Development of a Singleplex Real-Time Reverse Transcriptase PCR Assay for Pan-Dengue Virus Detection and Quantification
<p>Performance of 3′-UTR primers/probes. Quantification cycle (Cq) of 3′-UTR region (<b>a</b>–<b>e</b>) was determined using serial 10-fold diluted (10<sup>1</sup> to 10<sup>6</sup> RNA copies) in vitro transcript DENV1-4RNA as template. Coefficient of determination (R<sup>2</sup>) and PCR amplification efficiency (E) for DENV1 (<b>a</b>), DENV2 (<b>b</b>), DENV3 (<b>c</b>), and DENV4 (<b>d</b>) were analyzed from 8 independent experiments. The variation of Cq values at each RNA concentration among serotypes was analyzed by Bonferroni’s Multiple Comparison Test (<b>e</b>). RNAs extracted from cultured supernatants of Japanese encephalitis virus (JEV, Nakayama strain), yellow fever virus (YFV, 17D strain), zika virus (ZIKV, ZV0127 strain), DENV1 Hawaii, DENV2 16681, DENV3 H87, and DENV4 H241 were used as RNA templates to verify the specificity of our 3′-UTR primers/probes (<b>f</b>). Primers specific to E gene of JEV, YFV, or ZIKV were used to confirm the existence of RNA templates of each virus type. The sizes of PCR products for DENV1, DENV2, DENV3, DENV4, JEV, YFV, and ZIKV were 185, 187, 184, 189, 333, 306, and 365 base pairs, respectively. No RNA template (Neg) was used as a negative control. The PCR product was run in 2% agarose gel electrophoresis and was stained with gel red before visualization under UV light (<b>f</b>).</p> "> Figure 2
<p>Correlation of DENV genome levels quantified by RT-PCR specific to 3′-UTR and coding sequence. Quantification cycle (Cq) values or DENV genome levels (Log copies/mL) in various types of samples quantified by RT-PCR using the two types of probe/primer regions were compared and analyzed for correlation coefficient (R) and <span class="html-italic">p</span> values of linear regression. (<b>a</b>) A correlation plot showing Cq values from quantification of 144 samples of in vitro transcribed DENV1-4 RNA (ranging from 10<sup>1</sup>–10<sup>6</sup> copies/mL) from 6 independent experiments. (<b>b</b>) A correlation plot showing DENV genome levels (Log copies/mL) in DENV1-4 infected cell cultured supernatants (2–200,000 ffu/mL) from 15 independent experiments. (<b>c</b>) A correlation plot showing DENV genome levels (Log copies/mL) measured in plasma of 161 DENV infected patients collected since the first day of hospitalization to the day to defervescence (499 samples in total).</p> "> Figure 3
<p>Rate of DENV genome detection by real-time RT-PCR specific to only 3′-UTR or coding sequence. (<b>a</b>) Viral RNA levels in plasma of 161 DENV infected patients collected since the first day of hospitalization to the day to defervescence quantified by RT-PCR specific to 3′-UTR (gray circles) or coding sequence (white circles) were re-analyzed according to “Day to defervescence”. The number of samples are labeled on the top of each group. Detection rate of DENV detected by only 3′-UTR (gray bar) or only coding sequence (white bar) were analyzed according to “Day to defervescence” (<b>b</b>) or DENV serotypes (<b>c</b>).</p> "> Figure 4
<p>Efficiency of DENV genome detection. Data from <a href="#viruses-14-01271-f002" class="html-fig">Figure 2</a>c were re-analyzed according to “Day to defervescence”. Detection rates of 3′-UTR assay (black bar) or coding sequence assay (white bar) or detected by either assay (gray bar) were analyzed according to “Day to defervescence”. Proportions of DENV detection rates among groups were analyzed by McNemar’s test. Asterisks (** and ***) represent McNemar’s <span class="html-italic">p</span> values < 0.01 and <0.001, respectively.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Preparation of In Vitro RNA Standards
2.2. Preparation of Viruses from Cultured Cells
2.3. Clinical Specimens
2.4. Primers/Probes Design
2.5. RNA Extraction and Quantitative Real-Time RT-PCR
2.6. Statistical Analysis
3. Results
3.1. Performance of 3′-UTR Real-Time RT-PCR Assay
3.2. Efficiency of DENV Viral Genome Quantification of 3′-UTR Primers/Probes
3.3. Dual Region Detection Enhanced Sensitivity of qRT-PCR Assay
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Bhatt, S.; Gething, P.W.; Brady, O.J.; Messina, J.P.; Farlow, A.W.; Moyes, C.L.; Drake, J.M.; Brownstein, J.S.; Hoen, A.G.; Sankoh, O.; et al. The global distribution and burden of dengue. Nature 2013, 496, 504–507. [Google Scholar] [CrossRef] [PubMed]
- Messina, J.P.; Brady, O.J.; Golding, N.; Kraemer, M.U.G.; Wint, G.R.W.; Ray, S.E.; Pigott, D.M.; Shearer, F.M.; Johnson, K.; Earl, L.; et al. The current and future global distribution and population at risk of dengue. Nat. Microbiol. 2019, 4, 1508–1515. [Google Scholar] [CrossRef] [PubMed]
- Disease, G.B.D.; Injury, I.; Prevalence, C. Global, regional, and national incidence, prevalence, and years lived with disability for 354 diseases and injuries for 195 countries and territories, 1990–2017: A systematic analysis for the Global Burden of Disease Study 2017. Lancet 2018, 392, 1789–1858. [Google Scholar] [CrossRef]
- Dengue Haemorrhagic Fever: Diagnosis, Treatment, Prevention and Control; World Health Organization: Geneva, Switzerland, 1997.
