Simultaneous detection of three arboviruses using a triplex RT-PCR: enzyme hybridization assay.
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.
Prevention of Veterinary Medicine Department, College of Agriculture, Yanbian University, Yanji 133002, China.
Dan
D
Dong
Shi-hong
SH
Fu
Li-hua
LH
Wang
Zhi
Z
Lv
Tai-yuan
TY
Li
Guo-dong
GD
Liang
D000818:Animals; D001102:Arbovirus Infections / Q000175:diagnosis* / Q000821:virology*; D001103:Arboviruses / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D002460:Cell Line; D006224:Cricetinae; D017931:DNA Primers / Q000235:genetics; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors
D017931:DNA Primers; D012367:RNA, Viral