|Year : 2016 | Volume
| Issue : 3 | Page : 330-334
|Comparison of multiplex RT-PCR and real-time HybProbe assay for serotyping of dengue virus using reference strains and clinical samples from India
Anita Chakravarti1, Mayank Singh Chauhan1, Sayantan Banerjee2, Priyamvada Roy3
1 Department of Microbiology, Maulana Azad Medical College, New Delhi, India
2 Department of Microbiology, ESI-PGIMSR and ESIC Medical College, Kolkata, West Bengal, India
3 Department of Microbiology, University College of Medical Sciences and GTB Hospital, New Delhi, India
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|Date of Web Publication||10-Aug-2016|
| Abstract|| |
Background: Dengue virus serotyping is crucial from clinical management and epidemiological point of view. Aims: To compare efficacy of two molecular detection and typing methods, namely, multiplex reverse transcription polymerase chain reaction (RT-PCR) and real-time Hybprobe assay using a panel of known dilution of four reference Dengue virus strains and a panel of sera collected from clinically suspected dengue patients. Settings: This study was conducted at a tertiary-care teaching hospital in Delhi, India. Materials and Methods: Dengue serotype specific virus strains were used as prototypes for serotyping assays. Viral load was quantified by quantitative real time reverse transcription polymerase chain reaction (qRT-PCR). Acute phase serum samples were collected from 79 patients with clinically suspected Dengue fever on their first day of presentation during September-October 2012. Viral RNA from serum and cell culture supernatant was extracted. Reverse transcription was carried out. Quantitative detection of DENV RNA from reference strain culture supernatants and each of the 79 patient samples by real-time PCR was performed using light cycler Taqman master mix kit. Serotyping was done by multiplex RT-PCR assay and Hybprobe assay. Results: The multiplex RT-PCR assay, though found to be 100% specific, couldn't serotype either patient or reference strains with viral load less than 1000 RNA copies/ml. The Hybprobe assay was found to have 100% specificity and had a lower limit of serotype detection of merely 3.54 RNA copies/ml. Conclusions: HybProbe assay has an important role especially in situations where serotyping is to be performed in clinical samples with low viral load.
Keywords: Dengue, India, multiplex reverse transcription polymerase chain reaction, real-time HybProbe assay, serotyping
|How to cite this article:|
Chakravarti A, Chauhan MS, Banerjee S, Roy P. Comparison of multiplex RT-PCR and real-time HybProbe assay for serotyping of dengue virus using reference strains and clinical samples from India. Indian J Pathol Microbiol 2016;59:330-4
|How to cite this URL:|
Chakravarti A, Chauhan MS, Banerjee S, Roy P. Comparison of multiplex RT-PCR and real-time HybProbe assay for serotyping of dengue virus using reference strains and clinical samples from India. Indian J Pathol Microbiol [serial online] 2016 [cited 2020 May 29];59:330-4. Available from: http://www.ijpmonline.org/text.asp?2016/59/3/330/188141
| Introduction|| |
Dengue virus (DENV) belongs to family Flaviviridae, genus Flavivirus, and exists in antigenically distinct four serotypes (DENV-1 to DENV-4), each of which can infect human beings. WHO has estimated that about 2.5 billion people, i.e., 40% of the world's population, are at risk from dengue, with about 50 to 100 million cases of dengue infections occurring worldwide annually, despite significant under-reporting from the developing nations. The sequence of infections by different genotypes in primary and secondary infections has been seen to be significant in predicting complications.
During the last two decades, the epidemiology of dengue in India has been changing rapidly with approximately 6000–8000 dengue cases being reported annually. A 5 years study conducted between 2002 and 2006 at our hospital, provided clear-cut evidence for the presence of all four serotypes in Northern India, with DENV-2 and DENV-3 as the two most dominant ones. However, in contrast to the 2002–2006 findings, in the laboratory we observed that during the next 3 years, the incidence of DENV-1 surpassed the other serotypes, indicating a trend of cyclical replacements of strains. Studies have shown that DENV-2 and DENV-3 cause more severe disease than the other serotypes.,, Thus, it is clear that along with timely diagnosis of dengue, serotyping of the causative strain is also of immense importance from both the prognostic and epidemiological point of view. In this study, we have evaluated the performance of multiplex reverse transcription polymerase chain reaction (RT-PCR) and real-time HybProbe assay for serotyping DENV from both cultured reference viral strains and from serum samples of patients with clinically suspected dengue fever (DF).
