–8294
Publication Date (Web):July 3, 2018
https://doi.org/10.1021/acs.analchem.8b02062
Copyright © 2018 American Chemical Society
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Abstract

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Loop-mediated isothermal amplification (LAMP) is an extremely powerful tool for the detection of nucleic acids with high sensitivity and specificity. However, LAMP shows optimal performance at around 65 °C, which limits applications in point-of-care-testing (POCT). Here, we have developed a version of LAMP that uses phosphorothioated primers (PS-LAMP) to enable more efficient hairpin formation and extension at the termini of growing concatamers, and that therefore works at much lower temperatures. By including additional factors such as chaotropes (urea) and single-stranded DNA binding protein (SSB), the sensitivities and selectivities for amplicon detection with PS-LAMP at 40 °C were comparable with a regular LAMP reaction at 65 °C.

This publication is licensed for personal use by The American Chemical Society.

Loop-mediated isothermal amplification (LAMP) (1) is a rapid and sensitive method for nucleic acid amplification and has numerous applications in molecular diagnostics. (2−7) LAMP is typically carried out at a constant temperature (60–65 °C) (8−12) and can achieve 109-fold amplification within 1 h. A key to the underlying amplification scheme is the use of primers that generate foldback structures and their subsequent extension by a strand-displacing DNA polymerase such as the Bacillus stearothermophilus (Bst) DNA polymerase.
The applicability of LAMP, especially for point-of-care diagnostics, would be greatly improved if it could be performed at lower temperatures; this would potentially reduce both device complexity and power consumption. While there are other isothermal amplification methods that can be carried out at lower temperatures (around 40 °C), such as nucleic acid sequence-based amplification (NASBA), (13) strand displacement amplification (SDA), (14,15) rolling circle amplification (RCA), (16) helicase-dependent amplification (HDA), (17) and recombinase polymerase amplification (RPA), (18) these methods often require additional steps or enzymes relative to LAMP, which requires only a single polymerase. Moreover, these methods have lower specificity for their targets, since they all require only two primers, while LAMP is performed with from four to six specific primers.
In order to lower the operating temperature at which LAMP reactions can be carried out, we exploited a fundamental biophysical principle, that phosphorothioate residues destabilize helices. Boczkowska and co-workers have carried out thermodynamic studies on the stability of duplexes formed between phosphorothioate (PS)-modified ssDNA and complementary phosphodiester (PO)-modified ssDNA, (19) and reported that the PS modifications substantially reduced the melting temperature of PS–PO dsDNA. This in turn allows more breathing at the termini of dsDNA and should promote the formation of foldback hairpins for extension during LAMP. Based on this phenomenon, our lab has previously developed a different amplification method, phosphorothioated-terminal hairpin formation and self-priming extension (PS-THSP), (20) in which the incorporation of phosphorothioate (PS) modifications lead to improved self-folding efficiency of terminal hairpins. (21)
By now incorporating phosphorothioate (PS) modifications into the foldback primers used for LAMP we have created a more generic mechanism (PS-LAMP) for low temperature amplification. PS-LAMP can operate at temperatures as low as 40 °C with sensitivities that are similar to regular LAMP. The PS-terminated DNA should also display enhanced stability against degradation by various nucleases that may be present in biological samples, further enhancing the potential applicability of PS-LAMP for point-of-care (POC) diagnostics.

Results and Discussion

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Design of PS-LAMP Reactions

In a typical LAMP reaction (Figure 1), there are at least four primers: two inner primers (FIP and BIP) and two outer primers (F3 and B3). These primers are specific to six consecutive blocks of target sequences: B3, B2, B1, F1c, F2c, and F3c (from the 5′-end of the amplicon). Both the inner and outer primers anneal to the target template and are extended simultaneously. The extension of the outer primer with a strand displacing polymerase, such as Bst, therefore displaces the inner primer, which can then fold back on itself to create a dumbbell-shaped amplicon. The inner primers can hybridize to the single-stranded loops in the foldback structures and initiate another round of strand displacement synthesis, forming a concatamer amplicon with a self-priming 3′-hairpin. The ensuing exponential amplification during continuous strand displacement DNA synthesis generates increasingly complex, double-stranded concatamer amplicons. The single-stranded loops in these amplicons can be used to trigger sequence-specific strand exchange reporters, as we have previously described (Figure S1). (22,23) These reporters greatly reduce background signal and provide greater surety in the detection of LAMP amplicons.

Figure 1

Figure 1. Scheme of a phosphorothioated loop-mediated isothermal amplification (PS-LAMP).

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The loop structure is key to the amplification mechanism in LAMP reactions. We therefore attempted to improve the formation of the foldback structure in the extended dsDNA (red box) replication intermediate. All the internucleotide linkages in the F1c portion of the FIP primer (20 phosphates for MERS 1a and MERS 1b) and in the B1c portion of the BIP primer (23 and 21 phosphates for MERS 1a and MERS 1b, respectively) were modified with phosphorothioates (see also Figure 1). The F2 region in FIP and the B2 region in BIP were excluded from phosphorothioate modification as this might have reduced priming efficiency. The PS modifications should lead to great reductions in thermal stability at the terminus of the extended dsDNA replication intermediates, (20) and thus intrastrand hybridization (hairpin structure formation) should occur more readily, allowing more efficient exponential amplification and the execution of LAMP at lower reaction temperatures.
To verify that exponential amplification during PS-LAMP is more efficient than during regular LAMP, we compared amplification performance at two different temperatures, 60 and 65 °C). Amplification for LAMP and PS-LAMP was carried out identically except for the primers used and was monitored in real-time using a sequence-specific strand exchange reporter for 150 min in the absence or presence of target template (1.2 × 108 copies). Figure 2a shows that at 60 and 65 °C, LAMP and PS-LAMP could both detect 1.2 × 108 molecules within similar detection times, about 22 min. Interestingly, the fluorescence intensity of the PS-LAMP reaction at 150 min was 1.8 times higher than that of LAMP at 60 °C (Figure 2b). This signal improvement at lower temperatures is consistent with our hypothesis that phosphorothioates improve the self-folding of loops.

Figure 2

Figure 2. Comparison of R-LAMP and PS-LAMP at different temperatures for MERS 1b. (a) Under the same buffer condition, R-LAMP and PS-LAMP were performed at different temperatures (line 1, no template/R-LAMP/65 °C; line 2, 1.2 × 108 copies of template/R-LAMP/65 °C; line 3, no template/PS-LAMP/65 °C; line 4, 1.2 × 108 copies of template/PS-LAMP/65 °C; line 5, no template/R-LAMP/60 °C; line 6, 1.2 × 108 copies of template/R-LAMP/60 °C; line 7, no template/PS-LAMP/60 °C; line 8, 1.2 × 108 copies of template/PS-LAMP/60 °C). (b) Fluorescence intensities at 150 min for R-LAMP and PS-LAMP in the presence of 1.2 × 108 copies of template were compared at different temperatures. (c) R-LAMP and PS-LAMP were performed at 45 °C with an optimized buffer (12 U of Bst 2.0 DNA polymerase, 0.5 μg of ET SSB, and 2 mM of MgSO4).

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Optimization of Lower Temperature PS-LAMP Reactions

Given these results, we attempted to further optimize PS-LAMP for even lower temperatures, which would also further its potential use as a point-of-care diagnostic. When PS-LAMP was performed at progressively lower temperatures (60, 55, 50, and 45 °C), no amplification was eventually observed at 45 °C (Figure S2a). The buffer and reaction conditions (4 mM of MgSO4 and 8 U of Bst 2.0 DNA polymerase) were therefore further optimized at 45 °C to see if a signal could be generated. Magnesium ions are known to greatly impact self-folding, (24,25) and thus different concentrations of magnesium (0, 1, 2, 3, and 4 mM) were assessed. At 2 mM MgSO4, a small signal increase was observed (Figure S2b). The amount of Bst 2.0 DNA polymerase was then increased from 8 U to 12 U and a thermostable single-stranded DNA binding protein (ET SSB) was added (0.5 μg) to further destabilize duplexes and promote self-folding at the termini. (26−29) ET SSB proved more effective than an alternative single-stranded DNA binding protein, RecA (Figure S3).
Using the optimized reaction conditions (Figure S4), template amplification by PS-LAMP was possible at 45 °C, while normal LAMP reactions showed no amplification at this temperature (Figure 2c). That said, optimized PS-LAMP at 45 °C still took a longer time (63 min) to come to completion than did a normal LAMP reaction at a higher temperature (65 °C, 22 min).
To further lower the operational temperature of PS-LAMP, urea was added to the reaction mixture. Like heat and phosphorothioates, urea should disrupt base stacking and again improve the possibility of foldback priming. (30−32) Upon optimization, a urea concentration of 1.44 M yielded a workable PS-LAMP reaction at 40 °C (Figure 3a,b). Both urea and low temperatures decrease Bst 2.0 DNA polymerase activity, and thus the amount of Bst 2.0 DNA polymerase in the reaction was optimized once again, as shown in Figure 3c,d, and subsequent reactions contained 60 U of polymerase; higher concentrations (80 U and 120 U) inhibited the reaction. As it is known that the activity of Bst 2.0 DNA polymerase is reduced by about an order of magnitude at 37 °C relative to its activity at 65 °C, we added additional enzyme (60 U rather than 8 U) to the lower temperature reaction. At optimal urea and polymerase concentrations, ET SSB and MgSO4 were reoptimized (0.5 μg and 2 mM, respectively (Figure S5), further decreasing the detection time from approximately 117 to 70 min.

