A quantitative PCR assay for rapid detection of Shigella species in fresh produce.
A quantitative PCR (qPCR) assay with two primers and a TaqMan probe targeting conserved regions of the specific ipaH gene of Shigella species and enteroinvasive Escherichia coli (EIEC) were developed. This qPCR assay was used to identify 206 Shigella strains (including four Shigella species with all serotypes and two provisional Shigella species), 3 EIEC strains, and 113 non-Shigella strains with 100% accuracy. Pure cultures of six Shigella reference strains were used to derive standard curves to determine the detection limit and efficiency of the qPCR method. The ipaH qPCR assay had an equally low detection limit (0.12 to 0.74 CFU per PCR) for the four Shigella species tested. The average qPCR efficiency was 99.29% (95.36 to 103.92%). The detection limit of the qPCR assay tested on 15 varieties of inoculated fresh produce ranged from 0.4 to 16 CFU/100 ml of buffer rinse. This qPCR assay took the variation of wild-type nucleotides into consideration and was used successfully to screen fresh produce. This highly sensitive qPCR assay can be completed within 24 h and has potential use as a screening tool for all four Shigella species and EIEC in food samples.
Lin
Wen S
WS
U.S. Food and Drug Administration, Division of Field Science, 5600 Fishers Lane, Room 12-41, Rockville, Maryland 20857, USA. wen.lin@fda.hhs.gov
Cheng
Chorng-Ming
CM
Van
Khanh T
KT
D001483:Base Sequence; D015169:Colony Count, Microbial; D003257:Consumer Product Safety; D004269:DNA, Bacterial / Q000032:analysis*; D004926:Escherichia coli / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D005506:Food Contamination / Q000032:analysis*; D005516:Food Microbiology; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D012703:Serotyping; D012760:Shigella / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D013045:Species Specificity; D014675:Vegetables / Q000382:microbiology*
D004269:DNA, Bacterial