| Materials and Methods|| |
The dengue serotype-specific virus strains (serotype DENV-1: Hawaii; DENV-2: P-23085; DENV-3: 633798; and DENV-4: 642069) were obtained from the National Institute of Virology, Pune, Maharashtra, India. These viruses were used as prototypes for serotyping assays. Standard dilutions of virus strains were prepared. Viruses were propagated in C6/36 cells. After growth for 7 days at 28°C, cell culture supernatant was collected, clarified by centrifugation at 1500 rpm for 10 min and stored at −70°C until further use. Each virus strain in cell culture supernatants was diluted in RNAse free water, and viral load was quantified by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) by the method described below.
Standard 10-fold dilution of culture supernatants of each serotype of viral strains was prepared after RNA quantitation by qRT-PCR of original culture supernatant. The resultant dilutions in the range of ≥100–<101 virions/ml to ≥108–<109 virions/ml in serially diluted aliquots of culture supernatant, were incorporated for the study. The DENV being a single stranded RNA virus, each dilution was rechecked and adjusted accordingly by qRT-PCR, ranging from ≥100–<101 copies of RNA/ml to ≥108–<109 copies of RNA/ml.
Acute phase (<7 days of fever) serum samples were collected from 79 patients with clinically suspected DF or dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS) on their 1st day of presentation to the medical outpatient service or admission to the medical indoor ward of Lok Nayak Hospital, New Delhi (India) during September–October 2012. The clinical diagnosis of dengue was made by the attending clinical residents and physicians according to the standard WHO guidelines. Patients presenting with acute febrile illness of <7 days duration with two or more of the following manifestations: headache, retro-orbital pain, myalgia, arthralgia, rash were presumptively diagnosed as having DF. A probable or confirmed case of DF with hemorrhagic tendencies plus thrombocytopenia plus evidence of plasma leakage was clinically diagnosed to be DHF. Cases of DHF with evidence of circulatory failure were labeled as DSS. Serum was separated and stored at −70°C until further processed. The study protocol was approved by the Institutional Committee for Research Ethics. Written informed consent was obtained from all study subjects.
Viral RNA from serum and cell culture supernatant was extracted with High Pure Viral RNA Isolation Kit (Roche Diagnostics; Mannheim, Germany) according to manufacturer's instructions. Briefly, 200 µl of serum or cell culture supernatant was used for extraction of viral RNA. The RNA was eluted in 50 µl of elution buffer and used immediately in the following experiments.
Reverse transcription was carried out by heating 10 µl of extracted viral genome at 95°C for 5 min and chilled on ice for 5 min. Fifteen µl of RT reaction mixture was added, yielding a total reaction volume of 25 µl. It consisted of 50 mM Tris-HCl (pH 8.3), 4 mM MgCl2, 10 mM DTT, 50 mM KCL, a 0.4 mM concentration of each dNTP (Fermentas Life Sciences), 0.4 µM each of random hexamer (Invitrogen USA), and 100 units of RevertAid H Minus M-MuLV Reverse Transcriptase (MBI Fermentas, Life Sciences). Reverse transcription was allowed to proceed at 42°C for 60 min followed by inactivation of reverse transcriptase at 70°C for 15 min. The RT product was then held at 4°C.
Absolute quantification of dengue virus by real-time polymerase chain reaction (TaqMan assay)
Quantitative detection of DENV RNA from reference strain culture supernatants and each of the 79 patient samples by real-time reverse transcription polymerase chain reaction (PCR) was performed using LightCycler TaqMan Master Mix Kit on Roche LightCycler version 2.0 (Roche Diagnostic, GmbH, Mannheim, Germany). Earlier described TaqMan primers and probe were used in real-time TaqMan assay. Each reaction (20 µl) contained 2 µl of cDNA solution, 0.5 µM of each primer, 0.2 µM of TaqMan probe, and 4 µl of TaqMan Master Mix (FastStart Taq DNA Polymerase, reaction buffer, MgCl2, and deoxynucleoside triphosphates) (Roche Diagnostic, GmbH, Mannheim, Germany). The amplification program consisted of 1 cycle at 95°C with 10 min hold followed by 45 cycles of 95°C for 10 s, 56°C for 30 s, and 72°C for 1 s. A known quantity of internal standard was included in each preparation of real-time reverse transcription PCR. DENV negative control without cDNA was run with every PCR to assess specificity of the reaction. The experimental data were analyzed using the LightCycler software version 4.05 (Roche Diagnostic, GmbH, Mannheim, Germany). Nine 10-fold dilutions of each of the four reference strains were prepared by titration with RNA counts ranging from ≥100–<101 copies of RNA/ml to ≥108–<109 copies/ml. Each dilution was retested quantitatively.
Serotyping of dengue virus by multiplex RT-PCR Assay
PCR was performed as described elsewhere. Briefly, 1 µl of cDNA was added to 24 µl of PCR mixture consisting of 18 mMTris-HCl (pH 8.3), 45 mM KCL, 2 mM MgCl2, 0.2 mM concentration of each dNTP, 1 U of Taq DNA polymerase (Fermentas Life Sciences) and 0.4 µM of primer D1, TS1, TS2, TS3, and TS4. The PCR was performed by adding 1 µl of RT product into 24 µl of the PCR mix. PCR thermal profile was initial denaturation at 94°C for 3 min. followed by 40 cycles of 94°C for 30 s, 55°C for 60 s, 72°C for 120 s, and a final extension at 72°C for 10 min. The cDNA made from the viral RNA of DENV reference strains propagated in C6/36 cells was used as positive control in each run. Each of the nine dilutions for each reference strains was tested for serotyping. PCR products were subjected to electrophoresis on 2% agarose gel containing 0.5 µg/ml of ethidium bromide and observed under ultraviolet light. Specific segment sizes (482 bp for DENV-1, 119 bp for DENV-2, 290 bp for DENV-3, and 389 bp for DENV-4) of different dengue serotypes were observed.
Dengue serotyping by real-time HybProbe assay
HybProbe assay was performed for dengue serotyping. Serotype-specific primer pair and hybridization probes (based on fluorescence resonance energy transfer principle) were used as described previously. The hybridization probes specific for DENV-1 and DENV-3 were tagged with LC-Red 640 fluorescent dye while probes specific for DENV-2 and DENV-4 were labeled with LC-Red 705 fluorescent dye. Duplex real-time PCR was performed on each sample. Each sample was added to 2 LightCycler capillaries. One tube contained the HybProbe primers and probes specific for DENV-1 and DENV-2 serotypes while the other capillary tube contained the HybProbe primers and probes specific for DENV-3 and DENV-4 serotypes.
The real-time HybProbe serotyping assay was performed using the LightCycler LightCycler FastStart DNA MasterPLUS HybProbe kit on Roche LightCycler version 2.0 (Roche Diagnostics). Each reaction mixture (20 µl) contained 1 µl of cDNA solution, 0.5 µM of each primer and 0.2 µM of HybProbe probe and 4 µl of HybProbe Master Mix (Roche Diagnostic, GmbH, Mannheim, Germany). cDNA made from the viral RNA of DENV reference strains propagated in C6/36 cells was used as positive control in each run. Each of the nine dilutions for each four reference strains were tested for serotyping. DENV negative control without cDNA was run with every PCR to assess specificity of the reaction. The amplification program consisted of 1 cycle at 95°C with 10 min hold (enzyme activation) followed by 45 amplification cycles consisting of 95°C (denaturation) for 15 s, 56°C (annealing) for 15 s, and 72°C (extension) for 15 s. The fluorescence emitted was captured during the annealing phase of each cycle at 640 and 705 nm. After amplification, melting curve analysis was performed. It consisted of denaturation at 95°C without hold, annealing at 45°C for 30 s followed by continuous melting to 95°C with a ramp rate of 0.05°C/s. During melting, the fluorescence was continuously captured at 640 nm and 705 nm. The experimental data were analyzed using LightCycler software version 4.05 (Roche Diagnostic, GmbH, Mannheim, Germany).
| Results|| |
The performance of multiplex RT-PCR and real-time HybProbe assays were determined using cultured reference viral strains and 79 acute phase serum samples collected from clinically suspected dengue patients. DENV RNA was detected and quantified in 44 out of 79 patient samples by qRT-PCR.
