Simultaneous detection of three arboviruses using a triplex RT-PCR enzyme hybridization assay

• Published in: Virologica Sinica Vol. 27; no. 3; pp. 179 - 186
• Main Authors: Dong, Dan, Fu, Shi-hong, Wang, Li-hua, Lv, Zhi, Li, Tai-yuan, Liang, Guo-dong
• Format: Journal Article
• Language: English
• Published: Heidelberg Springer-Verlag 01-06-2012; SP Wuhan Institute of Virology, CAS; State Key Laboratory for Infectious Disease Prevention and Control,Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China%State Key Laboratory for Infectious Disease Prevention and Control,Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China%Prevention of Veterinary Medicine Department,College of Agriculture,Yanbian University,Yanji 133002,China; Prevention of Veterinary Medicine Department,College of Agriculture,Yanbian University,Yanji 133002,China
• Subjects: Agriculture; Animals; Arbovirus Infections - diagnosis; Arbovirus Infections - virology; arboviruses; Arboviruses - classification; Arboviruses - genetics; Arboviruses - isolation & purification; Biochemistry; Biomedical and Life Sciences; Biomedicine; Bunyaviridae; California encephalitis virus; Cell Line; Cerebrospinal fluid; Cricetinae; detection limit; DNA Primers - genetics; Enzymes; Flaviviridae; Getah virus; Humans; Medical Microbiology; Microbial Genetics and Genomics; Microbiology; Multiplex Polymerase Chain Reaction - economics; Multiplex Polymerase Chain Reaction - methods; Oncology; Polymerase chain reaction; Primers; Public health; Reverse Transcriptase Polymerase Chain Reaction - economics; Reverse Transcriptase Polymerase Chain Reaction - methods; RNA, Viral - genetics; RNA, Viral - isolation & purification; Sensitivity and Specificity; Tahyna virus; Time Factors; Togaviridae; Virology; Tahyna Virus (TAHV); Multiplex RT-PCR; Enzyme Hybridization; Japanese Encephalitis virus (JEV); Getah Virus (GETV); Getah Virus(GETV); Tahyna Virus(TAHV); Japanese Encephalitis virus(JEV)
• ISSN: 1674-0769; 1995-820X
• DOI: 10.1007/s12250-012-3246-9

Arboviruses represent a serious problem to public health and agriculture worldwide. Fast, accurate identification of the viral agents of arbovirus-associated disease is essential for epidemiological surveillance and laboratory investigation. We developed a cost-effective, rapid, and highly sensitive one-step “triplex RT-PCR enzyme hybridization” assay for simultaneous detections of Japanese Encephallitis virus (JEV, Flaviviridae), Getah virus (GETV, Togaviridae), and Tahyna virus (TAHV, Bunyaviridae) using three pairs of primers to amplify three target sequences in one RT-PCR reaction. The analytical sensitivity of this assay was 1 PFU/mL for JEV, 10 PFU/mL for GETV, and 10 PFU/mL for TAHV. This assay is significantly more rapid and less expensive than the traditional serological detection and single RT-PCR reaction methods. When “triplex RT-PCR enzyme hybridization” was applied to 29 cerebrospinal fluid (CSF) samples that were JEV-positive by normal RT-PCR assay, all samples were strongly positive for JEV, but negative for GETV and TAHV, demonstrating a good sensitivity, specificity, and performance at CSF specimen detection.