An Improved Multiplex Real-Time SYBR Green PCR Assay for Analysis of 24 Target Genes from 16 Bacterial Species in Fecal DNA Samples from Patients with Foodborne Illnesses

Japanese Journal of Infectious Diseases

Yoshiki Etoh⁶  Keiji Yamaguchi³  Hiroshi Fukushima⁴  Komei Shirabe²  Masanori Watahiki¹  Mitsuhiro Kameyama²  Ryoichi Goto³  Kazumi Horikawa⁶  Jun Kawase⁵  Tetsuya Ikeda³  Tomoko Shima¹ 

[1] Toyama Institute of Health;Yamaguchi Prefectural Institute of Public Health and Environment;Hokkaido Institute of Public Health;Shimane Prefectural Livestock Technology Center;Shimane Prefectural Institute of Public Health and Environmental Science;Fukuoka Institute of Health and Environmental Sciences

关键词: real-time PCR;    foodborne outbreak;    EHEC;    Salmonella;    Campylobacter;   

DOI  :  10.7883/yoken.JJID.2015.027

学科分类：传染病学

来源: National Institute of Infectious Diseases

【 摘 要 】

References(36)Here, we developed a new version of our original screening system (Rapid Foodborne Bacterial Screening 24; RFBS24), which can simultaneously detect 24 genes of foodborne pathogens in fecal DNA samples. This new version (RFBS24 ver. 5) detected all known stx2 subtypes, enterotoxigenic Escherichia coli (STh genotype), and Vibrio parahaemolyticus (trh2), which were not detected by the original RFBS24 assay. The detection limits of RFBS24 ver. 5 were approximately 5.6 × 10−2–5.6 × 10−5 (ng DNA)/reaction, significantly lower (10- to 100-fold) than those of the original RFBS24 for the 22 target genes analyzed here. We also tested the new assay on fecal DNA samples from patients infected with Salmonella, Campylobacter, or enterohemorrhagic E. coli. The number of bacterial target genes detected by RFBS24 ver. 5 was greater than that detected by RFBS24. RFBS24 ver. 5 combined with an Ultra Clean Fecal DNA Isolation Kit showed adequate performance (sensitivity and specificity 89% and 100%, respectively, for Salmonella spp. and 100% and 83%, respectively, for Campylobacter jejuni) in terms of rapid detection of a causative pathogen during foodborne-illness outbreaks. Thus, RFBS24 ver. 5 is more useful than the previous assay system for detection of foodborne pathogens and offers quick simultaneous analysis of many targets and thus facilitates rapid dissemination of information to public health officials.

【 授权许可】

Unknown