Development of a multiplex real-time RT-PCR assay for simultaneous detection of dengue and chikungunya viruses

Cecilia, D.; Kakade, Mahadeo; Alagarasu, Kalichamy; Patil, J.A.; Salunke, Asha; Parashar, Deepti; Shah, P.S.

doi:10.1007/s00705-014-2217-x

Cited by 59 publications

(46 citation statements)

References 13 publications

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“…Some of these assays are not quantitative or use plasmid DNA as a standard for quantitation (36,37). Another study recently described the development of a multiplex real-time RT-PCR for the simultaneous detection of DENV and CHIKV with a detection limit of 1 to 50 PFU and an assay cutoff value C T of 32, due to nonspecific signal observed in the nontemplate control (40). In this study, we describe the development and validation of a more sensitive, rapid, single-reaction, one-step, multiplex realtime RT-PCR for the detection, quantitation, and differentiation of DENV-1 to -4 and CHIK in patient samples.…”

Section: Discussionmentioning

confidence: 99%

Development and Validation of a Quantitative, One-Step, Multiplex, Real-Time Reverse Transcriptase PCR Assay for Detection of Dengue and Chikungunya Viruses

et al. 2016

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Dengue virus (DENV) and chikungunya virus (CHIKV) are important human pathogens with common transmission vectors and similar clinical presentations. Patient care may be impacted by the misdiagnosis of DENV and CHIKV in areas where both viruses cocirculate. In this study, we have developed and validated a one-step multiplex reverse transcriptase PCR (RT-PCR) to simultaneously detect, quantify, and differentiate between four DENV serotypes (pan-DENV) and chikungunya virus. The assay uses TaqMan technology, employing two forward primers, three reverse primers, and four fluorophore-labeled probes in a single-reaction format. Coextracted and coamplified RNA was used as an internal control (IC), and in vitro-transcribed DENV and CHIKV RNAs were used to generate standard curves for absolute quantification. The diagnostic 95% limits of detection (LOD) within the linear range were 50 and 60 RNA copies/reaction for DENV (serotypes 1 to 4) and CHIKV, respectively. Our assay was able to detect 53 different strains of DENV, representing four serotypes, and six strains of CHIKV. No cross-reactivity was observed with related flaviviruses and alphaviruses, To evaluate diagnostic sensitivity and specificity, 89 clinical samples positive or negative for DENV (serotypes 1 to 4) and CHIKV by the standard virus isolation method were tested in our assay. The multiplex RT-PCR assay showed 95% sensitivity and 100% specificity for DENV and 100% sensitivity and specificity for CHIKV. With an assay turnaround time of less than 2 h, including extraction of RNA, the multiplex quantitative RT-PCR assay provides rapid diagnosis for the differential detection of two clinically indistinguishable diseases, whose geographical occurrence is increasingly overlapping. Dengue is a mosquito-borne viral infection in humans that is of global importance. An estimated 390 million infections occur each year, of which 96 million result in clinically apparent infections, and in some epidemics the mortality rate may reach 5% (1, 2). Dengue viruses (DENVs) are members of the family Flaviviridae and consist of four antigenically distinct serotypes which exhibit 65% to 70% sequence homology (3). Disease manifestations range from mild undifferentiated acute dengue fever (DF) to severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) (4, 5). After an incubation period of 2 to 10 days, a primary infection presents with the acute onset of high fever (Ն40°C), accompanied by headache, retroorbital pain, generalized myalgias, arthralgias, and malaise. The early phase of DHF or DSS has similar clinical features; however, after defervescence, new abdominal pain, nausea, and vomiting, followed by thrombocytopenia and a rise in hematocrit, may progress to shock leading to multiorgan dysfunction, life-threatening hemorrhage, and death. DF symptoms may be clinically indistinguishable from acute febrile illness due to other infectious diseases such as influenza, malaria, measles, and chikungunya virus (CHIKV) infections. CHIKVs are alphaviruses belonging to the ...

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Section: Discussionmentioning

confidence: 99%

Development and Validation of a Quantitative, One-Step, Multiplex, Real-Time Reverse Transcriptase PCR Assay for Detection of Dengue and Chikungunya Viruses

et al. 2016

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“…In short, we describe the first real-time RT-PCR with a broad specificity to alphavirus infections, compared to previously assays that primarily focused on CHIKV, EEEV, and Sindbis virus, alone or in combination with other arboviruses [2,10,25,37,39]. Therefore, our assay may be very useful for the preliminary diagnosis and screening of infections by alphaviruses, during outbreaks, and in general surveillance studies.…”

Section: Discussionmentioning

confidence: 99%

A real-time RT-PCR for rapid detection and quantification of mosquito-borne alphaviruses

et al. 2016

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Mosquito-borne alphaviruses are widely distributed throughout the world, causing important human illnesses. Therefore, the development of methods to enable early diagnosis of infections by alphavirus is essential. We show here the development and evaluation of a quantitative real-time RT-PCR using genus-specific primers to the nsP1 viral gene of all mosquito-borne alphaviruses. The specificity and sensitivity of the assay were tested using seven alphaviruses and RNA transcribed from Venezuelan equine encephalitis virus. The detection limits of real-time RT-PCR ranged from 10 to 76 copies per ml. The melting temperature (T M ) values for amplification of the alphavirus genomes were 83.05°C and 85.28°C. Interestingly, the assay suggested the possibility the arthritogenic alphaviruses with T M peaks of 84.83 to 85.28°C and encephalitic alphaviruses of 83.34°C to 84.68°C could be discriminated both diseases. Real-time RT-PCR may prove very useful for the screening and preliminary diagnosis in outbreaks and surveillance studies as well as for measuring the viral load in pathogenesis studies.

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“…The sensitivity and specificity of genetic methods developed for CHIKV diagnosis can reach 100%. Moreover, real-time RT-PCR combined with sequence analysis was used to identify CHIKV genotypes, while multiplex PCR were utilized to detect CHIKV and dengue infection simultaneously in one tube (Edwards et al, 2007;Cecilia et al, 2015). Although detection of viral RNA is most useful for rapid identification of the infectious virus, it is still limited by the narrow detection window of first week of illness.…”

Section: Genetic Methodsmentioning

confidence: 99%

Recent progress on chikungunya virus research

Cao

et al. 2017

Virol. Sin.

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Development of a multiplex real-time RT-PCR assay for simultaneous detection of dengue and chikungunya viruses

Cited by 59 publications

References 13 publications

Development and Validation of a Quantitative, One-Step, Multiplex, Real-Time Reverse Transcriptase PCR Assay for Detection of Dengue and Chikungunya Viruses

Development and Validation of a Quantitative, One-Step, Multiplex, Real-Time Reverse Transcriptase PCR Assay for Detection of Dengue and Chikungunya Viruses

A real-time RT-PCR for rapid detection and quantification of mosquito-borne alphaviruses

Recent progress on chikungunya virus research

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