- Halstead, S.B.; Russell, P.K. Protective and immunological behavior of chimeric yellow fever dengue vaccine. Vaccine 2016, 34, 1643–1647. [Google Scholar] [CrossRef]
- Hunsperger, E.A.; Yoksan, S.; Buchy, P.; Nguyen, V.C.; Sekaran, S.D.; Enria, D.A.; Pelegrino, J.L.; Vazquez, S.; Artsob, H.; Drebot, M.; et al. Evaluation of commercially available anti-dengue virus immunoglobulin M tests. Emerg. Infect. Dis. 2009, 15, 436–440. [Google Scholar] [CrossRef]
- Peeling, R.W.; Artsob, H.; Pelegrino, J.L.; Buchy, P.; Cardosa, M.J.; Devi, S.; Enria, D.A.; Farrar, J.; Gubler, D.J.; Guzman, M.G.; et al. Evaluation of diagnostic tests: Dengue. Nat. Rev. Microbiol. 2010, 8, S30–S37. [Google Scholar] [CrossRef]
- Dussart, P.; Petit, L.; Labeau, B.; Bremand, L.; Leduc, A.; Moua, D.; Matheus, S.; Baril, L. Evaluation of two new commercial tests for the diagnosis of acute dengue virus infection using NS1 antigen detection in human serum. PLoS Negl. Trop. Dis. 2008, 2, e280. [Google Scholar] [CrossRef]
- Namekar, M.; Ellis, E.M.; O’Connell, M.; Elm, J.; Gurary, A.; Park, S.Y.; Imrie, A.; Nerurkar, V.R. Evaluation of a new commercially available immunoglobulin M capture enzyme-linked immunosorbent assay for diagnosis of dengue virus infection. J. Clin. Microbiol. 2013, 51, 3102–3106. [Google Scholar] [CrossRef]
- Anderson, N.W.; Jespersen, D.J.; Rollins, L.; Seaton, B.; Prince, H.E.; Theel, E.S. Detection of the dengue virus NS1 antigen using an enzyme immunoassay. Diagn. Microbiol. Infect. Dis. 2014, 79, 194–197. [Google Scholar] [CrossRef]
- Pal, S.; Dauner, A.L.; Mitra, I.; Forshey, B.M.; Garcia, P.; Morrison, A.C.; Halsey, E.S.; Kochel, T.J.; Wu, S.J. Evaluation of dengue NS1 antigen rapid tests and ELISA kits using clinical samples. PLoS ONE 2014, 9, e113411. [Google Scholar] [CrossRef] [PubMed]
- Vivek, R.; Ahamed, S.F.; Kotabagi, S.; Chandele, A.; Khanna, I.; Khanna, N.; Nayak, K.; Dias, M.; Kaja, M.K.; Shet, A. Evaluation of a pan-serotype point-of-care rapid diagnostic assay for accurate detection of acute dengue infection. Diagn. Microbiol. Infect. Dis. 2017, 87, 229–234. [Google Scholar] [CrossRef]
- Kabir, M.A.; Zilouchian, H.; Younas, M.A.; Asghar, W. Dengue Detection: Advances in Diagnostic Tools from Conventional Technology to Point of Care. Biosensors 2021, 11, 206. [Google Scholar] [CrossRef]
- Lau, Y.L.; Lai, M.Y.; Teoh, B.T.; Abd-Jamil, J.; Johari, J.; Sam, S.S.; Tan, K.K.; AbuBakar, S. Colorimetric Detection of Dengue by Single Tube Reverse-Transcription-Loop-Mediated Isothermal Amplification. PLoS ONE 2015, 10, e0138694. [Google Scholar] [CrossRef]
- Neeraja, M.; Lakshmi, V.; Lavanya, V.; Priyanka, E.N.; Parida, M.M.; Dash, P.K.; Sharma, S.; Rao, P.V.; Reddy, G. Rapid detection and differentiation of dengue virus serotypes by NS1 specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay in patients presenting to a tertiary care hospital in Hyderabad, India. J. Virol. Methods 2015, 211, 22–31. [Google Scholar] [CrossRef]