Figure 3

Figure 3. Effects of urea and Bst 2.0 DNA polymerase on PS-LAMP at 40 °C for MERS 1b. Fluorescence intensities were monitored with different amounts of urea (line 1, 0 M/no template; line 2, 0 M/1.2 × 108 copies of template; line 3, 0.48 M/no template; line 4, 0.48 M/1.2 × 108 copies of template; line 5, 0.96 M/no template; line 6, 0.96 M/1.2 × 108 copies of template; line 7, 1.2 M/no template; line 8, 1.2 M/1.2 × 108 copies of template; line 9, 1.44 M/no template; line 10, 1.44 M/1.2 × 108 copies of template) in the buffer (12 U of Bst 2.0 DNA polymerase, 0.5 μg of ET SSB, and 2 mM of MgSO4) (a) or Bst 2.0 DNA polymerase (line 1, 20 U/no template; line 2, 20 U/1.2 × 108 copies of template; line 3, 40 U/no template; line 4, 40 U/1.2 × 108 copies of template; line 5, 60 U/no template; line 6, 60 U/1.2 × 108 copies of template; line 7, 80 U/no template; line 8, 80 U/1.2 × 108 copies of template; line 9, 120 U/no template; line 10, 120 U/1.2 × 108 copies of template) in the buffer (1.44 M of urea, 0.5 μg of ET SSB, and 2 mM of MgSO4) (c) during PS-LAMP at 40 °C. DT values were plotted with the concentrations of urea (b) and units of Bst 2.0 DNA polymerase (d).

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Selectivity

To validate the sequence-specificity of PS-LAMP, various nontarget templates (MERS 1a, NRP2 and human genomic DNA) were tested in parallel with the target template (MERS 1b) using a MERS 1b primer set. As indicated in Figure S6, at template concentrations of 500 pg (1.2 × 108 copies) PS-LAMP produced negligible responses with noncognate templates. When the target amplicon (MERS 1b) was mixed with the noncomplementary templates, no diminution in positive signal was observed (Figure S6).

Quantitation by PS-LAMP

The quantitative behavior of PS-LAMP at 40 °C in the presence of urea and under fully optimized conditions was analyzed by monitoring the changes in the fluorescence intensity of the OSD reporter as a function of template concentration. As shown in Figure 4, when plasmids bearing the MERS 1b and MERS 1a genes were used as targets, as few as 4 800 molecules and 12 molecules, respectively, could be successfully detected within 110 and 80 min by PS-LAMP. However, there was slight variability in time-to-detection (Cq, redefined as DT (detection time, min) by multiplying Cq by 3 in this paper) between assays. For instance, DT for 1.2 × 108 template copies ranged from 65 to 85 min between different experiments (see Figures 3c and 4a and Figure S5c). This variability is expected since LAMP is a complex continuous amplification process. Other studies have also reported that LAMP has lower precision and dynamic range of quantitation when compared to quantitative qPCR. (33) As a result, LAMP is not often used for quantitation but rather serves as a sensitive method for detecting nucleic acid analytes. Overall, our PS-LAMP assays performed with reproducible sensitivity and specificity comparable to other reported LAMP assays. The detection of the MERS 1a amplicon was roughly comparable with regular LAMP at 65 °C, but detection of the MERS 1b amplicon with PS-LAMP at 40 °C was less sensitive than for regular LAMP at 65 °C (24 molecules within 30 min) (Figure S7). This is not surprising, as there can be wide variation in the detection limits for different amplicons even with regular LAMP, depending upon the sequences of primers and templates, (4,12,23) and as the PS-LAMP technique is further developed it should be possible to identify comparable optimization rules for amplicon choice and primer design.

Figure 4

Figure 4. Quantitative analysis of PS-LAMP for different templates. The fluorescence intensities were monitored for PS-LAMP at 40 °C in the buffer (1.44 M of urea, 60 U of Bst 2.0 DNA polymerase, 0.5 μg of ET SSB, and 2 mM of MgSO4) with titrated MERS 1b (a) and MERS 1a (b) plasmids.

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Conclusions

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LAMP is an ultrasensitive nucleic acid amplification method that can often detect small numbers DNA or RNA templates within roughly an hour. However, the requirement for high temperatures limits it applicability, and we have therefore relied on a fundamental understanding of nucleic acid hybridization chemistry, the disruption of helical stability by phosphorothioates, to develop PS-LAMP, which shows comparable sensitivities even down to 40 °C. Operating at lower temperatures should inherently reduce device complexity and power consumption when adapting molecular diagnostics to microscale or portable devices.
In addition to being able to operate at more moderate temperatures, PS-LAMP may better enable the use of degenerate primer sets to capture a wider range of phylogenetic variants into amplicons. Improving amplification at lower temperatures will also enable the use of AT-rich primers and probes, which have previously proven problematic for LAMP. (9)
Overall, PS-LAMP may now allow mesothermal amplification and concomitant applications in molecular diagnostics that should be on par with similar techniques, such as RPA. We anticipate that future versions of PS-LAMP may be even more readily adapted to POC devices. While we have used an initial denaturation step of 95 °C, it is also possible to perform LAMP without this step, (34−36) and the application of similar procedures to PS-LAMP would similarly enable a one-step reaction protocol, especially since the reagents used should still allow a modest initial heat pulse of 60 °C. As the technique is further developed, the development of primer design rules that accommodate lower temperatures and phosphorothioates will increase its overall applicability. It may be possible to move reaction temperatures even lower, potentially allowing the body or other nonpowered environments to serve as a heat source for amplification. In addition, the ability to “dial in” amplicon acquisition via the number of phosphorothioates, the amount of urea present, and the temperature of the reaction will provide much greater experimental control over the amplification and detection of specific targets.

Supporting Information

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.8b02062.

  • Scheme of one-step strand displacement (OSD); effects of MgSO4, RecA, and ET SSB at different temperatures; selectivity analysis of PS-LAMP for different templates; quantitative analysis of regular LAMP for different templates; and oligonucleotide sequences used in this study (PDF)

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Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

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  • Corresponding Author
    • Andrew D. Ellington - Institute for Cellular and Molecular Biology, Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas 78712, United States;  Email: [email protected]
  • Authors
    • Sheng Cai - Laboratory of Pharmaceutical Analysis and Drug Metabolism, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, Zhejiang University, Hangzhou, Zhejiang 310058, China
    • Cheulhee Jung - Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 02841, Republic of Korea;  Orcidhttp://orcid.org/0000-0001-7392-5862
    • Sanchita Bhadra - Institute for Cellular and Molecular Biology, Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas 78712, United States
  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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This work was supported by National Aeronautics and Space Administration Astrobiology Institute Cooperative Agreement Notice (NASA NAI CAN) [Grant NNX15AF46G] and Welch [Grant F-1654]. This publication was also made possible through the support of a grant from the John Templeton Foundation [Grant 54466]. The opinions expressed in this publication are those of the authors and do not necessarily reflect the views of the John Templeton Foundation. Funding for the open access was provided by the John Templeton Foundation.