Serotyping by multiplex RT-PCR and real-time HybProbe assays
The multiplex RT-PCR assay could serotype only 20 out of 36 known serial dilutions of the four reference viral strains and 8 out of 44 DENV RNA positive patient samples. All the 36 serial dilutions of the four reference strains and 43 out of 44 DENV RNA positive patient samples were successfully serotyped by the HybProbe assay [Table 1]. Forty-two DENV RNA positive samples were found to be DENV-1 and one was DENV-2 by the real-time HybProbe assay. Only 1 sample positive for DENV RNA could not be serotyped by real-time HybProbe Assay. All the 35 samples negative for DENV RNA by qRT-PCR did not show any presence of viral RNA by multiplex RT-PCR and real-time HybProbe assay.
|Table 1: Comparison of viral load and serotyping ability of multiplex RT-PCR assay and real-time HybProbe assay|
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Performance of multiplex RT-PCR and real-time HybProbe assays for dengue virus serotyping
The serotyping limit in terms of RNA copies was determined for both multiplex RT-PCR and real-time HybProbe assays. While assessing each of the panel of nine serial dilutions for the four reference viral strains, it was found that the real-time HybProbe assay was able to identify the serotype in all strains ranging from dilutions ≥100–<101 copies/ml to ≥108–109 copies/ml, thus identifying all the 36 dilutions from four strains. However, multiplex RT-PCR assay could serotype only 20 out of 36 known serial dilutions of the four reference viral strains ranging from dilutions from 104 to 109 copies/ml [Table 1]. The real-time HybProbe assay serotyped 97.73% (43 out of 44) of the clinical specimens while multiplex RT-PCR assay serotyped 18.18% (8 out of 44) samples. Serotyping result obtained by both assays was in 100% agreement with each other. All the samples negative for dengue viral RNA turned out to be negative by both assays suggesting 100% specificity of each assay. The results and comparison of serotyping by both assays have been summarized in [Table 1] and [Table 2].
|Table 2: Comparison of performance of multiplex RT-PCR and real-time HybProbe assay for serotyping of dengue virus|
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Serotype detection limit of multiplex RT-PCR and real-time HybProbe assay
The lowest detection limit of both the assays was determined in terms of RNA copies/ml. The presence of at least 104 RNA copies/ml was found to be the lowest detection limit for serotyping DENV by multiplex RT-PCR assay. It was unable to serotype both the diluted reference strains and the patients' samples that were found to contain lower levels of virus by qRT-PCR. In contrast, the detection limit of real-time HybProbe assay was found to be as low as ≥1–<10 RNA copies/ml, both for the reference strains as well as the patients' samples. One case with 100 RNA copies was serotyped as DENV-2 by real-time HybProbe assay [Table 1].
Days of fever and serotype detection by multiplex RT-PCR and real-time HybProbe assay
The 44 samples in which DENV RNA was detected and quantified by qRT-PCR were from patients with fever of 1–5 days duration. Multiplex RT-PCR assay was unable to serotype samples from patients with fever of more than 3 days whereas real-time HybProbe assay could serotype all samples except 1 from a patient with 5 days of fever [Table 3].
|Table 3: Comparison of days of fever and serotyping ability of both assays|
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The cost per unit tested, were estimated for both the assays and expressed in US dollars (USD) and Indian Rupees (INR). The average cost of dengue diagnosis and serotyping by the multiplex RT-PCR was 2.73 USD (178 INR) while that by the HybProbe assay was 10.90 USD (711 INR) per sample tested.
| Discussion|| |
At present, laboratory diagnosis and typing of DENV infection is based on virus isolation, virus genome detection by molecular techniques and detection of DENV specific antigen and antibodies. Although virus isolation is considered as the gold standard for dengue detection, it is time-consuming (7–10 days), labor exhaustive and requires trained personnel. Among conventional serological techniques, plaque reduction neutralization technique provides information about virus serotypes, but its use is limited due to high cost and time consumption.