- Gao, M.; Waggoner, J.J.; Hecht, S.M.; Chen, S. Selective Detection of Dengue Virus Serotypes Using Tandem Toehold-Mediated Displacement Reactions. ACS Infect. Dis. 2019, 5, 1907–1914. [Google Scholar] [CrossRef]
- Gao, M.; Daniel, D.; Zou, H.; Jiang, S.; Lin, S.; Huang, C.; Hecht, S.M.; Chen, S. Rapid detection of a dengue virus RNA sequence with single molecule sensitivity using tandem toehold-mediated displacement reactions. Chem. Commun. 2018, 54, 968–971. [Google Scholar] [CrossRef]
- Shu, P.Y.; Chang, S.F.; Kuo, Y.C.; Yueh, Y.Y.; Chien, L.J.; Sue, C.L.; Lin, T.H.; Huang, J.H. Development of group- and serotype-specific one-step SYBR green I-based real-time reverse transcription-PCR assay for dengue virus. J. Clin. Microbiol. 2003, 41, 2408–2416. [Google Scholar] [CrossRef]
- Johnson, B.W.; Russell, B.J.; Lanciotti, R.S. Serotype-specific detection of dengue viruses in a fourplex real-time reverse transcriptase PCR assay. J. Clin. Microbiol. 2005, 43, 4977–4983. [Google Scholar] [CrossRef]
- Kong, Y.Y.; Thay, C.H.; Tin, T.C.; Devi, S. Rapid detection, serotyping and quantitation of dengue viruses by TaqMan real-time one-step RT-PCR. J. Virol. Methods 2006, 138, 123–130. [Google Scholar] [CrossRef]
- Hue, K.D.; Tuan, T.V.; Thi, H.T.; Bich, C.T.; Anh, H.H.; Wills, B.A.; Simmons, C.P. Validation of an internally controlled one-step real-time multiplex RT-PCR assay for the detection and quantitation of dengue virus RNA in plasma. J. Virol. Methods 2011, 177, 168–173. [Google Scholar] [CrossRef]
- Srikiatkhachorn, A.; Wichit, S.; Gibbons, R.V.; Green, S.; Libraty, D.H.; Endy, T.P.; Ennis, F.A.; Kalayanarooj, S.; Rothman, A.L. Dengue viral RNA levels in peripheral blood mononuclear cells are associated with disease severity and preexisting dengue immune status. PLoS ONE 2012, 7, e51335. [Google Scholar] [CrossRef]
- Waggoner, J.J.; Abeynayake, J.; Sahoo, M.K.; Gresh, L.; Tellez, Y.; Gonzalez, K.; Ballesteros, G.; Pierro, A.M.; Gaibani, P.; Guo, F.P.; et al. Single-reaction, multiplex, real-time rt-PCR for the detection, quantitation, and serotyping of dengue viruses. PLoS Negl. Trop. Dis. 2013, 7, e2116. [Google Scholar] [CrossRef]
- Najioullah, F.; Viron, F.; Cesaire, R. Evaluation of four commercial real-time RT-PCR kits for the detection of dengue viruses in clinical samples. Virol. J. 2014, 11, 164. [Google Scholar] [CrossRef]
- Alm, E.; Lindegren, G.; Falk, K.I.; Lagerqvist, N. One-step real-time RT-PCR assays for serotyping dengue virus in clinical samples. BMC Infect. Dis. 2015, 15, 493. [Google Scholar] [CrossRef]
- Waggoner, J.J.; Ballesteros, G.; Gresh, L.; Mohamed-Hadley, A.; Tellez, Y.; Sahoo, M.K.; Abeynayake, J.; Balmaseda, A.; Harris, E.; Pinsky, B.A. Clinical evaluation of a single-reaction real-time RT-PCR for pan-dengue and chikungunya virus detection. J. Clin. Virol. 2016, 78, 57–61. [Google Scholar] [CrossRef]
- Yang, L.; Liang, W.; Jiang, L.; Li, W.; Cao, W.; Wilson, Z.A.; Zhang, D. A novel universal real-time PCR system using the attached universal duplex probes for quantitative analysis of nucleic acids. BMC Mol. Biol. 2008, 9, 54. [Google Scholar] [CrossRef]
- Brownie, J.; Shawcross, S.; Theaker, J.; Whitcombe, D.; Ferrie, R.; Newton, C.; Little, S. The elimination of primer-dimer accumulation in PCR. Nucleic Acids Res. 1997, 25, 3235–3241. [Google Scholar] [CrossRef]
- Polz, M.F.; Cavanaugh, C.M. Bias in template-to-product ratios in multitemplate PCR. Appl. Environ. Microbiol. 1998, 64, 3724–3730. [Google Scholar] [CrossRef]
- Prada-Arismendy, J.; Rincon, V.; Castellanos, J.E. Comparative evaluation of permissiveness to dengue virus serotype 2 infection in primary rodent macrophages. J. Trop. Med. 2012, 2012, 950303. [Google Scholar] [CrossRef]
- Yenchitsomanus, P.T.; Sricharoen, P.; Jaruthasana, I.; Pattanakitsakul, S.N.; Nitayaphan, S.; Mongkolsapaya, J.; Malasit, P. Rapid detection and identification of dengue viruses by polymerase chain reaction (PCR). S. Asian J. Trop. Med. Public Health 1996, 27, 228–236. [Google Scholar]
- Punyadee, N.; Mairiang, D.; Thiemmeca, S.; Komoltri, C.; Pan-Ngum, W.; Chomanee, N.; Charngkaew, K.; Tangthawornchaikul, N.; Limpitikul, W.; Vasanawathana, S.; et al. Microparticles provide a novel biomarker to predict severe clinical outcomes of dengue virus infection. J. Virol. 2015, 89, 1587–1607. [Google Scholar] [CrossRef]
- Lin, C.W.; Wu, S.C. A functional epitope determinant on domain III of the Japanese encephalitis virus envelope protein interacted with neutralizing-antibody combining sites. J. Virol. 2003, 77, 2600–2606. [Google Scholar] [CrossRef]
- Faye, O.; Faye, O.; Dupressoir, A.; Weidmann, M.; Ndiaye, M.; Alpha Sall, A. One-step RT-PCR for detection of Zika virus. J. Clin. Virol. 2008, 43, 96–101. [Google Scholar] [CrossRef]
- Innis, B.L.; Nisalak, A.; Nimmannitya, S.; Kusalerdchariya, S.; Chongswasdi, V.; Suntayakorn, S.; Puttisri, P.; Hoke, C.H. An enzyme-linked immunosorbent assay to characterize dengue infections where dengue and Japanese encephalitis co-circulate. Am. J. Trop. Med. Hyg. 1989, 40, 418–427. [Google Scholar] [CrossRef]
- Puttikhunt, C.; Prommool, T.; Nathaporn, U.; Ong-ajchaowlerd, P.; Yoosook, K.; Tawilert, C.; Duangchinda, T.; Jairangsri, A.; Tangthawornchaikul, N.; Malasit, P.; et al. The development of a novel serotyping-NS1-ELISA to identify serotypes of dengue virus. J. Clin. Virol. 2011, 50, 314–319. [Google Scholar] [CrossRef]
- Hall, T.A. Bioedit: A user-friendly biological sequence alignment editor and analysis program for windows 95/98/NT. Nucleic Acids Symp. Ser. 1999, 41, 95–98. [Google Scholar]
- Untergasser, A.; Cutcutache, I.; Koressaar, T.; Ye, J.; Faircloth, B.C.; Remm, M.; Rozen, S.G. Primer3--new capabilities and interfaces. Nucleic Acids Res. 2012, 40, e115. [Google Scholar] [CrossRef]
- R Core Team. R: A Language and Environment for Statistical Computing. Available online: https://www.R-project.org/ (accessed on 4 May 2018).