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    Establishment and application of a real-time loop-mediated isothermal amplification system for the detection of CYP2C19 polymorphisms
    Zhang, Chao; Yao, Yao; Zhu, Juan-Li; Zhang, Si-Nong; Zhang, Shan-Shan; Wei, Hua; Hui, Wen-Li; Cui, Ya-Li
    Scientific Reports (2016), 6 (), 26533CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)
    Single-nucleotide polymorphisms (SNPs) represent the most widespread type of genetic variation (approx. 90%) in the human genome, and the demand to overcome such variation has received more attention now than ever before. The capacity to rapidly assess SNPs that correlate with disease predisposition, drug efficacy and drug toxicity is a key step for the development of personalized medicine. In this work, a rapid one-step SNP detection method, real-time loop-mediated isothermal amplification (RT-LAMP), was first applied for CYP2C19 polymorphisms testing. The optimized method was established with specifically designed primers for target amplification by real-time detection in approx. 30 min under isothermal conditions. RT-LAMP amplified few copies of template to produce significant amts. of product and quant. detected human DNA with compatible specificity and sensitivity. The success in the establishment of this RT-LAMP protocol for CYP2C19 polymorphism testing is significant for the extension of this technique for the detection of other SNPs, which will further facilitate the development of personalized medicine.
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  8. 8
    Nagamine, K.; Kuzuhara, Y.; Notomi, T. Isolation of Single-Stranded DNA from Loop-Mediated Isothermal Amplification Products. Biochem. Biophys. Res. Commun. 2002, 290 (4), 1195– 1198,  DOI: 10.1006/bbrc.2001.6334
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  9. 9
    Tomita, N.; Mori, Y.; Kanda, H.; Notomi, T. Loop-Mediated Isothermal Amplification (LAMP) of Gene Sequences and Simple Visual Detection of Products. Nat. Protoc. 2008, 3 (5), 877– 882,  DOI: 10.1038/nprot.2008.57
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    9
    Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products
    Tomita, Norihiro; Mori, Yasuyoshi; Kanda, Hidetoshi; Notomi, Tsugunori
    Nature Protocols (2008), 3 (5), 877-882CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)
    As the human genome is decoded and its involvement in diseases is being revealed through postgenome research, increased adoption of genetic testing is expected. Crit. to such testing methods is the ease of implementation and comprehensible presentation of amplification results. Loop-mediated isothermal amplification (LAMP) is a simple, rapid, specific and cost-effective nucleic acid amplification method when compared to PCR, nucleic acid sequence-based amplification, self-sustained sequence replication and strand displacement amplification. This protocol details an improved simple visual detection system for the results of the LAMP reaction. In LAMP, a large amt. of DNA is synthesized, yielding a large pyrophosphate ion byproduct. Pyrophosphate ion combines with divalent metallic ion to form an insol. salt. Adding manganous ion and calcein, a fluorescent metal indicator, to the reaction soln. allows a visualization of substantial alteration of the fluorescence during the one-step amplification reaction, which takes 30-60 min. As the signal recognition is highly sensitive, this system enables visual discrimination of results without costly specialized equipment. This detection method should be helpful in basic research on medicine and pharmacy, environmental hygiene, point-of-care testing and more.
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  10. 10
    Nagamine, K.; Hase, T.; Notomi, T. Accelerated Reaction by Loop-Mediated Isothermal Amplification Using Loop Primers. Mol. Cell. Probes 2002, 16 (3), 223– 229,  DOI: 10.1006/mcpr.2002.0415
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    10
    Accelerated reaction by loop-mediated isothermal amplification using loop primers
    Nagamine, K.; Hase, T.; Notomi, T.
    Molecular and Cellular Probes (2002), 16 (3), 223-229CODEN: MCPRE6; ISSN:0890-8508. (Elsevier Science Ltd.)
    Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method that amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions using a set of four specially designed primers and a DNA polymerase with strand displacement activity. We have developed a method that accelerates the LAMP reaction by using addnl. primers, termed loop primers. Loop primers hybridize to the stem-loops, except for the loops that are hybridized by the inner primers, and prime strand displacement DNA synthesis. Although both inner and loop primers react via the loops, they do so by different mechanisms. The LAMP method presented here uses loop primers to achieve reaction times of less than half that of the original LAMP method. Since the total time of anal. including detection is less than 1 h, this new method should facilitate genetic anal., including genetic diagnosis in the clin. lab.
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  11. 11
    Mair, G.; Vilei, E. M.; Wade, A.; Frey, J.; Unger, H. Isothermal Loop-Mediated Amplification (Lamp) for Diagnosis of Contagious Bovine Pleuro-Pneumonia. BMC Vet. Res. 2013, 9, 108,  DOI: 10.1186/1746-6148-9-108
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    11
    Isothermal loop-mediated amplification (LAMP) for diagnosis of contagious bovine pleuro-pneumonia
    Mair, Georg; Vilei, Edy M.; Wade, Abel; Frey, Joachim; Unger, Hermann
    BMC Veterinary Research (2013), 9 (), 108, 8 pp.CODEN: BVRMA9; ISSN:1746-6148. (BioMed Central Ltd.)
    Contagious Bovine Pleuropneumonia (CBPP) is the most important chronic pulmonary disease of cattle on the African continent causing severe economic losses. The disease, caused by infection with Mycoplasma mycoides subsp. mycoides is transmitted by animal contact and develops slowly into a chronic form preventing an early clin. diagnosis. Because available vaccines confer a low protection rate and short-lived immunity, the rapid diagnosis of infected animals combined with traditional curbing measures is seen as the best way to control the disease. While traditional labor-intensive bacteriol. methods for the detection of M. mycoides subsp. mycoides have been replaced by mol. genetic techniques in the last two decades, these latter approaches require well-equipped labs. and specialized personnel for the diagnosis. This is a handicap in areas where CBPP is endemic and early diagnosis is essential. We present a rapid, sensitive and specific diagnostic tool for M. mycoides subsp. mycoides detection based on isothermal loop-mediated amplification (LAMP) that is applicable to field conditions. The primer set developed is highly specific and sensitive enough to diagnose clin. cases without prior cultivation of the organism. The LAMP assay detects M. mycoides subsp. mycoides DNA directly from crude samples of pulmonary/pleural fluids and serum/plasma within an hour using a simple diln. protocol. A photometric detection of LAMP products allows the real-time visualization of the amplification curve and the application of a melting curve/re-assocn. anal. presents a means of quality assurance based on the predetd. strand-inherent temp. profile supporting the diagnosis. The CBPP LAMP developed in a robust kit format can be run on a battery-driven mobile device to rapidly detect M. mycoides subsp. mycoides infections from clin. or post mortem samples. The stringent innate quality control allows a conclusive on-site diagnosis of CBPP such as during farm or slaughter house inspections.
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  12. 12
    Tanner, N. A.; Zhang, Y.; Evans, T. C. Simultaneous Multiple Target Detection in Real-Time Loop-Mediated Isothermal Amplification. BioTechniques 2012, 53 (2), 81– 89,  DOI: 10.2144/0000113902
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    12
    Simultaneous multiple target detection in real-time loop-mediated isothermal amplification
    Tanner, Nathan A.; Zhang, Yinhua; Evans, Thomas C., Jr.
    BioTechniques (2012), 53 (2), 81-82, 84, 86, 88-89CODEN: BTNQDO; ISSN:0736-6205. (Informa Healthcare)
    Loop-mediated isothermal amplification (LAMP) is a rapid and reliable sequence-specific isothermal nucleic acid amplification technique. To date, all reported real-time detection methods for LAMP have been restricted to single targets, limiting the utility of this technique. Here, we adapted std. LAMP primers to contain a quencher-fluorophore duplex region that upon strand sepn. results in a gain of fluorescent signal. This approach permitted the real-time detection of 1-4 target sequences in a single LAMP reaction tube utilizing a std. real-time fluorometer. The methodol. was highly reproducible and sensitive, detecting below 100 copies of human genomic DNA. It was also robust, with a 7-order of magnitude dynamic range of detectable targets. Furthermore, using a new strand-displacing DNA polymerase or its warm-start version, Bst 2.0 or Bst 2.0 WarmStart DNA polymerases, resulted in 50% faster amplification signals than wild-type Bst DNA polymerase, large fragment in this new multiplex LAMP procedure. The coupling of this new multiplex technique with next generation isothermal DNA polymerases should increase the utility of the LAMP method for mol. diagnostics.
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  13. 13
    Guatelli, J. C.; Whitfield, K. M.; Kwoh, D. Y.; Barringer, K. J.; Richman, D. D.; Gingeras, T. R. Isothermal, in Vitro Amplification of Nucleic Acids by a Multienzyme Reaction Modeled after Retroviral Replication. Proc. Natl. Acad. Sci. U. S. A. 1990, 87 (5), 1874– 1878,  DOI: 10.1073/pnas.87.5.1874
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    Walker, G. T.; Little, M. C.; Nadeau, J. G.; Shank, D. D. Isothermal in Vitro Amplification of DNA by a Restriction Enzyme/DNA Polymerase System. Proc. Natl. Acad. Sci. U. S. A. 1992, 89 (1), 392– 396,  DOI: 10.1073/pnas.89.1.392
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    14
    Isothermal in vitro amplification of DNA by a restriction enzyme/DNA polymerase system
    Walker, G. Terrance; Little, Michael C.; Nadeau, James G.; Shank, Daryl D.
    Proceedings of the National Academy of Sciences of the United States of America (1992), 89 (1), 392-6CODEN: PNASA6; ISSN:0027-8424.
    An isothermal in vitro DNA amplification method was developed based upon the following sequence of reaction events. Restriction enzyme cleavage and subsequent heat denaturation of a DNA sample generates 2 single-stranded target DNA fragment (T1 and T2). Present in excess are 2 DNA amplification primers (P1 and P2). The 3' end of P1 binds to the 3' and of T1, forming a duplex with 5' overhangs. Likewise, P2 binds to T2. The 5'overhangs of P1 and P2 contain a recognition sequence (5'-GTTGAC-3') for the restriction enzyme HincII. An exonuclease-deficient form of the large fragment of Escherichia coli DNA polymerase I (exo- Klenow polymerase) [Derbyshire, V., et al., (1988)] extends the 3' ends of the duplexes using dGTP, dCTP, TTP, and deoxyadenosine 5'-[α-thio]triphosphate, which produces hemiphosphorothioate recognition sites on P1·T1 and P2·T2. HincII nicks the unprotected primer strands of the hemiphosphorothioate recognition sites, leaving intact the modified complementary strands. The exo- Klenow polymerase extends the 3' end at the nick on P1·T1 and displaces the downstream strand that is functionally equiv. to T2. Likewise, extension at the nick on P2·T2 results in displacement of a downstream strand functionally equiv. to T1. Nicking and polymn./displacement steps cycle continuously on P1·T1 and P2·T2 because extension at a nick regenerates a nickable HincII recognition site. Target amplification is exponential because strands displaced from P1·T1 serve as targets for P2 and strands displaced from P2·T2 serve as targets for P1. A 106-fold amplification of a genomic sequence from Mycobacterium tuberculosis or M. bovis was achieved in 4 h at 37°.
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  15. 15
    Walker, G. T.; Fraiser, M. S.; Schram, J. L.; Little, M. C.; Nadeau, J. G.; Malinowski, D. P. Strand Displacement Amplification–an Isothermal, in Vitro DNA Amplification Technique. Nucleic Acids Res. 1992, 20 (7), 1691– 1696,  DOI: 10.1093/nar/20.7.1691
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    Fire, A.; Xu, S. Q. Rolling Replication of Short DNA Circles. Proc. Natl. Acad. Sci. U. S. A. 1995, 92 (10), 4641– 4645,  DOI: 10.1073/pnas.92.10.4641
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    Rolling replication of short DNA circles
    Fire, Andrew; Xu, Si-Qun
    Proceedings of the National Academy of Sciences of the United States of America (1995), 92 (10), 4641-5CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Natural genes and proteins often contain tandemly repeated sequence motifs that dramatically increase physiol. specificity and activity. Given the selective value of such repeats, it is likely that several different mechanisms have been responsible for their generation. One mechanism that has been shown to generate relatively long tandem repeats (in the kilobase range) in rolling circle replication. In this communication, we demonstrate that rolling circle synthesis in a simple enzymic system can produce tandem repeats of monomers as short as 34 bp. In addn. to suggesting possible origins for natural tandem repeats, these observations provide a facile means for constructing libraries of repeated motifs for use in "in vitro evolution" expts. designed to selected mols. with defined biol. or chem. properties.
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    Vincent, M.; Xu, Y.; Kong, H. Helicase-Dependent Isothermal DNA Amplification. EMBO Rep. 2004, 5 (8), 795– 800,  DOI: 10.1038/sj.embor.7400200
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    17
    Helicase-dependent isothermal DNA amplification
    Vincent, Myriam; Xu, Yan; Kong, Huimin
    EMBO Reports (2004), 5 (8), 795-800CODEN: ERMEAX; ISSN:1469-221X. (Nature Publishing Group)
