Quenching of Unincorporated Amplification Signal Reporters in Reverse-Transcription Loop-Mediated Isothermal Amplification Enabling Bright, Single-Step, Closed-Tube, and Multiplexed Detection of RNA VirusesClick to copy article linkArticle link copied!
Abstract
Reverse-transcription-loop-mediated isothermal amplification (RT-LAMP) has frequently been proposed as an enabling technology for simplified diagnostic tests for RNA viruses. However, common detection techniques used for LAMP and RT-LAMP have drawbacks, including poor discrimination capability, inability to multiplex targets, high rates of false positives, and (in some cases) the requirement of opening reaction tubes postamplification. Here, we present a simple technique that allows closed-tube, target-specific detection, based on inclusion of a dye-labeled primer that is incorporated into a target-specific amplicon if the target is present. A short, complementary quencher hybridizes to unincorporated primer upon cooling down at the end of the reaction, thereby quenching fluorescence of any unincorporated primer. Our technique, which we term QUASR (for quenching of unincorporated amplification signal reporters, read “quasar”), does not significantly reduce the amplification efficiency or sensitivity of RT-LAMP. Equipped with a simple LED excitation source and a colored plastic gel filter, the naked eye or a camera can easily discriminate between positive and negative QUASR reactions, which produce a difference in signal of approximately 10:1 without background subtraction. We demonstrate that QUASR detection is compatible with complex sample matrices such as human blood, using a novel LAMP primer set for bacteriophage MS2 (a model RNA virus particle). Furthermore, we demonstrate single-tube duplex detection of West Nile virus (WNV) and chikungunya virus (CHIKV) RNA.
Materials and Methods
LAMP Primer Design
Viral Templates
QUASR Primer Design
RT-LAMP Assays
Results and Discussion
Figure 1
Figure 1. Principle of QUASR detection in LAMP or RT-LAMP. One of the loop primers (LF or LB) or inner primers (FIP or BIP) is labeled with a dye. The reaction mixture also contains a short probe, labeled with a dark quencher at the 3′ end, and complementary to 7–13 bases at the 5′ end of the dye labeled primer. The quench probe is present at slight excess relative to the labeled primer and has Tm > 10 °C below the temperature of the LAMP reaction, such that it remains dissociated during the amplification. After incubation, the reaction is cooled to ambient temperature, resulting in dark quenching of fluorescent primers (negative reactions) or highly fluorescent amplicons (positive reactions).
Figure 2
Figure 2. QUASR improves end point discrimination between positive and negative reactions compared to an intercalating dye: (A) comparison of room temperature end point detection with QUASR versus the intercalating dye SYTO 62 for RT-LAMP amplification of MS2 phage in PCR tubes. The top row of tubes shows positive reactions and the bottom row of tubes shows negative reactions. The four pairs of reactions on the left utilize QUASR via FIP-Cy5 with varying amounts of complementary quenching probe, FIPc, for detection. The two pairs of reactions on the right utilize SYTO 62 for detection. (B) Annealing curves for QUASR (1.6 μM FIP-Cy5 with 2.4 μM FIPc) and SYTO 62 (4 μM) reactions postamplification, by monitoring fluorescence in the Cy5 channel, while cooling from 85 to 25 °C in a real-time PCR machine. (C) Signal discrimination (positive/negative fluorescent signal) as reactions cool from 85 to 25 °C.
Figure 3
Figure 3. QUASR enables room temperature discrimination between positive and negative RT-LAMP reactions in 10% whole blood. In contrast, discrimination with SYTO 62 is completely lost in the presence of whole blood. Comparison by Tukey’s test within 10% blood group following ANOVA, P < 0.0001. Other differences were statistically significant but not shown.
Figure 4
Figure 4. Multiplexed visual detection of WNV/CHIKV by QUASR RT-LAMP. 100 PFU equivalent of each viral RNA was used in each reaction where indicated by a plus sign. No template controls are indicated with a negative sign. WNV positives appear bright red when excited with green light (A), and CHIKV positives appear bright green when excited with blue light (B). A composite overlay of the images shows that the combination appears yellow (C). The image from an iPhone 6 using an unfiltered blue LED excitation source and a plastic theater gel as an emission filter confirms multiplexed detection (D).
Figure 5
Figure 5. QUASR LAMP and DARQ LAMP exist on a continuum. (A) Real-time fluorescence detection of 10 000 PFU equivalent WNV RNA per 10 μL of reaction by RT-LAMP using FIP-ROX primer. Increasing the melting temperature of the FIP-complementary quencher probe decreases background fluorescence but dramatically slows amplification time. Quencher probes with internal base pair mismatches are denoted with the letter “m” at the end of their name. A full list of quencher probes is provided in Table S2. The arrow demonstrates the transition from QUASR RT-LAMP to DARQ RT-LAMP, represented by the full-length quenching probe FIPc-25. (B) The time to positivity, determined by real time monitoring with SYTO dye in a separate fluorescence channel, increases dramatically as the FIP/FIPc complex melting temperature approaches and surpasses the reaction temperature for RT-LAMP. Melting temperature is far more important than even a 1 000-fold change in WNV template RNA concentration.