Molecular techniques targeting identification of virus genome offer rapid diagnosis and typing of DENV and are gradually replacing virus isolation as the new gold standard for DENV diagnosis in acute-phase serum samples. The most commonly used is nested RT-PCR, which was originally developed by Lanciotti et al., and later modified into single tube multiplex PCR by Harris et al., Recently, several investigators have reported highly sensitive qRT-PCR approaches for the detection and serotyping of DENV. Different fluorescence chemistries (SYBR green, TaqMan and HybProbe) have been utilized by different researchers., Other techniques such as transcription mediated amplification, RT-LAMP assay, and dry format quantitative RT-PCR assay have also been developed for the diagnosis of dengue infection., The assays also vary between singleplex, duplex, and fourplex assay systems.,,
We detected DENV-1 and DENV-2 serotypes in the present study while none of the samples tested positive for DENV-3 or DENV-4. This is in accordance with another study in Delhi by Afreen et al. The HybProbe assay was found to be faster and less prone to cross contamination than multiplex RT-PCR. It also measures the viremia level which is not possible in case of multiplex RT-PCR. The HybProbe assay has its own advantages over the other commonly used TaqMan and SYBR green based real-time approaches. In comparison to the SYBR green (nonspecifically binds to double stranded DNA), the HybProbe assay is found to be more specific. In SYBR green based assay, detection of the desired amplicon is performed by melting curve analysis which is only possible once real-time amplification is over. In addition to the real-time detection, offline melting curve analysis is also possible in HybProbe assay simultaneously, which makes the result analysis of HybProbe assay more specific than the TaqMan based assay. In this study, real-time HybProbe assay was able to detect serotypes in more samples than multiplex RT-PCR. The detection limit was as low as 1–10 RNA copies by HybProbe assay, and it was able to identify serotypes in the samples with the viral load as low as 3.54 × 100 RNA copies. Multiplex RT-PCR could not serotype 81.82% (36 out of 44) samples, probably due to the low viral load (<1000 RNA copies) in them. It was observed that multiplex RT-PCR could serotype samples from patients with 1 to 3 days of fever while HybProbe assay serotyped even samples from patients with 5 days of fever. This may be due to increase antibody levels with time, which in turn decreases the viral load. These findings suggest that HybProbe assay is more effective in the detection and typing of DENV with low viral load and can also be used for a longer duration than multiplex RT-PCR. This study supports the fact that qRT-PCR is more sensitive (1000 times in our study) than conventional multiplex RT-PCR. These findings are consistent with those reported previously by Chakravarti et al. We have observed certain other plus points also for the HybProbe RT-PCR assay. It is faster than multiplex RT-PCR. In the latter, serotyping of DENV can be accomplished in approximately 6 h after cDNA synthesis (time required for PCR reaction setup, PCR run, and agarose gel electrophoresis), while in HybProbe assay the serotyping can be accomplished in just 1½ h. HybProbe assay also offers amplification and detection of dengue serotypes in a closed capillary system, thus minimizing the risk of cross contamination of samples and exposure to carcinogenic chemicals, as this could possibly occur in the case of multiplex RT-PCR assay.
| Conclusions|| |
To conclude, we compared multiplex RT-PCR and HybProbe assay for the detection and serotyping of DENV in diluted culture supernatant of reference DENV strains as well as clinical samples. Both the assays were found to be highly specific. The advantage with HybProbe assay was its ability to detect and serotype DENVs in samples with low viral load, which could not be serotyped using multiplex RT-PCR assay. Often during the acute phase of infection, the viral load comes down to low levels, for which the HybProbe assay will prove to be more useful than multiplex RT-PCR. The main disadvantage of HybProbe assay in comparison to multiplex RT-PCR is its fourfold higher cost per unit sample tested. Considering the rising number of DF cases in India and the neighboring countries, we suggest incorporation of an easy to perform and accurate molecular tool for serotyping the prevalent DENV strains, for which HybProbe assay may be considered as a fair alternative.
The authors acknowledge with gratitude the immense help extended by Mr. Suman Kumar, PhD student, Virology Laboratory, Department of Microbiology, Maulana Azad Medical College, during all stages of the work.
Financial support and sponsorship
This work was financially supported by the Indian Council of Medical Research, New Delhi, Government of India.