    Polymerase chain reaction is the most widely used method for in vitro DNA amplification. However, it requires thermocycling to sep. two DNA strands. In vivo, DNA is replicated by DNA polymerases with various accessory proteins, including a DNA helicase that acts to sep. duplex DNA. The authors have devised a new in vitro isothermal DNA amplification method by mimicking this in vivo mechanism. Helicase-dependent amplification (HDA) utilizes a DNA helicase to generate single-stranded templates for primer hybridization and subsequent primer extension by a DNA polymerase. HDA does not require thermocycling. In addn., it offers several advantages over other isothermal DNA amplification methods by having a simple reaction scheme and being a true isothermal reaction that can be performed at one temp. for the entire process. These properties offer a great potential for the development of simple portable DNA diagnostic devices to be used in the field and at the point-of-care.
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    Piepenburg, O.; Williams, C. H.; Stemple, D. L.; Armes, N. A. DNA Detection Using Recombination Proteins. PLoS Biol. 2006, 4 (7), e204,  DOI: 10.1371/journal.pbio.0040204
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    Boczkowska, M.; Guga, P.; Stec, W. J. Stereodefined Phosphorothioate Analogues of DNA: Relative Thermodynamic Stability of the Model PS-DNA/DNA and PS-DNA/RNA Complexes†. Biochemistry 2002, 41 (41), 12483– 12487,  DOI: 10.1021/bi026225z
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    Jung, C.; Ellington, A. D. A Primerless Molecular Diagnostic: Phosphorothioated-Terminal Hairpin Formation and Self-Priming Extension (PS-THSP). Anal. Bioanal. Chem. 2016, 408 (30), 8583– 8591,  DOI: 10.1007/s00216-016-9479-y
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    20
    A primerless molecular diagnostic: phosphorothioated-terminal hairpin formation and self-priming extension (PS-THSP)
    Jung, Cheulhee; Ellington, Andrew D.
    Analytical and Bioanalytical Chemistry (2016), 408 (30), 8583-8591CODEN: ABCNBP; ISSN:1618-2642. (Springer)
    There are various ways that priming can occur in nucleic acid amplification reactions. While most reactions rely on a primer to initiate amplification, a mechanism for DNA amplification has been developed in which hairpin sequences at the 3' terminus of a single-stranded oligonucleotide fold on themselves to initiate priming. Unfortunately, this method is less useful for diagnostic applications because the self-folding efficiency is low and only works over a narrow range of reaction temps. In order to adapt this strategy for anal. applications the authors have developed a variant that the authors term phosphorothioated-terminal hairpin formation and self-priming extension (PS-THSP). In PS-THSP a phosphorothioate (PS) modification is incorporated into the DNA backbone, leading to a redn. in the thermal stability of dsDNA and increased self-folding of terminal hairpins. By optimizing the no. of PS linkages that are included in the initial template, the authors greatly increased self-folding efficiency and the range of reaction temps., ultimately achieving a detection limit of 1 pM. This improved method was readily adapted to the detection of single nucleotide polymorphisms and to the detection of non-nucleic acid analytes, such as alk. phosphatase, which was quant. detected at a limit of 0.05 mU/mL, ∼10-fold better than com. assays.
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    LaPlanche, L. A.; James, T. L.; Powell, C.; Wilson, W. D.; Uznanski, B.; Stec, W. J.; Summers, M. F.; Zon, G. Phosphorothioate-Modified Oligodeoxyribonucleotides. III. NMR and UV Spectroscopic Studies of the Rp-Rp, Sp-Sp, and Rp-Sp Duplexes, [d(GGSAATTCC)]2, Derived from Diastereomeric O-Ethyl Phosphorothioates. Nucleic Acids Res. 1986, 14 (22), 9081– 9093,  DOI: 10.1093/nar/14.22.9081
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    Jiang, Y. S.; Bhadra, S.; Li, B.; Wu, Y. R.; Milligan, J. N.; Ellington, A. D. Robust Strand Exchange Reactions for the Sequence-Specific, Real-Time Detection of Nucleic Acid Amplicons. Anal. Chem. 2015, 87 (6), 3314– 3320,  DOI: 10.1021/ac504387c
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    Robust strand exchange reactions for the sequence-specific, real-time detection of nucleic acid amplicons
    Jiang, Yu Sherry; Bhadra, Sanchita; Li, Bingling; Wu, Yuefeng Rose; Milligan, John N.; Ellington, Andrew D.
    Analytical Chemistry (Washington, DC, United States) (2015), 87 (6), 3314-3320CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Loop-mediated isothermal amplification (LAMP) of DNA is a powerful isothermal nucleic acid amplification method that can generate upward of 109 copies from less than 100 copies of template DNA within an hour. Unfortunately, although the amplification reactions are extremely powerful, real-time and specific detection of LAMP products remains anal. challenging. In order to both improve the specificity of LAMP detection and to make readout simpler and more reliable, we have replaced the intercalating dye typically used for monitoring in real-time fluorescence with a toehold-mediated strand exchange reaction termed one-step strand displacement (OSD). Due to the inherent sequence specificity of toehold-mediated strand exchange, the OSD reporter could successfully distinguish side products from true amplicons arising from templates corresponding to the biomedically relevant M. tuberculosis RNA polymerase (rpoB) and the melanoma-related biomarker BRAF. OSD allowed the Yes/No detection of rpoB in a complex mixt. such as synthetic sputum and also demonstrated single nucleotide specificity in Yes/No detection of a mutant BRAF allele (V600E) in the presence of 20-fold more of the wild-type gene. Real-time detection of different genes in multiplex LAMP reactions also proved possible. The development of simple, readily designed, modular equiv. of TaqMan probes for isothermal amplification reactions should generally improve the applicability of these reactions and may eventually assist with the development of point-of-care tests.
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    Bhadra, S.; Jiang, Y. S.; Kumar, M. R.; Johnson, R. F.; Hensley, L. E.; Ellington, A. D. Real-Time Sequence-Validated Loop-Mediated Isothermal Amplification Assays for Detection of Middle East Respiratory Syndrome Coronavirus (MERS-CoV). PLoS One 2015, 10 (4), e0123126,  DOI: 10.1371/journal.pone.0123126
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    23
    Real-time sequence-validated loop-mediated isothermal amplification assays for detection of middle east respiratory syndrome coronavirus (MERS-CoV)
    Bhadra, Sanchita; Jiang, Yu Sherry; Kumar, Mia R.; Johnson, Reed F.; Hensley, Lisa E.; Ellington, Andrew D.
    PLoS One (2015), 10 (4), e0123126/1-e0123126/21CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
    The Middle East respiratory syndrome coronavirus (MERS-CoV), an emerging human coronavirus, causes severe acute respiratory illness with a 35% mortality rate. In light of the recent surge in reported infections we have developed asym. five-primer reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for detection of MERS-CoV. Isothermal amplification assays will facilitate the development of portable point-of-care diagnostics that are crucial for management of emerging infections. The RT-LAMP assays are designed to amplify MERS-CoV genomic loci located within the open reading frame (ORF)1a and ORF1b genes and upstream of the E gene. Addnl. we applied one-step strand displacement probes (OSD) for real-time sequence-specific verification of LAMP amplicons. Asym. amplification effected by incorporating a single loop primer in each assay accelerated the time-to-result of the OSD-RT-LAMP assays. The resulting assays could detect 0.02 to 0.2 plaque forming units (PFU) (5 to 50 PFU/mL) of MERS-CoV in infected cell culture supernatants within 30 to 50 min and did not cross-react with common human respiratory pathogens.
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    Zahran, M.; Berezniak, T.; Imhof, P.; Smith, J. C. Role of Magnesium Ions in DNA Recognition by the EcoRV Restriction Endonuclease. FEBS Lett. 2011, 585 (17), 2739– 2743,  DOI: 10.1016/j.febslet.2011.07.036
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    Sissi, C.; Palumbo, M. Effects of Magnesium and Related Divalent Metal Ions in Topoisomerase Structure and Function. Nucleic Acids Res. 2009, 37 (3), 702– 711,  DOI: 10.1093/nar/gkp024
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    Chang, Y.; Gong, L.; Yuan, W.; Li, X.; Chen, G.; Li, X.; Zhang, Q.; Wu, C. Replication Protein A (RPA1a) Is Required for Meiotic and Somatic DNA Repair But Is Dispensable for DNA Replication and Homologous Recombination in Rice. Plant Physiol. 2009, 151 (4), 2162– 2173,  DOI: 10.1104/pp.109.142877
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    Zhu, Z.; Ravelet, C.; Perrier, S.; Guieu, V.; Fiore, E.; Peyrin, E. Single-Stranded DNA Binding Protein-Assisted Fluorescence Polarization Aptamer Assay for Detection of Small Molecules. Anal. Chem. 2012, 84 (16), 7203– 7211,  DOI: 10.1021/ac301552e
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    27
    Single-Stranded DNA Binding Protein-Assisted Fluorescence Polarization Aptamer Assay for Detection of Small Molecules
    Zhu, Zhenyu; Ravelet, Corinne; Perrier, Sandrine; Guieu, Valerie; Fiore, Emmanuelle; Peyrin, Eric
    Analytical Chemistry (Washington, DC, United States) (2012), 84 (16), 7203-7211CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Here, we describe a new fluorescence polarization aptamer assay (FPAA) strategy which is based on the use of the single-stranded DNA binding (SSB) protein from Escherichia coli as a strong FP signal enhancer tool. This approach relied on the unique ability of the SSB protein to bind the nucleic acid aptamer in its free state but not in its target-bound folded one. Such a feature was exploited by using the antiadenosine (Ade)-DNA aptamer (Apt-A) as a model functional nucleic acid. Two fluorophores (fluorescein and Texas Red) were introduced into different sites of Apt-A to design a dozen fluorescent tracers. In the absence of the Ade target, the binding of the labeled aptamers to SSB governed a very high fluorescence anisotropy increase (in the 0.130-0.200 range) as the consequence of (i) the large global diffusion difference between the free and SSB-bound tracers and (ii) the restricted movement of the dye in the SSB-bound state. When the analyte was introduced into the reaction system, the formation of the folded tertiary structure of the Ade-Apt-A complex triggered the release of the labeled nucleic acids from the protein, leading to a strong decrease in the fluorescence anisotropy. The key factors involved in the fluorescence anisotropy change were considered through the development of a competitive displacement model, and the optimal tracer candidate was selected for the Ade assay under buffer and realistic (dild. human serum) conditions. The SSB-assisted principle was found to operate also with another aptamer system, i.e., the antiargininamide DNA aptamer, and a different biosensing configuration, i.e., the sandwich-like design, suggesting the broad usefulness of the present approach. This sensing platform allowed generation of a fluorescence anisotropy signal for aptamer probes which did not operate under the direct format and greatly improved the assay response relative to that of the most previously reported small target FPAA.
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    Shlyakhtenko, L. S.; Lushnikov, A. Y.; Miyagi, A.; Lyubchenko, Y. L. Specificity of Binding of Single-Stranded DNA-Binding Protein to Its Target. Biochemistry 2012, 51 (7), 1500– 1509,  DOI: 10.1021/bi201863z
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    Rogers, K.; Hobgood, M.; Nance, J.; Cline, D.; Browning, S.; Eason, M.; Eversburg, A.; Lawson, N.; Campbell, L.; Wilhelm, D. Structural Modeling of Gene 32 Protein and SSB’s Roles in DNA Replication, Recombination and Repair. FASEB J. 2010, 24 (1 Supplement), lb48– lb48
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    Singer, A.; Kuhn, H.; Frank-Kamenetskii, M.; Meller, A. Detection of Urea-Induced Internal Denaturation of DsDNA Using Solid-State Nanopores. J. Phys.: Condens. Matter 2010, 22 (45), 454111,  DOI: 10.1088/0953-8984/22/45/454111
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    30
    Detection of urea-induced internal denaturation of dsDNA using solid-state nanopores
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    Microfluidic continuous flow digital loop-mediated isothermal amplification (LAMP)
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    Lab on a Chip (2015), 15 (3), 776-782CODEN: LCAHAM; ISSN:1473-0189. (Royal Society of Chemistry)
    Digital nucleic acid detection is rapidly becoming a popular technique for ultra-sensitive and quant. detection of nucleic acid mols. in a wide range of biomedical studies. Digital polymerase chain reaction (PCR) remains the most popular way of conducting digital nucleic acid detection. However, due to the need for thermocycling, digital PCR is difficult to implement in a streamlined manner on a single microfluidic device, leading to complex fragmented workflows and multiple sep. devices and instruments. Loop-mediated isothermal amplification (LAMP) is an excellent isothermal alternative to PCR with potentially better specificity than PCR because of the use of multiple primer sets for a nucleic acid target. Here we report a microfluidic droplet device implementing all the steps required for digital nucleic acid detection including droplet generation, incubation and in-line detection for digital LAMP. As compared to microchamber or droplet array-based digital assays, the continuous flow operation of this device eliminates the constraints on the no. of total reactions imposed by the footprint of the device and the anal. throughput caused by the time for lengthy incubation and transfer of materials between instruments.
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  • Abstract

