Universal Primer‐Multiplex PCR Approach for Simultaneous Detection of Escherichia coli, Listeria monocytogenes, and Salmonella spp. in Food Samples

Abstract

Escherichia coli, Listeria monocytogenes, and Salmonella spp. are 3 kinds of the most important food-borne human pathogens. Traditional microbiological analysis is labor-intensive, time-consuming, and easily contaminated, thus producing false positive signals; it also involves much subjectivity judgments. Multiplex-PCR could be applied to detect multiple target organisms simultaneously to save time and labor, but there is always disproportionate amplification resulting from the disparity of different primers. To gain a rapid and sensitive method, a universal primer-multiplex PCR system (UP-M-PCR) was developed and applied for simultaneous detection of the 3 organisms. This method simplified traditional multiplex-PCR reaction system and overcame its amplification disparities among different primers; moreover, it got a high specificity and sensitivity (85, 155, and 104 copies/reaction for E. coli O157, L. monocytogenes, and Salmonella spp., respectively). Compared with the time-consuming and laborious microbiological analysis, UP-M-PCR had a lower risk of cross-contamination without inoculation and incubation. Test results for 36 food samples showed that UP-M-PCR method got a relative accuracy of 91.77% when compared with traditional microbiological analysis. It could serve as a rapid screening method for pathogen detection and could detect target genes even in dead pathogenic cells. In addition, it has the potential to be performed in an automation mode and might find broader application in simultaneous detection of other multiple pathogens.

Molecular characterization of some food borne pathogens in soft cheese samples collected from Jeddah, Saudi Arabia

“…Our results also showed that the DNA templates from the three contaminated cheese samples (sample 2, 4, 13) can be efficiently amplified using specific primer sets of ESC-F/R, LIS-F/R, and SAL -F/R This result validates the specificity and efficiency of UP-PCR amplification for the detection of the three expected DNA fragments for each pathogen on agarose gels. This result was in accordance with the conclusion of universal multiplex PCR (Yuan et al, 2009). These results confirm that invA, prfA, eaeA gene sequences targeted are specific for S. enterica , L. monocytogenes , E. coli O157:H7 and the combination of 16 h enrichment and PCR assay is sufficiently discriminatory to enable detection of these three pathogens in food.…”

“…This result referred the high specificity and sensitivity of universal primer-Multiplex -PCR in multiple pathogen detection which also confirmed that compound specific primer pairs had a high sensitivity in a single PCR. Yuan et al, (2009) confirmed the efficiency of this multiplex in detection of the three pathogens in artificial contaminates food. The presence of the three pathogenic bacteria in the twenty tested cheese samples was evaluated using Up-M-PCR.…”

“…The primer sets selected for the multiplex PCR assay are revealed in Table 2. Universal primer and compound specific primer pairs of Ecoli706-F/R, LM440-F/R, and Sal320-F/R, designed based on the presence of highly conserved sequence in all previous genes were used for the specific detection of E. coli O157:H7, L. monocytogenes, and S. enterica (Yuan et al, 2009). Furthermore 16S rRNA gene was also targeted as an internal control of the existence of amplifiable bacterial DNA.…”

Triplex PCR for the Simultaneous Detection of Pseudomonas aeruginosa, Helicobacter hepaticus, and Salmonella typhimurium

“…Yuan et al reported that 0.5 pg DNA from Escherichia coli, Listeria monocytogenes, and Salmonella spp. was suffi cient for detection by a triplex PCR method [17]. The level of sensitivity described by them suggests that the triplex PCR assay described in this study will be useful for the detection of gastroenteric diseases in rodents.…”

A new 7-plex PCR assay for simultaneous detection of shiga toxin-producing Escherichia coli O157 and Salmonella Enteritidis in meat products

“…in food or carcasses (Yuan et al 2009;Kim et al 2006;Omiccioli et al 2009), serological identification of Salmonella spp., (Fitzgerald et al 2003;Shanmugasundaram et al 2009) or toxin producing type identification in E. coli (Fujioka et al 2009;Kim et al 2005). However, to our knowledge, none of these multiplex assays could detect E. coli O157 along with shiga toxin-producing type and serotype Enteritidis of Salmonella simultaneously.…”