Conflicts of interest
There are no conflicts of interest.
| References|| |
Lindenbach BD, Rice CM. Molecular biology of flaviviruses. Adv Virus Res 2003;59:23-61.
McBride WJ, Bielefeldt-Ohmann H. Dengue viral infections; pathogenesis and epidemiology. Microbes Infect 2000;2:1041-50.
Chakravarti A, Arora R, Luxemburger C. Fifty years of dengue in India. Trans R Soc Trop Med Hyg 2012;106:273-82.
Kumaria R. Correlation of disease spectrum among four dengue serotypes: A five years hospital based study from India. Braz J Infect Dis 2010;14:141-6.
Matlani M, Chakravarti A. Changing trends of dengue disease: A brief report from a tertiary care hospital in New Delhi. Braz J Infect Dis 2011;15:184-5.
Endy TP, Nisalak A, Chunsuttiwat S, Libraty DH, Green S, Rothman AL, et al.
Spatial and temporal circulation of dengue virus serotypes: A prospective study of primary school children in Kamphaeng Phet, Thailand. Am J Epidemiol 2002;156:52-9.
Nisalak A, Endy TP, Nimmannitya S, Kalayanarooj S, Thisayakorn U, Scott RM, et al.
Serotype-specific dengue virus circulation and dengue disease in Bangkok, Thailand from 1973 to 1999. Am J Trop Med Hyg 2003;68:191-202.
World Health Organization. Dengue: Guidelines for Diagnosis, Treatment, Prevention and Control – New Edition. Geneva: World Health Organization; 2009.
Drosten C, Göttig S, Schilling S, Asper M, Panning M, Schmitz H, et al.
Rapid detection and quantification of RNA of Ebola and Marburg viruses, Lassa virus, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, dengue virus, and yellow fever virus by real-time reverse transcription-PCR. J Clin Microbiol 2002;40:2323-30.
Chakravarti A, Kar P, Kumaria R, Batra VV, Verma V. Improved detection of dengue virus serotypes from serum samples – Evaluation of single tube multiplex RT-PCR with cell culture. WHO Dengue Bull 2006;30:133-40.
Lai YL, Chung YK, Tan HC, Yap HF, Yap G, Ooi EE, et al.
Cost-effective real-time reverse transcriptase PCR (RT-PCR) to screen for dengue virus followed by rapid single-tube multiplex RT-PCR for serotyping of the virus. J Clin Microbiol 2007;45:935-41.
Kao CL, King CC, Chao DY, Wu HL, Chang GJ. Laboratory diagnosis of dengue virus infection: Current and future perspectives in clinical diagnosis and public health. J Microbiol Immunol Infect 2005;38:5-16.
Guzmán MG, Kourí G. Dengue diagnosis, advances and challenges. Int J Infect Dis 2004;8:69-80.
Lanciotti RS, Calisher CH, Gubler DJ, Chang GJ, Vorndam AV. Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction. J Clin Microbiol 1992;30:545-51.
Harris E, Roberts TG, Smith L, Selle J, Kramer LD, Valle S, et al.
Typing of dengue viruses in clinical specimens and mosquitoes by single-tube multiplex reverse transcriptase PCR. J Clin Microbiol 1998;36:2634-9.
Johnson BW, Russell BJ, Lanciotti RS. Serotype-specific detection of dengue viruses in a fourplex real-time reverse transcriptase PCR assay. J Clin Microbiol 2005;43:4977-83.
Muñoz-Jordán JL, Collins CS, Vergne E, Santiago GA, Petersen L, Sun W, et al.
Highly sensitive detection of dengue virus nucleic acid in samples from clinically ill patients. J Clin Microbiol 2009;47:927-31.
Parida M, Horioke K, Ishida H, Dash PK, Saxena P, Jana AM, et al.
Rapid detection and differentiation of dengue virus serotypes by a real-time reverse transcription-loop-mediated isothermal amplification assay. J Clin Microbiol 2005;43:2895-903.
Afreen N, Naqvi IH, Broor S, Ahmed A, Parveen S. Phylogenetic and molecular clock analysis of dengue serotype 1 and 3 from New Delhi, India. PLoS One 2015;10:e0141628.
Dr. Anita Chakravarti
Department of Microbiology, Maulana Azad Medical College, New Delhi - 110 002
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2], [Table 3]
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