Conclusion
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b04054.
Primer and quencher sequences; viral culture methods; characterization of RT-LAMP primer set for MS2 phage; reduction of false positives by QUASR RT-LAMP; and additional fluorescence images of duplex QUASR RT-LAMP (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.
Acknowledgment
This work was supported by Sandia National Laboratories’ Laboratory-Directed Research and Development (LDRD) Program, Grant 173111 (PI: Meagher). Sandia National Laboratories is a multiprogram laboratory managed and operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corporation, for the U.S. Department of Energy’s National Nuclear Security Administration under Contract DE-AC04-94AL85000.
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Abstract
Figure 1
Figure 1. Principle of QUASR detection in LAMP or RT-LAMP. One of the loop primers (LF or LB) or inner primers (FIP or BIP) is labeled with a dye. The reaction mixture also contains a short probe, labeled with a dark quencher at the 3′ end, and complementary to 7–13 bases at the 5′ end of the dye labeled primer. The quench probe is present at slight excess relative to the labeled primer and has Tm > 10 °C below the temperature of the LAMP reaction, such that it remains dissociated during the amplification. After incubation, the reaction is cooled to ambient temperature, resulting in dark quenching of fluorescent primers (negative reactions) or highly fluorescent amplicons (positive reactions).
Figure 2
Figure 2. QUASR improves end point discrimination between positive and negative reactions compared to an intercalating dye: (A) comparison of room temperature end point detection with QUASR versus the intercalating dye SYTO 62 for RT-LAMP amplification of MS2 phage in PCR tubes. The top row of tubes shows positive reactions and the bottom row of tubes shows negative reactions. The four pairs of reactions on the left utilize QUASR via FIP-Cy5 with varying amounts of complementary quenching probe, FIPc, for detection. The two pairs of reactions on the right utilize SYTO 62 for detection. (B) Annealing curves for QUASR (1.6 μM FIP-Cy5 with 2.4 μM FIPc) and SYTO 62 (4 μM) reactions postamplification, by monitoring fluorescence in the Cy5 channel, while cooling from 85 to 25 °C in a real-time PCR machine. (C) Signal discrimination (positive/negative fluorescent signal) as reactions cool from 85 to 25 °C.
Figure 3
Figure 3. QUASR enables room temperature discrimination between positive and negative RT-LAMP reactions in 10% whole blood. In contrast, discrimination with SYTO 62 is completely lost in the presence of whole blood. Comparison by Tukey’s test within 10% blood group following ANOVA, P < 0.0001. Other differences were statistically significant but not shown.
Figure 4
Figure 4. Multiplexed visual detection of WNV/CHIKV by QUASR RT-LAMP. 100 PFU equivalent of each viral RNA was used in each reaction where indicated by a plus sign. No template controls are indicated with a negative sign. WNV positives appear bright red when excited with green light (A), and CHIKV positives appear bright green when excited with blue light (B). A composite overlay of the images shows that the combination appears yellow (C). The image from an iPhone 6 using an unfiltered blue LED excitation source and a plastic theater gel as an emission filter confirms multiplexed detection (D).
Figure 5
Figure 5. QUASR LAMP and DARQ LAMP exist on a continuum. (A) Real-time fluorescence detection of 10 000 PFU equivalent WNV RNA per 10 μL of reaction by RT-LAMP using FIP-ROX primer. Increasing the melting temperature of the FIP-complementary quencher probe decreases background fluorescence but dramatically slows amplification time. Quencher probes with internal base pair mismatches are denoted with the letter “m” at the end of their name. A full list of quencher probes is provided in Table S2. The arrow demonstrates the transition from QUASR RT-LAMP to DARQ RT-LAMP, represented by the full-length quenching probe FIPc-25. (B) The time to positivity, determined by real time monitoring with SYTO dye in a separate fluorescence channel, increases dramatically as the FIP/FIPc complex melting temperature approaches and surpasses the reaction temperature for RT-LAMP. Melting temperature is far more important than even a 1 000-fold change in WNV template RNA concentration.
References
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- 20Naze, F.; Le Roux, K.; Schuffenecker, I.; Zeller, H.; Staikowsky, F.; Grivard, P.; Michault, A.; Laurent, P. J. Virol. Methods 2009, 162 (1–2) 1– 7 DOI: 10.1016/j.jviromet.2009.03.00620https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXht1GgtbbJ&md5=985ad28aa3837e83b3de0acef69fa45eSimultaneous detection and quantitation of Chikungunya, Dengue and West Nile viruses by multiplex RT-PCR assays and Dengue virus typing using High Resolution MeltingNaze, F.; Le Roux, K.; Schuffenecker, I.; Zeller, H.; Staikowsky, F.; Grivard, P.; Michault, A.; Laurent, P.Journal of Virological Methods (2009), 162 (1-2), 1-7CODEN: JVMEDH; ISSN:0166-0934. (Elsevier B.V.)Chikungunya (CHIKV), Dengue (DENV) and West Nile (WNV) viruses are arthropod-borne viruses that are able to emerge or re-emerge in many regions due to climatic changes and increase in travel. Since these viruses produce similar clin. signs it is important for physicians and epidemiologists to differentiate them rapidly. A mol. method was developed for their detection and quantitation in plasma samples and a DENV typing technique were developed. The method consisted in performing two multiplex real-time one-step RT-PCR assays, to detect and quantify the three viruses. Both assays were conducted in a single run, from a single RNA ext. contg. a unique coextd. and coamplified composite internal control. The quantitation results were close to the best detection thresholds obtained with simplex RT-PCR techniques. The differentiation of DENV types was performed using a High Resoln. Melting technique. The assays enable the early diagnosis of the three arboviruses during viremia, including cases of coinfection. The method is rapid, specific and highly sensitive with a potential for clin. diagnosis and epidemiol. surveillance. A DENV pos. sample can be typed conveniently using the High Resoln. Melting technique using the same app.