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    Figure 1

    Figure 1. Scheme of a phosphorothioated loop-mediated isothermal amplification (PS-LAMP).

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    Figure 2

    Figure 2. Comparison of R-LAMP and PS-LAMP at different temperatures for MERS 1b. (a) Under the same buffer condition, R-LAMP and PS-LAMP were performed at different temperatures (line 1, no template/R-LAMP/65 °C; line 2, 1.2 × 108 copies of template/R-LAMP/65 °C; line 3, no template/PS-LAMP/65 °C; line 4, 1.2 × 108 copies of template/PS-LAMP/65 °C; line 5, no template/R-LAMP/60 °C; line 6, 1.2 × 108 copies of template/R-LAMP/60 °C; line 7, no template/PS-LAMP/60 °C; line 8, 1.2 × 108 copies of template/PS-LAMP/60 °C). (b) Fluorescence intensities at 150 min for R-LAMP and PS-LAMP in the presence of 1.2 × 108 copies of template were compared at different temperatures. (c) R-LAMP and PS-LAMP were performed at 45 °C with an optimized buffer (12 U of Bst 2.0 DNA polymerase, 0.5 μg of ET SSB, and 2 mM of MgSO4).

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    Figure 3

    Figure 3. Effects of urea and Bst 2.0 DNA polymerase on PS-LAMP at 40 °C for MERS 1b. Fluorescence intensities were monitored with different amounts of urea (line 1, 0 M/no template; line 2, 0 M/1.2 × 108 copies of template; line 3, 0.48 M/no template; line 4, 0.48 M/1.2 × 108 copies of template; line 5, 0.96 M/no template; line 6, 0.96 M/1.2 × 108 copies of template; line 7, 1.2 M/no template; line 8, 1.2 M/1.2 × 108 copies of template; line 9, 1.44 M/no template; line 10, 1.44 M/1.2 × 108 copies of template) in the buffer (12 U of Bst 2.0 DNA polymerase, 0.5 μg of ET SSB, and 2 mM of MgSO4) (a) or Bst 2.0 DNA polymerase (line 1, 20 U/no template; line 2, 20 U/1.2 × 108 copies of template; line 3, 40 U/no template; line 4, 40 U/1.2 × 108 copies of template; line 5, 60 U/no template; line 6, 60 U/1.2 × 108 copies of template; line 7, 80 U/no template; line 8, 80 U/1.2 × 108 copies of template; line 9, 120 U/no template; line 10, 120 U/1.2 × 108 copies of template) in the buffer (1.44 M of urea, 0.5 μg of ET SSB, and 2 mM of MgSO4) (c) during PS-LAMP at 40 °C. DT values were plotted with the concentrations of urea (b) and units of Bst 2.0 DNA polymerase (d).

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    Figure 4

    Figure 4. Quantitative analysis of PS-LAMP for different templates. The fluorescence intensities were monitored for PS-LAMP at 40 °C in the buffer (1.44 M of urea, 60 U of Bst 2.0 DNA polymerase, 0.5 μg of ET SSB, and 2 mM of MgSO4) with titrated MERS 1b (a) and MERS 1a (b) plasmids.

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  • References

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    This article references 36 other publications.