- Avirutnan, P.; Punyadee, N.; Noisakran, S.; Komoltri, C.; Thiemmeca, S.; Auethavornanan, K.; Jairungsri, A.; Kanlaya, R.; Tangthawornchaikul, N.; Puttikhunt, C.; et al. Vascular leakage in severe dengue virus infections: A potential role for the nonstructural viral protein NS1 and complement. J. Infect. Dis. 2006, 193, 1078–1088. [Google Scholar] [CrossRef]
- Pfaffl, M.W.; Horgan, G.W.; Dempfle, L. Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res. 2002, 30, e36. [Google Scholar] [CrossRef]
- Brooks, E.M.; Sheflin, L.G.; Spaulding, S.W. Secondary structure in the 3’ UTR of EGF and the choice of reverse transcriptases affect the detection of message diversity by RT-PCR. Biotechniques 1995, 19, 806–812. [Google Scholar]
- Aaskov, J.; Buzacott, K.; Thu, H.M.; Lowry, K.; Holmes, E.C. Long-term transmission of defective RNA viruses in humans and Aedes mosquitoes. Science 2006, 311, 236–238. [Google Scholar] [CrossRef] [PubMed]
- Li, D.; Lott, W.B.; Lowry, K.; Jones, A.; Thu, H.M.; Aaskov, J. Defective interfering viral particles in acute dengue infections. PLoS ONE 2011, 6, e19447. [Google Scholar] [CrossRef]
- Liu, R.; Yue, L.; Li, X.; Yu, X.; Zhao, H.; Jiang, Z.; Qin, E.; Qin, C. Identification and characterization of small sub-genomic RNAs in dengue 1-4 virus-infected cell cultures and tissues. Biochem. Biophys. Res. Commun. 2010, 391, 1099–1103. [Google Scholar] [CrossRef] [PubMed]
- Li, D.; Aaskov, J. Sub-genomic RNA of defective interfering (D.I.) dengue viral particles is replicated in the same manner as full length genomes. Virology 2014, 468–470, 248–255. [Google Scholar] [CrossRef]
- Pesko, K.N.; Fitzpatrick, K.A.; Ryan, E.M.; Shi, P.Y.; Zhang, B.; Lennon, N.J.; Newman, R.M.; Henn, M.R.; Ebel, G.D. Internally deleted WNV genomes isolated from exotic birds in New Mexico: Function in cells, mosquitoes, and mice. Virology 2012, 427, 10–17. [Google Scholar] [CrossRef]
- Sun, Y.; Jain, D.; Koziol-White, C.J.; Genoyer, E.; Gilbert, M.; Tapia, K.; Panettieri, R.A., Jr.; Hodinka, R.L.; Lopez, C.B. Immunostimulatory Defective Viral Genomes from Respiratory Syncytial Virus Promote a Strong Innate Antiviral Response during Infection in Mice and Humans. PLoS Pathog. 2015, 11, e1005122. [Google Scholar] [CrossRef]
- Vasilijevic, J.; Zamarreno, N.; Oliveros, J.C.; Rodriguez-Frandsen, A.; Gomez, G.; Rodriguez, G.; Perez-Ruiz, M.; Rey, S.; Barba, I.; Pozo, F.; et al. Reduced accumulation of defective viral genomes contributes to severe outcome in influenza virus infected patients. PLoS Pathog. 2017, 13, e1006650. [Google Scholar] [CrossRef]
- Li, Q.; Tong, Y.; Xu, Y.; Niu, J.; Zhong, J. Genetic Analysis of Serum-Derived Defective Hepatitis C Virus Genomes Revealed Novel Viral cis Elements for Virus Replication and Assembly. J. Virol. 2018, 92, e02182-17. [Google Scholar] [CrossRef]
- Simon, O.; Williams, T.; Caballero, P.; Lopez-Ferber, M. Dynamics of deletion genotypes in an experimental insect virus population. Proc. Biol. Sci. 2006, 273, 783–790. [Google Scholar] [CrossRef]
