A two-tube multiplex reverse transcription PCR assay for simultaneous detection of viral and bacterial pathogens of infectious diarrhea.
Diarrhea caused by viral and bacterial infections is a major health problem in developing countries. The purpose of this study is to develop a two-tube multiplex PCR assay using automatic electrophoresis for simultaneous detection of 13 diarrhea-causative viruses or bacteria, with an intended application in provincial Centers for Diseases Control and Prevention, China. The assay was designed to detect rotavirus A, norovirus genogroups GI and GII, human astrovirus, enteric adenoviruses, and human bocavirus (tube 1), and Salmonella, Vibrio parahaemolyticus, diarrheagenic Escherichia coli, Campylobacter jejuni, Shigella, Yersinia, and Vibrio cholera (tube 2). The analytical specificity was examined with positive controls for each pathogen. The analytical sensitivity was evaluated by performing the assay on serial tenfold dilutions of in vitro transcribed RNA, recombinant plasmids, or bacterial culture. A total of 122 stool samples were tested by this two-tube assay and the results were compared with those obtained from reference methods. The two-tube assay achieved a sensitivity of 20-200 copies for a single virus and 10(2)-10(3) CFU/mL for bacteria. The clinical performance demonstrated that the two-tube assay had comparable sensitivity and specificity to those of reference methods. In conclusion, the two-tube assay is a rapid, cost-effective, sensitive, specific, and high throughput method for the simultaneous detection of enteric bacteria and virus.
Key Laboratory for Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, No. 155, Changbai Road, Changping District, Beijing 102206, China.
Ji
J
Wang
Key Laboratory for Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, No. 155, Changbai Road, Changping District, Beijing 102206, China.
Ziqian
Z
Xu
Key Laboratory for Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, No. 155, Changbai Road, Changping District, Beijing 102206, China.
Peihua
P
Niu
Key Laboratory for Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, No. 155, Changbai Road, Changping District, Beijing 102206, China.
Chen
C
Zhang
National Institute for Infectious Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China.
Jingyun
J
Zhang
Key Laboratory for Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, No. 155, Changbai Road, Changping District, Beijing 102206, China.
Li
L
Guan
National Institute for Infectious Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China.
Biao
B
Kan
Key Laboratory for Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, No. 155, Changbai Road, Changping District, Beijing 102206, China.
Zhaojun
Z
Duan
Key Laboratory for Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, No. 155, Changbai Road, Changping District, Beijing 102206, China.
Xuejun
0000-0001-5891-5260
X
Ma
D001419:Bacteria / Q000145:classification / Q000302:isolation & purification*; D004403:Dysentery / Q000175:diagnosis / Q000382:microbiology* / Q000473:pathology; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014780:Viruses / Q000145:classification / Q000302:isolation & purification* / Q000472:pathogenicity