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      We have developed a novel method, termed loop-mediated isothermal amplification (LAMP), that amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions. This method employs a DNA polymerase and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA. An inner primer contg. sequences of the sense and anti-sense strands of the target DNA initiates LAMP. The following strand displacement DNA synthesis primed by an outer primer releases a single-stranded DNA. This serves as template for DNA synthesis primed by the second inner and outer primers that hybridize to the other end of the target, which produces a stem-loop DNA structure. In subsequent LAMP cycling one inner primer hybridizes to the loop on the product and initiates displacement DNA synthesis, yielding the original stem-loop DNA and a new stem-loop DNA with a stem twice as long. The cycling reaction continues with accumulation of 109 copies of target in less than an hour. The final products are stem-loop DNAs with several inverted repeats of the target and cauliflower-like structures with multiple loops formed by annealing between alternately inverted repeats of the target in the same strand. Because LAMP recognizes the target by six distinct sequences initially and by four distinct sequences afterwards, it is expected to amplify the target sequence with high selectivity.
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      Leptospirosis has been one of the worldwide zoonotic diseases caused by pathogenicLeptospira spp. Many mol. techniques have consecutively been developed to detect such pathogen including loop-mediated isothermal amplification method (LAMP). The objectives of this study were to evaluate the diagnostic accuracy of LAMP assay and real-time PCR using bacterial culture as the gold std. and to assess the agreement among these three tests using Cohen's kappa statistics. In total, 533 urine samples were collected from 266 beef and 267 dairy cattle reared in central region of Thailand. Sensitivity and specificity of LAMP were 96.8% (95% CI 81.5-99.8) and 97.0% (95% CI 94.9-98.2), resp. The accuracy of LAMP (97.0%) was significantly higher than that of real-time PCR (91.9%) at 95% CI. With Cohen's kappa statistics, culture method and LAMP were substantially agreed with each other (77.4%), whereas real-time PCR only moderately agreed with culture (47.7%) and LAMP (45.3%), resp. Consequently, LAMP was more effective than real-time PCR in detecting Leptospira spp. in the urine of cattle. Besides, LAMP had less cost and was simpler than real-time PCR. Thus, LAMP was an excellent alternative for routine surveillance of leptospirosis in cattle.
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      Molecular and Cellular Probes (2016), 30 (4), 205-210CODEN: MCPRE6; ISSN:0890-8508. (Elsevier Ltd.)
      In the present study 28 Trueperella pyogenes strains isolated from various origins could successfully be identified with a newly designed loop-mediated isothermal amplification (LAMP) assay based on gene cpn60 encoding chaperonin. No cross reaction could be obsd. with control strains representing four species of genus Trueperella and seven species of closely related genus Arcanobacterium. The present cpn60 LAMP assay might allow a reliable and low cost identification of T. pyogenes also in labs. with less specified equipment.
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      The recent outbreak of Zika virus (ZIKV) infection in the Americas and its devastating impact on fetal development have prompted the World Health Organization (WHO) to declare the ZIKV pandemic as a Public Health Emergency of International Concern. Rapid and reliable diagnostics for ZIKV are vital because ZIKV-infected individuals display no symptoms or nonspecific symptoms similar to other viral infections. Because immunoassays lack adequate sensitivity and selectivity and are unable to identify active state of infection, mol. diagnostics are an effective means to detect ZIKV soon after infection and throughout pregnancy. We report on a highly sensitive reverse-transcription loop-mediated, isothermal amplification (RT-LAMP) assay for rapid detection of ZIKV and its implementation in a simple, easy-to-use, inexpensive, point-of-care (POC) disposable cassette that carries out all the unit operations from sample introduction to detection. For thermal control of the cassette, we use a chem. heated cup without a need for elec. power. Amplification products are detected with leuco crystal violet (LCV) dye by eye without a need for instrumentation. We demonstrated the utility of our POC diagnostic system by detecting ZIKV in oral samples with sensitivity of 5 plaque-forming units (PFU) in less than 40 min. Our system is particularly suitable for resource-poor settings, where centralized lab. facilities, funds, and trained personnel are in short supply, and for use in doctors' offices, clinics, and at home.
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      Scientific Reports (2016), 6 (), 28935CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)
      A rapid LAMP (loop-mediated isothermal amplification) detection method was developed on the basis of the ITS sequence of P. viticola, the major causal agent of grape downy mildew. Among the 38 fungal and oomycete species tested, DNA isolated exclusively from P. viticola resulted in a specific product after LAMP amplification. This assay had high sensitivity and was able to detect the presence of less than 33 fg of genomic DNA per 25-μL reaction within 30 min. The infected leaves may produce sporangia that serve as a secondary inoculum. The developed LAMP assay is efficient for estg. the latent infection of grape leaves by P. viticola. When combined with the rapid and simple DNA extn. method, this assay's total detection time is shortened to approx. one hour; therefore it is suitable for on-site detection of latent infection in the field. The sporangia levels in the air are strongly assocd. with disease severity. The LAMP method was also demonstrated to be able to est. the level of sporangia released in the air in a certain period. This assay should make disease forecasting more accurate and rapid and should be helpful in decision-making regarding the control of grape downy mildew.
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      Scientific Reports (2016), 6 (), 26533CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)
      Single-nucleotide polymorphisms (SNPs) represent the most widespread type of genetic variation (approx. 90%) in the human genome, and the demand to overcome such variation has received more attention now than ever before. The capacity to rapidly assess SNPs that correlate with disease predisposition, drug efficacy and drug toxicity is a key step for the development of personalized medicine. In this work, a rapid one-step SNP detection method, real-time loop-mediated isothermal amplification (RT-LAMP), was first applied for CYP2C19 polymorphisms testing. The optimized method was established with specifically designed primers for target amplification by real-time detection in approx. 30 min under isothermal conditions. RT-LAMP amplified few copies of template to produce significant amts. of product and quant. detected human DNA with compatible specificity and sensitivity. The success in the establishment of this RT-LAMP protocol for CYP2C19 polymorphism testing is significant for the extension of this technique for the detection of other SNPs, which will further facilitate the development of personalized medicine.
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      As the human genome is decoded and its involvement in diseases is being revealed through postgenome research, increased adoption of genetic testing is expected. Crit. to such testing methods is the ease of implementation and comprehensible presentation of amplification results. Loop-mediated isothermal amplification (LAMP) is a simple, rapid, specific and cost-effective nucleic acid amplification method when compared to PCR, nucleic acid sequence-based amplification, self-sustained sequence replication and strand displacement amplification. This protocol details an improved simple visual detection system for the results of the LAMP reaction. In LAMP, a large amt. of DNA is synthesized, yielding a large pyrophosphate ion byproduct. Pyrophosphate ion combines with divalent metallic ion to form an insol. salt. Adding manganous ion and calcein, a fluorescent metal indicator, to the reaction soln. allows a visualization of substantial alteration of the fluorescence during the one-step amplification reaction, which takes 30-60 min. As the signal recognition is highly sensitive, this system enables visual discrimination of results without costly specialized equipment. This detection method should be helpful in basic research on medicine and pharmacy, environmental hygiene, point-of-care testing and more.
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      Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method that amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions using a set of four specially designed primers and a DNA polymerase with strand displacement activity. We have developed a method that accelerates the LAMP reaction by using addnl. primers, termed loop primers. Loop primers hybridize to the stem-loops, except for the loops that are hybridized by the inner primers, and prime strand displacement DNA synthesis. Although both inner and loop primers react via the loops, they do so by different mechanisms. The LAMP method presented here uses loop primers to achieve reaction times of less than half that of the original LAMP method. Since the total time of anal. including detection is less than 1 h, this new method should facilitate genetic anal., including genetic diagnosis in the clin. lab.
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      Contagious Bovine Pleuropneumonia (CBPP) is the most important chronic pulmonary disease of cattle on the African continent causing severe economic losses. The disease, caused by infection with Mycoplasma mycoides subsp. mycoides is transmitted by animal contact and develops slowly into a chronic form preventing an early clin. diagnosis. Because available vaccines confer a low protection rate and short-lived immunity, the rapid diagnosis of infected animals combined with traditional curbing measures is seen as the best way to control the disease. While traditional labor-intensive bacteriol. methods for the detection of M. mycoides subsp. mycoides have been replaced by mol. genetic techniques in the last two decades, these latter approaches require well-equipped labs. and specialized personnel for the diagnosis. This is a handicap in areas where CBPP is endemic and early diagnosis is essential. We present a rapid, sensitive and specific diagnostic tool for M. mycoides subsp. mycoides detection based on isothermal loop-mediated amplification (LAMP) that is applicable to field conditions. The primer set developed is highly specific and sensitive enough to diagnose clin. cases without prior cultivation of the organism. The LAMP assay detects M. mycoides subsp. mycoides DNA directly from crude samples of pulmonary/pleural fluids and serum/plasma within an hour using a simple diln. protocol. A photometric detection of LAMP products allows the real-time visualization of the amplification curve and the application of a melting curve/re-assocn. anal. presents a means of quality assurance based on the predetd. strand-inherent temp. profile supporting the diagnosis. The CBPP LAMP developed in a robust kit format can be run on a battery-driven mobile device to rapidly detect M. mycoides subsp. mycoides infections from clin. or post mortem samples. The stringent innate quality control allows a conclusive on-site diagnosis of CBPP such as during farm or slaughter house inspections.