- Tapia, K.; Kim, W.K.; Sun, Y.; Mercado-Lopez, X.; Dunay, E.; Wise, M.; Adu, M.; Lopez, C.B. Defective viral genomes arising in vivo provide critical danger signals for the triggering of lung antiviral immunity. PLoS Pathog. 2013, 9, e1003703. [Google Scholar] [CrossRef]
- Parameswaran, P.; Wang, C.; Trivedi, S.B.; Eswarappa, M.; Montoya, M.; Balmaseda, A.; Harris, E. Intrahost Selection Pressures Drive Rapid Dengue Virus Microevolution in Acute Human Infections. Cell Host Microbe 2017, 22, 400–410.e5. [Google Scholar] [CrossRef] [PubMed]
- Poirier, E.Z.; Goic, B.; Tome-Poderti, L.; Frangeul, L.; Boussier, J.; Gausson, V.; Blanc, H.; Vallet, T.; Loyd, H.; Levi, L.I.; et al. Dicer-2-Dependent Generation of Viral DNA from Defective Genomes of RNA Viruses Modulates Antiviral Immunity in Insects. Cell Host Microbe 2018, 23, 353–365.e8. [Google Scholar] [CrossRef] [PubMed]
- Linder, A.; Bothe, V.; Linder, N.; Schwarzlmueller, P.; Dahlstrom, F.; Bartenhagen, C.; Dugas, M.; Pandey, D.; Thorn-Seshold, J.; Boehmer, D.F.R.; et al. Defective Interfering Genomes and the Full-Length Viral Genome Trigger RIG-I After Infection With Vesicular Stomatitis Virus in a Replication Dependent Manner. Front. Immunol. 2021, 12, 595390. [Google Scholar] [CrossRef] [PubMed]
Characteristics | Patients (n = 161) |
---|---|
Gender (male:female) | 95:66 |
Age [mean ± SD (min–max)] | 10.6 ± 2.6 (5–15) |
First date of collection [median (min–max)] | |
Day of illness | 3 (0–7) |
Day to defervescence * | −2 (−3–−1) |
Last date of collection [median (min–max)] | |
Day of illness | 6 (1–9) |
Day to defervescence | 0 (0) |
Severity (DF:DHF1:DHF2:DHF3) # | 80:22:53:6 |
DENV infection (primary:secondary) | 0:161 |
DENV1 (collected from year 2008 to 2013) | 39 |
DENV2 (collected from year 2006 to 2012) | 39 |
DENV3 (collected from year 2010 to 2013) | 39 |
DENV4 (collected from year 2006 to 2013) | 44 |
No. | Primers/Probes | Nucleotide Sequences (5′–3′) | Nucleotide No. (Region) | |
---|---|---|---|---|
1 | F primer | GGTTAGAGGAGACCCCTCCC | 10424–10443 (all DENV-3′-UTR) | |
R primer | GGCGY # TCTGTGCCTGGA | 10596–10612 (all DENV-3′-UTR) | ||
Probe | 6-FAM-CAGGATCTCTGGTCTY # TCCCAGCGT–BHQ | 10553–10577 (all DENV-3′-UTR) | ||
2 | F primer | CAAAAGGAAGTCGTGCAATA | 8974–8993 (DENV1-NS5) | |
R primer | CTGAGTGAATTCTCTCTR $ CTGAACC | 9061–9085 (DENV1-NS5) | ||
Probe | 6-FAM-CATGTGGTTGGGAGCACGC–BHQ | 8999–9017 (DENV1-NS5) | ||
3 | F primer | CAGGTTATGGCACY # GTCACR $ AT | 1463–1484 (DENV2-E) | |
R primer | CCATCTGCAGCAACACCATCTC | 1519–1540 (DENV2-E) | ||
Probe | 6-FAM-CTCY # CCGAGAACAGGCCTCGACTTCAA–BHQ | 1491–1517 (DENV2-E) | ||
4 | F primer | GGACTGGACACACGCACY # CA | 740–759 (DENV3-M) | |
R primer | CATGTCTCTACCTTCTCGACTTGTCT | 788–813 (DENV3-M) | ||
Probe | 6-FAM-ACCTGGATGTCGGCY # GAAGGAGCTTG–BHQ | 761–786 (DENV3-M) | ||
5 | F primer | TTGTY # CTAATGATGCTN & GTCG | 896–916 (DENV4-M/E) | |