- 21Sun, B.; Shen, F.; McCalla, S. E.; Kreutz, J. E.; Karymov, M. A.; Ismagilov, R. F. Anal. Chem. 2013, 85 (3) 1540– 1546 DOI: 10.1021/ac3037206There is no corresponding record for this reference.
- 22Curtis, K. A.; Rudolph, D. L.; Owen, S. M. J. Virol. Methods 2008, 151 (2) 264– 270 DOI: 10.1016/j.jviromet.2008.04.01122https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXosVymsb0%253D&md5=143d792b41c1a94327f643872bebc2e4Rapid detection of HIV-1 by reverse-transcription, loop-mediated isothermal amplification (RT-LAMP)Curtis, Kelly A.; Rudolph, Donna L.; Owen, S. MicheleJournal of Virological Methods (2008), 151 (2), 264-270CODEN: JVMEDH; ISSN:0166-0934. (Elsevier B.V.)A rapid, cost-effective diagnostic or confirmatory test for the detection of early HIV-1 infection is highly desired, esp. for use in resource-poor or point-of-care settings. The reverse-transcription loop-mediated isothermal amplification (RT-LAMP) technol. has been evaluated for the detection of HIV-1 DNA and RNA, using six RT-LAMP primers designed against highly conserved sequences located within the protease and p24 gene regions. Amplification from lab-adapted HIV-1 DNA and RNA was detected as early as 30 min, with max. sensitivity of 10 and 100 copies per reaction, resp., reached at 60 min. Comparable sensitivity was obsd. with extd. nucleic acid from plasma and blood samples of HIV-1-infected individuals. Furthermore, the RT-LAMP procedure was modified for the direct detection of HIV-1 nucleic acid in plasma and blood samples, eliminating the need for an addnl. nucleic acid extn. step and reducing the overall procedure time to approx. 90 min.
- 23Dauner, A. L.; Mitra, I.; Gilliland, T., Jr.; Seales, S.; Pal, S.; Yang, S.-C.; Guevara, C.; Chen, J.-H.; Liu, Y.-C.; Kochel, T. J.; Wu, S.-J. L. Diagn. Microbiol. Infect. Dis. 2015, 83 (1) 30– 36 DOI: 10.1016/j.diagmicrobio.2015.05.00423https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXpt1Kqs7g%253D&md5=481144b5894b0a9972e9c3391ea8129bDevelopment of a pan-serotype reverse transcription loop-mediated isothermal amplification assay for the detection of dengue virusDauner, Allison L.; Mitra, Indrani; Gilliland, Theron, Jr.; Seales, Sajeewane; Pal, Subhamoy; Yang, Shih-Chun; Guevara, Carolina; Chen, Jiann-Hwa; Liu, Yung-Chuan; Kochel, Tadeusz J.; Wu, Shuenn-Jue L.Diagnostic Microbiology and Infectious Disease (2015), 83 (1), 30-36CODEN: DMIDDZ; ISSN:0732-8893. (Elsevier)During dengue outbreaks, acute diagnosis at the patient's point of need followed by appropriate supportive therapy reduces morbidity and mortality. To facilitate needed diagnosis, we developed and optimized a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay that detects all 4 serotypes of dengue virus (DENV). We used a quencher to reduce nonspecific amplification. The assay does not require expensive thermocyclers, utilizing a simple water bath to maintain the reaction at 63 °C. Results can be visualized using UV fluorescence, handheld readers, or lateral flow immunochromatog. tests. We report a sensitivity of 86.3% (95% confidence interval [CI], 72.7-94.8%) and specificity of 93.0% (95% CI, 83.0-98.1%) using a panel of clin. specimens characterized by DENV quant. reverse transcription-polymerase chain reaction. This pan-serotype DENV RT-LAMP can be adapted to field-expedient formats where it can provide actionable diagnosis near the patient's point of need.
- 24Rudolph, D. L.; Sullivan, V.; Owen, S. M.; Curtis, K. A. PLoS One 2015, 10 (5) e0126609– e0126613 DOI: 10.1371/journal.pone.0126609There is no corresponding record for this reference.
Supporting Information
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b04054.
Primer and quencher sequences; viral culture methods; characterization of RT-LAMP primer set for MS2 phage; reduction of false positives by QUASR RT-LAMP; and additional fluorescence images of duplex QUASR RT-LAMP (PDF)
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