Simultaneous Screening of 24 Target Genes of Foodborne Pathogens in 35 Foodborne Outbreaks Using Multiplex Real-Time SYBR Green PCR Analysis

Abstract

A set of 8 multiplex real-time SYBR Green PCR (SG-PCR) assays including 3 target primers and an internal amplification control (IAC) primer was simultaneously evaluated in 3 h or less with regard to detection of 24 target genes of 23 foodborne pathogens in 7 stool specimens of foodborne outbreak using a 96-well reaction plate. This assay, combined with DNA extraction (QIAamp DNA Stool Mini kit), offered detection of greater than 103-104 foodborne pathogens per g in stool specimens. The products formed were identified using melting point temperature (Tm) curve analysis. This assay was evaluated for the detection of foodborne pathogens in 33 out of 35 cases of foodborne outbreak, using 4 different PCR instruments in 5 different laboratories. No interference from the multiplex real-time SG-PCR assay, including IAC, was observed in stool specimens in any analysis. We found multiplex real-time SG-PCR assay for simultaneous detection of 24 target genes of foodborne pathogens to be comprehensive, rapid, inexpensive, accurate, of high selectivity, and good for screening probability.

A Rapid and Simple Real-Time PCR Assay for Detecting Foodborne Pathogenic Bacteria in Human Feces

“…In addition, we determined the LOD of the real-time PCR assay using the DAPM and the QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany), which is commonly used to extract and purify DNA with a column from stool samples (10)(11)(12). Then, the 2 LODs were compared.…”

Rapid and Accurate Diagnosis Based on Real-Time PCR Cycle Threshold Value for the Identification of <i>Campylobacter jejuni</i>, <i>astA</i> Gene-Positive <i>Escherichia coli</i>, and <i>eae</i> Gene-Positive <i>E. coli</i>

“…Although real-time polymerase chain reaction (PCR) assays detect viral pathogens within a few hours, bacterial culture methods (BCs), used to identify the causative pathogen, are time-consuming and laborious (3-7 days) (4-6). We developed a SYBR Green I-multiplex real-time PCR (SG-mPCR) screening system that simultaneously (within 3.5-4 h) analyzes 6 types of diarrheagenic Escherichia coli and 15 bacterial foodborne pathogens (1,7). This screening system, designated as Rapid Foodborne Bacterial Screening 24 (RFBS24 ver.5) includes 8 sets (Sets A-H) of SG-mPCR assays, each comprised of 6-7 primers for 3 genes and a competitive internal amplification control (IAC).…”

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

“…Fukushima et al (6) described an expedited screening system that can simultaneously analyze 24 bacterial target genes using multiplex real-time SYBR Green polymerase chain reaction (SG-PCR). This screening system, named Rapid Foodborne Bacterial Screening 24 (RFBS24), includes 8 sets of multiplex real-time SG-PCR assays, with each set comprising 4 primer pairs for 3 target genes and an internal amplification control (IAC).…”

“…RFBS24 can identify foodborne pathogens more quickly than traditional methods can, thus leading to quicker dissemination of relevant information to public health officials. Nonetheless, Fukushima et al (6) reported that the detection limit of RFBS24 for Salmonella spp. is worse than that for other pathogens and that identification of some bacterial pathogens is difficult due to small differences in melting temperatures (T m ) of PCR products between some target genes.…”

“…is worse than that for other pathogens and that identification of some bacterial pathogens is difficult due to small differences in melting temperatures (T m ) of PCR products between some target genes. Moreover, RFBS24 does not detect genotype STh of enterotoxigenic Escherichia coli (ETEC) (6). These drawbacks may be attributed to poor design of primers for the detection of Salmonella spp., interactions among primers, and/or suboptimal PCR conditions; however, none of these possible causes have been investigated sufficiently.…”