R primer | TCCACCTGAGACTCCTTCY # A | 965–984 (DENV4-M/E) | ||
Probe | 6-FAM-TY # CCY # ACTCCTACGCATCGCATTCCG–BHQ | 927–952 (DENV4-M/E) |
Serotypes | Copies/Reaction | Positive/Total | Detection Rate (%) | Mean ± SD of Ct Values | % CV of Ct Values |
---|---|---|---|---|---|
DENV1 | 100 | 8/8 | 100 | 33.2 ± 0.5 | 1.6 |
10 * | 8/8 | 100 * | 36.3 ± 0.8 | 2.3 # | |
5 | 0/8 | 0 | UD 1 | - | |
DENV2 | 100 | 8/8 | 100 | 31.6 ± 0.3 | 1.0 |
10 * | 8/8 | 100 * | 35.2 ± 0.5 | 1.5 # | |
5 | 0/8 | 0 | UD | - | |
DENV3 | 100 | 8/8 | 100 | 32.8 ± 1.2 | 3.5 |
10 * | 8/8 | 100 * | 36.9 ± 1.0 | 2.7 # | |
5 | 0/8 | 0 | UD | - | |
DENV4 | 100 | 8/8 | 100 | 33.1 ± 1.7 | 5.3 |
10 * | 8/8 | 100 * | 35.8 ± 1.6 | 4.4 # | |
5 | 0/8 | 0 | UD | - |
DENV Serotypes | Total Patient Number | DENV Positive Number | Sensitivity of Detection (%) |
---|---|---|---|
All serotypes | 161 | 156 | 96.9 |
DENV1 | 39 | 39 | 100.0 |
DENV2 | 39 | 39 | 100.0 |
DENV3 | 39 | 39 | 100.0 |
DENV4 | 44 | 39 | 88.6 |
Day of Fever | Total Specimens | DENV Positive Specimens | Sensitivity of Detection (%) |
---|---|---|---|
≤2 | 79 | 79 | 100 |
3 | 112 | 98 | 87.5 |
4 | 145 | 91 | 62.8 |
≥5 | 192 | 52 | 27.1 |
Serotypes | Copies/Reaction | Positive/Total | Positivity Rate (%) | Mean ± SD of Ct Values | % CV of Ct Values |
---|---|---|---|---|---|
DENV1 | 100 | 8/8 | 100 | 29.3 ± 1.6 | 5.4 |
10 | 8/8 | 100 * | 32.6 ± 1.5 | 4.7 # | |
5 | 5/8 | 63 | NA 1 | - | |
DENV2 | 100 | 8/8 | 100 | 31.6 ± 0.3 | 2.8 |
10 | 8/8 | 100 * | 35.2 ± 0.5 | 3.1 # | |
5 | 1/8 | 13 | NA | - | |
DENV3 | 100 | 8/8 | 100 | 32.8 ± 1.2 | 2.4 |
10 | 8/8 | 100 * | 36.9 ± 1.0 | 2.4 # | |
5 | 7/8 | 88 | NA | - | |
DENV4 | 100 | 8/8 | 100 | 33.1 ± 1.7 | 4.2 |
10 | 8/8 | 100 * | 35.8 ± 1.6 | 2.0 # | |
5 | 7/8 | 88 | NA | - |
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Songjaeng, A.; Thiemmeca, S.; Mairiang, D.; Punyadee, N.; Kongmanas, K.; Hansuealueang, P.; Tangthawornchaikul, N.; Duangchinda, T.; Mongkolsapaya, J.; Sriruksa, K.; et al. Development of a Singleplex Real-Time Reverse Transcriptase PCR Assay for Pan-Dengue Virus Detection and Quantification. Viruses 2022, 14, 1271. https://doi.org/10.3390/v14061271
Songjaeng A, Thiemmeca S, Mairiang D, Punyadee N, Kongmanas K, Hansuealueang P, Tangthawornchaikul N, Duangchinda T, Mongkolsapaya J, Sriruksa K, et al. Development of a Singleplex Real-Time Reverse Transcriptase PCR Assay for Pan-Dengue Virus Detection and Quantification. Viruses. 2022; 14(6):1271. https://doi.org/10.3390/v14061271
Chicago/Turabian StyleSongjaeng, Adisak, Somchai Thiemmeca, Dumrong Mairiang, Nuntaya Punyadee, Kessiri Kongmanas, Prachya Hansuealueang, Nattaya Tangthawornchaikul, Thaneeya Duangchinda, Juthathip Mongkolsapaya, Kanokwan Sriruksa, and et al. 2022. "Development of a Singleplex Real-Time Reverse Transcriptase PCR Assay for Pan-Dengue Virus Detection and Quantification" Viruses 14, no. 6: 1271. https://doi.org/10.3390/v14061271