Development and Validation of a Quantitative, One-Step, Multiplex, Real-Time Reverse Transcriptase PCR Assay for Detection of Dengue and Chikungunya Viruses.
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.
Viral and Rickettsial Diseases Department, Naval Medical Research Center, Silver Spring, Maryland, USA monika.simmons.civ@mail.mil.
Monika
M
Simmons
Viral and Rickettsial Diseases Department, Naval Medical Research Center, Silver Spring, Maryland, USA.
Todd
T
Myers
Virology and Emerging Infections Department, U.S. Naval Medical Research Unit No. 6, Lima, Peru.
Carolina
C
Guevara
St. George's University School of Medicine, St. George, Grenada.
Donald
D
Jungkind
Viral and Rickettsial Diseases Department, Naval Medical Research Center, Silver Spring, Maryland, USA.
Maya
M
Williams
Division of Viral Diseases, Walter Reed Army Institute of Research, Silver Spring, Maryland, USA.
Huo-Shu
HS
Houng
D065632:Chikungunya Fever / Q000175:diagnosis*; D002646:Chikungunya virus / Q000302:isolation & purification*; D003715:Dengue / Q000175:diagnosis*; D003716:Dengue Virus / Q000302:isolation & purification*; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors