Chen Zhang1, Peihua Niu1, Yanying Hong2, Ji Wang1, Jingyun Zhang3, Xuejun Ma4. 1. 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. 2. Beijing Traditional Chinese Medicine Hospital, Capital Medical University Medical Laboratory, Beijing, China. 3. National Institute for Infectious Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China. 4. 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.
Abstract
OBJECTIVES: 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. CONCLUSION: This assay proved to be a cost-effective, sensitive and reliable method for simultaneous detection of enteric viruses and bacteria.
OBJECTIVES: 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. CONCLUSION: This assay proved to be a cost-effective, sensitive and reliable method for simultaneous detection of enteric viruses and bacteria.
Authors: Jungwon Hyun; Dae Hyun Ko; Su Kyung Lee; Han Sung Kim; Jae Seok Kim; Wonkeun Song; Hyun Soo Kim Journal: Ann Lab Med Date: 2018-05 Impact factor: 3.464