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      Loop-mediated isothermal amplification (LAMP) is a rapid and reliable sequence-specific isothermal nucleic acid amplification technique. To date, all reported real-time detection methods for LAMP have been restricted to single targets, limiting the utility of this technique. Here, we adapted std. LAMP primers to contain a quencher-fluorophore duplex region that upon strand sepn. results in a gain of fluorescent signal. This approach permitted the real-time detection of 1-4 target sequences in a single LAMP reaction tube utilizing a std. real-time fluorometer. The methodol. was highly reproducible and sensitive, detecting below 100 copies of human genomic DNA. It was also robust, with a 7-order of magnitude dynamic range of detectable targets. Furthermore, using a new strand-displacing DNA polymerase or its warm-start version, Bst 2.0 or Bst 2.0 WarmStart DNA polymerases, resulted in 50% faster amplification signals than wild-type Bst DNA polymerase, large fragment in this new multiplex LAMP procedure. The coupling of this new multiplex technique with next generation isothermal DNA polymerases should increase the utility of the LAMP method for mol. diagnostics.
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      Rolling replication of short DNA circles
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      Natural genes and proteins often contain tandemly repeated sequence motifs that dramatically increase physiol. specificity and activity. Given the selective value of such repeats, it is likely that several different mechanisms have been responsible for their generation. One mechanism that has been shown to generate relatively long tandem repeats (in the kilobase range) in rolling circle replication. In this communication, we demonstrate that rolling circle synthesis in a simple enzymic system can produce tandem repeats of monomers as short as 34 bp. In addn. to suggesting possible origins for natural tandem repeats, these observations provide a facile means for constructing libraries of repeated motifs for use in "in vitro evolution" expts. designed to selected mols. with defined biol. or chem. properties.
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      EMBO Reports (2004), 5 (8), 795-800CODEN: ERMEAX; ISSN:1469-221X. (Nature Publishing Group)
      Polymerase chain reaction is the most widely used method for in vitro DNA amplification. However, it requires thermocycling to sep. two DNA strands. In vivo, DNA is replicated by DNA polymerases with various accessory proteins, including a DNA helicase that acts to sep. duplex DNA. The authors have devised a new in vitro isothermal DNA amplification method by mimicking this in vivo mechanism. Helicase-dependent amplification (HDA) utilizes a DNA helicase to generate single-stranded templates for primer hybridization and subsequent primer extension by a DNA polymerase. HDA does not require thermocycling. In addn., it offers several advantages over other isothermal DNA amplification methods by having a simple reaction scheme and being a true isothermal reaction that can be performed at one temp. for the entire process. These properties offer a great potential for the development of simple portable DNA diagnostic devices to be used in the field and at the point-of-care.
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      Jung, C.; Ellington, A. D. A Primerless Molecular Diagnostic: Phosphorothioated-Terminal Hairpin Formation and Self-Priming Extension (PS-THSP). Anal. Bioanal. Chem. 2016, 408 (30), 8583– 8591,  DOI: 10.1007/s00216-016-9479-y
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      A primerless molecular diagnostic: phosphorothioated-terminal hairpin formation and self-priming extension (PS-THSP)
      Jung, Cheulhee; Ellington, Andrew D.
      Analytical and Bioanalytical Chemistry (2016), 408 (30), 8583-8591CODEN: ABCNBP; ISSN:1618-2642. (Springer)
      There are various ways that priming can occur in nucleic acid amplification reactions. While most reactions rely on a primer to initiate amplification, a mechanism for DNA amplification has been developed in which hairpin sequences at the 3' terminus of a single-stranded oligonucleotide fold on themselves to initiate priming. Unfortunately, this method is less useful for diagnostic applications because the self-folding efficiency is low and only works over a narrow range of reaction temps. In order to adapt this strategy for anal. applications the authors have developed a variant that the authors term phosphorothioated-terminal hairpin formation and self-priming extension (PS-THSP). In PS-THSP a phosphorothioate (PS) modification is incorporated into the DNA backbone, leading to a redn. in the thermal stability of dsDNA and increased self-folding of terminal hairpins. By optimizing the no. of PS linkages that are included in the initial template, the authors greatly increased self-folding efficiency and the range of reaction temps., ultimately achieving a detection limit of 1 pM. This improved method was readily adapted to the detection of single nucleotide polymorphisms and to the detection of non-nucleic acid analytes, such as alk. phosphatase, which was quant. detected at a limit of 0.05 mU/mL, ∼10-fold better than com. assays.
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      LaPlanche, L. A.; James, T. L.; Powell, C.; Wilson, W. D.; Uznanski, B.; Stec, W. J.; Summers, M. F.; Zon, G. Phosphorothioate-Modified Oligodeoxyribonucleotides. III. NMR and UV Spectroscopic Studies of the Rp-Rp, Sp-Sp, and Rp-Sp Duplexes, [d(GGSAATTCC)]2, Derived from Diastereomeric O-Ethyl Phosphorothioates. Nucleic Acids Res. 1986, 14 (22), 9081– 9093,  DOI: 10.1093/nar/14.22.9081
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      Jiang, Y. S.; Bhadra, S.; Li, B.; Wu, Y. R.; Milligan, J. N.; Ellington, A. D. Robust Strand Exchange Reactions for the Sequence-Specific, Real-Time Detection of Nucleic Acid Amplicons. Anal. Chem. 2015, 87 (6), 3314– 3320,  DOI: 10.1021/ac504387c
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      Robust strand exchange reactions for the sequence-specific, real-time detection of nucleic acid amplicons
      Jiang, Yu Sherry; Bhadra, Sanchita; Li, Bingling; Wu, Yuefeng Rose; Milligan, John N.; Ellington, Andrew D.
      Analytical Chemistry (Washington, DC, United States) (2015), 87 (6), 3314-3320CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Loop-mediated isothermal amplification (LAMP) of DNA is a powerful isothermal nucleic acid amplification method that can generate upward of 109 copies from less than 100 copies of template DNA within an hour. Unfortunately, although the amplification reactions are extremely powerful, real-time and specific detection of LAMP products remains anal. challenging. In order to both improve the specificity of LAMP detection and to make readout simpler and more reliable, we have replaced the intercalating dye typically used for monitoring in real-time fluorescence with a toehold-mediated strand exchange reaction termed one-step strand displacement (OSD). Due to the inherent sequence specificity of toehold-mediated strand exchange, the OSD reporter could successfully distinguish side products from true amplicons arising from templates corresponding to the biomedically relevant M. tuberculosis RNA polymerase (rpoB) and the melanoma-related biomarker BRAF. OSD allowed the Yes/No detection of rpoB in a complex mixt. such as synthetic sputum and also demonstrated single nucleotide specificity in Yes/No detection of a mutant BRAF allele (V600E) in the presence of 20-fold more of the wild-type gene. Real-time detection of different genes in multiplex LAMP reactions also proved possible. The development of simple, readily designed, modular equiv. of TaqMan probes for isothermal amplification reactions should generally improve the applicability of these reactions and may eventually assist with the development of point-of-care tests.
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    23. 23
      Bhadra, S.; Jiang, Y. S.; Kumar, M. R.; Johnson, R. F.; Hensley, L. E.; Ellington, A. D. Real-Time Sequence-Validated Loop-Mediated Isothermal Amplification Assays for Detection of Middle East Respiratory Syndrome Coronavirus (MERS-CoV). PLoS One 2015, 10 (4), e0123126,  DOI: 10.1371/journal.pone.0123126
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      Real-time sequence-validated loop-mediated isothermal amplification assays for detection of middle east respiratory syndrome coronavirus (MERS-CoV)
      Bhadra, Sanchita; Jiang, Yu Sherry; Kumar, Mia R.; Johnson, Reed F.; Hensley, Lisa E.; Ellington, Andrew D.
      PLoS One (2015), 10 (4), e0123126/1-e0123126/21CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
      The Middle East respiratory syndrome coronavirus (MERS-CoV), an emerging human coronavirus, causes severe acute respiratory illness with a 35% mortality rate. In light of the recent surge in reported infections we have developed asym. five-primer reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for detection of MERS-CoV. Isothermal amplification assays will facilitate the development of portable point-of-care diagnostics that are crucial for management of emerging infections. The RT-LAMP assays are designed to amplify MERS-CoV genomic loci located within the open reading frame (ORF)1a and ORF1b genes and upstream of the E gene. Addnl. we applied one-step strand displacement probes (OSD) for real-time sequence-specific verification of LAMP amplicons. Asym. amplification effected by incorporating a single loop primer in each assay accelerated the time-to-result of the OSD-RT-LAMP assays. The resulting assays could detect 0.02 to 0.2 plaque forming units (PFU) (5 to 50 PFU/mL) of MERS-CoV in infected cell culture supernatants within 30 to 50 min and did not cross-react with common human respiratory pathogens.
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      Zahran, M.; Berezniak, T.; Imhof, P.; Smith, J. C. Role of Magnesium Ions in DNA Recognition by the EcoRV Restriction Endonuclease. FEBS Lett. 2011, 585 (17), 2739– 2743,  DOI: 10.1016/j.febslet.2011.07.036
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      Sissi, C.; Palumbo, M. Effects of Magnesium and Related Divalent Metal Ions in Topoisomerase Structure and Function. Nucleic Acids Res. 2009, 37 (3), 702– 711,  DOI: 10.1093/nar/gkp024
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      Chang, Y.; Gong, L.; Yuan, W.; Li, X.; Chen, G.; Li, X.; Zhang, Q.; Wu, C. Replication Protein A (RPA1a) Is Required for Meiotic and Somatic DNA Repair But Is Dispensable for DNA Replication and Homologous Recombination in Rice. Plant Physiol. 2009, 151 (4), 2162– 2173,  DOI: 10.1104/pp.109.142877
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      Zhu, Z.; Ravelet, C.; Perrier, S.; Guieu, V.; Fiore, E.; Peyrin, E. Single-Stranded DNA Binding Protein-Assisted Fluorescence Polarization Aptamer Assay for Detection of Small Molecules. Anal. Chem. 2012, 84 (16), 7203– 7211,  DOI: 10.1021/ac301552e
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      Single-Stranded DNA Binding Protein-Assisted Fluorescence Polarization Aptamer Assay for Detection of Small Molecules
      Zhu, Zhenyu; Ravelet, Corinne; Perrier, Sandrine; Guieu, Valerie; Fiore, Emmanuelle; Peyrin, Eric
