A probe-free four-tube real-time PCR assay for simultaneous detection of twelve enteric viruses and bacteria.
OBJECTIVE
We aim to develop a multiplex real-time PCR assay to detect the most common pathogens causing community outbreaks of diarrhea.


METHODS
Four reaction systems of fluorescence dye-based real-time PCR assay were performed to amplify genes of norovirus, sapovirus, rotavirus, astrovirus, adenovirus, Campylobacter jejuni, Yersinia enterocolitica, Vibrio parahaemolyticus, Salmonella spp., Escherichia coli, and Shigella spp. PCR products of each pathogen were identified by characteristic peaks in melting curves.


RESULTS
The assay was able to achieve detection limit of 50 copies/reaction for each individual virus target, and 140-500CFU/mL for each individual bacterium target. A total of 122 clinical specimens from hospitalized children with acute diarrhea were used to evaluate the assay. The clinical sensitivity was very similar to that of reference methods. Norovirus genogroup II revealed the highest detectable rate (45/122, 36.9%). Coinfection was found in 28 out of 122 (23%) clinical specimens.


CONCLUSIONS
This assay proved to be a cost-effective, sensitive and reliable method for simultaneous detection of enteric viruses and bacteria.
Key Laboratory for Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China.
Chen
C
Zhang
Key Laboratory for Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China.
Peihua
P
Niu
Beijing Traditional Chinese Medicine Hospital, Capital Medical University Medical Laboratory, Beijing, China.
Yanying
Y
Hong
Key Laboratory for Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China.
Ji
J
Wang
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, Beijing 102206, China. Electronic address: maxj@ivdc.chinacdc.cn.
Xuejun
X
Ma
D001419:Bacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D002675:Child, Preschool; D017714:Community-Acquired Infections / Q000175:diagnosis* / Q000382:microbiology / Q000821:virology; D003362:Cost-Benefit Analysis; D003967:Diarrhea / Q000175:diagnosis* / Q000382:microbiology / Q000821:virology; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D025202:Molecular Diagnostic Techniques / Q000191:economics / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D012680:Sensitivity and Specificity; D044366:Transition Temperature; D014780:Viruses / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*
Bacteria; Diarrhea; Melting curve; Real-time PCR; Virus