      Analytical Chemistry (Washington, DC, United States) (2012), 84 (16), 7203-7211CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Here, we describe a new fluorescence polarization aptamer assay (FPAA) strategy which is based on the use of the single-stranded DNA binding (SSB) protein from Escherichia coli as a strong FP signal enhancer tool. This approach relied on the unique ability of the SSB protein to bind the nucleic acid aptamer in its free state but not in its target-bound folded one. Such a feature was exploited by using the antiadenosine (Ade)-DNA aptamer (Apt-A) as a model functional nucleic acid. Two fluorophores (fluorescein and Texas Red) were introduced into different sites of Apt-A to design a dozen fluorescent tracers. In the absence of the Ade target, the binding of the labeled aptamers to SSB governed a very high fluorescence anisotropy increase (in the 0.130-0.200 range) as the consequence of (i) the large global diffusion difference between the free and SSB-bound tracers and (ii) the restricted movement of the dye in the SSB-bound state. When the analyte was introduced into the reaction system, the formation of the folded tertiary structure of the Ade-Apt-A complex triggered the release of the labeled nucleic acids from the protein, leading to a strong decrease in the fluorescence anisotropy. The key factors involved in the fluorescence anisotropy change were considered through the development of a competitive displacement model, and the optimal tracer candidate was selected for the Ade assay under buffer and realistic (dild. human serum) conditions. The SSB-assisted principle was found to operate also with another aptamer system, i.e., the antiargininamide DNA aptamer, and a different biosensing configuration, i.e., the sandwich-like design, suggesting the broad usefulness of the present approach. This sensing platform allowed generation of a fluorescence anisotropy signal for aptamer probes which did not operate under the direct format and greatly improved the assay response relative to that of the most previously reported small target FPAA.
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      Shlyakhtenko, L. S.; Lushnikov, A. Y.; Miyagi, A.; Lyubchenko, Y. L. Specificity of Binding of Single-Stranded DNA-Binding Protein to Its Target. Biochemistry 2012, 51 (7), 1500– 1509,  DOI: 10.1021/bi201863z
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      Rogers, K.; Hobgood, M.; Nance, J.; Cline, D.; Browning, S.; Eason, M.; Eversburg, A.; Lawson, N.; Campbell, L.; Wilhelm, D. Structural Modeling of Gene 32 Protein and SSB’s Roles in DNA Replication, Recombination and Repair. FASEB J. 2010, 24 (1 Supplement), lb48– lb48
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      Singer, A.; Kuhn, H.; Frank-Kamenetskii, M.; Meller, A. Detection of Urea-Induced Internal Denaturation of DsDNA Using Solid-State Nanopores. J. Phys.: Condens. Matter 2010, 22 (45), 454111,  DOI: 10.1088/0953-8984/22/45/454111
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      Detection of urea-induced internal denaturation of dsDNA using solid-state nanopores
      Singer, Alon; Kuhn, Heiko; Frank-Kamenetskii, Maxim; Meller, Amit
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      The ability to detect and measure dsDNA thermal fluctuations is of immense importance in understanding the underlying mechanisms responsible for transcription and replication regulation. The authors describe here the ability of solid-state nanopores to detect sub-nanometer changes in DNA structure as a result of chem. enhanced thermal fluctuations. In this study, the authors investigate the subtle changes in the mean effective diam. of a dsDNA mol. with 3-5 nm solid-state nanopores as a function of urea concn. and the DNA's AT content. The authors' studies reveal an increase in the mean effective diam. of a DNA mol. of approx. 0.6 nm at 8.7 M urea. In agreement with the mechanism of DNA local denaturation, the authors observe a sigmoid dependence of these effects on urea concn. The authors find that the translocation times in urea are markedly slower than would be expected if the dynamics were governed primarily by viscous effects. Furthermore, the authors find that the sensitivity of the nanopore is sufficient to statistically differentiate between DNA mols. of nearly identical lengths differing only in sequence and AT content when placed in 3.5 M urea. The authors' results demonstrate that nanopores can detect subtle structural changes and are thus a valuable tool for detecting differences in biomols.' environment.
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      Conway, B. E. Effect of Urea on the Viscosity of Deoxyribonucleic Acid Solutions. J. Polym. Sci. 1956, 20 (95), 299– 306,  DOI: 10.1002/pol.1956.120209506
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      Effect of urea on the viscosity of deoxyribonucleic acid solutions
      Conway, B. E.
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      The viscosity of urea-treated deoxyribonucleic acid (DNA) was measured in H2O and in 0.1M NaCl at low concns. and low rates of shear. The apparent intrinsic viscosity of aq. DNA solns. increased on treatment with urea. In NaCl soln. the viscosity was decreased. No change in the viscosity of the NaCl soln. occurred after removal of the urea by dialysis. The results are consistent with a dimeric mol. structure in which the 2 halves of the mol. are held together by H bonds. The mol. thus undergoes scission in the presence of urea to form flexible fragments.
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      Schwinefus, J. J.; Engelsgjerd, S.; Mangold, K.; Thompson, P. Urea Induced DNA Denaturation. Biophys. J. 2013, 104 (2), 425a,  DOI: 10.1016/j.bpj.2012.11.2364
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      Nixon, G. J.; Svenstrup, H. F.; Donald, C. E.; Carder, C.; Stephenson, J. M.; Morris-Jones, S.; Huggett, J. F.; Foy, C. A. A Novel Approach for Evaluating the Performance of Real Time Quantitative Loop-Mediated Isothermal Amplification-Based Methods. Biomol. Detect. Quantif. 2014, 2, 4– 10,  DOI: 10.1016/j.bdq.2014.11.001
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      A novel approach for evaluating the performance of real time quantitative loop-mediated isothermal amplification-based methods
      Nixon Gavin J; Donald Carol E; Huggett Jim F; Foy Carole A; Svenstrup Helle F; Carder Caroline; Stephenson Judith M; Morris-Jones Stephen
      Biomolecular detection and quantification (2014), 2 (), 4-10 ISSN:2214-7535.
      Molecular diagnostic measurements are currently underpinned by the polymerase chain reaction (PCR). There are also a number of alternative nucleic acid amplification technologies, which unlike PCR, work at a single temperature. These 'isothermal' methods, reportedly offer potential advantages over PCR such as simplicity, speed and resistance to inhibitors and could also be used for quantitative molecular analysis. However there are currently limited mechanisms to evaluate their quantitative performance, which would assist assay development and study comparisons. This study uses a sexually transmitted infection diagnostic model in combination with an adapted metric termed isothermal doubling time (IDT), akin to PCR efficiency, to compare quantitative PCR and quantitative loop-mediated isothermal amplification (qLAMP) assays, and to quantify the impact of matrix interference. The performance metric described here facilitates the comparison of qLAMP assays that could assist assay development and validation activities.
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      Tzeling, J. M. W.; Yean, C. Y. A Shelf-Stable Fluorogenic Isothermal Amplification Assay for the Detection of Burkholderia Pseudomallei. Analyst 2016, 141 (4), 1246– 1249,  DOI: 10.1039/C5AN01741F
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      Sun, Y.; Quyen, T. L.; Hung, T. Q.; Chin, W. H.; Wolff, A.; Bang, D. D. A Lab-on-a-Chip System with Integrated Sample Preparation and Loop-Mediated Isothermal Amplification for Rapid and Quantitative Detection of Salmonella Spp. in Food Samples. Lab Chip 2015, 15 (8), 1898– 1904,  DOI: 10.1039/C4LC01459F
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      35
      A lab-on-a-chip system with integrated sample preparation and loop-mediated isothermal amplification for rapid and quantitative detection of Salmonella spp. in food samples
      Sun, Yi; Quyen, Than Linh; Hung, Tran Quang; Chin, Wai Hoe; Wolff, Anders; Bang, Dang Duong
      Lab on a Chip (2015), 15 (8), 1898-1904CODEN: LCAHAM; ISSN:1473-0189. (Royal Society of Chemistry)
      Foodborne disease is a major public health threat worldwide. Salmonellosis, an infectious disease caused by Salmonella spp., is one of the most common foodborne diseases. Isolation and identification of Salmonella by conventional bacterial culture or mol.-based methods are time consuming and usually take a few hours to days to complete. In response to the demand for rapid on line or on site detection of pathogens, in this study, we describe for the first time an eight-chamber lab-on-a-chip (LOC) system with integrated magnetic bead-based sample prepn. and loop-mediated isothermal amplification (LAMP) for rapid and quant. detection of Salmonella spp. in food samples. The whole diagnostic procedures including DNA isolation, isothermal amplification, and real-time detection were accomplished in a single chamber. Up to eight samples could be handled simultaneously and the system was capable to detect Salmonella at concn. of 50 cells per test within 40 min. The simple design, together with high level of integration, isothermal amplification, and quant. anal. of multiple samples in short time, will greatly enhance the practical applicability of the LOC system for rapid on-site screening of Salmonella for applications in food safety control, environmental surveillance, and clin. diagnostics.
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    36. 36
      Rane, T. D.; Chen, L.; Zec, H. C.; Wang, T.-H. Microfluidic Continuous Flow Digital Loop-Mediated Isothermal Amplification (LAMP). Lab Chip 2015, 15 (3), 776– 782,  DOI: 10.1039/C4LC01158A
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      Microfluidic continuous flow digital loop-mediated isothermal amplification (LAMP)
      Rane, Tushar D.; Chen, Liben; Zec, Helena C.; Wang, Tza-Huei
      Lab on a Chip (2015), 15 (3), 776-782CODEN: LCAHAM; ISSN:1473-0189. (Royal Society of Chemistry)
      Digital nucleic acid detection is rapidly becoming a popular technique for ultra-sensitive and quant. detection of nucleic acid mols. in a wide range of biomedical studies. Digital polymerase chain reaction (PCR) remains the most popular way of conducting digital nucleic acid detection. However, due to the need for thermocycling, digital PCR is difficult to implement in a streamlined manner on a single microfluidic device, leading to complex fragmented workflows and multiple sep. devices and instruments. Loop-mediated isothermal amplification (LAMP) is an excellent isothermal alternative to PCR with potentially better specificity than PCR because of the use of multiple primer sets for a nucleic acid target. Here we report a microfluidic droplet device implementing all the steps required for digital nucleic acid detection including droplet generation, incubation and in-line detection for digital LAMP. As compared to microchamber or droplet array-based digital assays, the continuous flow operation of this device eliminates the constraints on the no. of total reactions imposed by the footprint of the device and the anal. throughput caused by the time for lengthy incubation and transfer of materials between instruments.
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    The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.8b02062.

    • Scheme of one-step strand displacement (OSD); effects of MgSO4, RecA, and ET SSB at different temperatures; selectivity analysis of PS-LAMP for different templates; quantitative analysis of regular LAMP for different templates; and oligonucleotide sequences used in this study (PDF)


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