Real-Time TaqMan PCR for Yersinia enterocolitica Detection Based on theailandfoxAGenes

Abstract

Y ersinia enterocolitica, a cause of emerging enteric infections, is a foodborne pathogen associated with various enteric and systemic syndromes, e.g., diarrhea, enteritis, and enterocolitis. Therefore, the detection of this pathogen has important significance. Previous real-time PCR for detection of Y. enterocolitica was primarily based on the ail gene; biotype 1A nonpathogenic strains were not included (1-3). However, recent studies (4,5) showed that biotype 1A ystB-positive strains are potentially pathogenic and related outbreaks were reported. We therefore designed a TaqMan real-time PCR method for detection of both pathogenic and nonpathogenic Y. enterocolitica strains.Using data from sequence analysis of the ail and foxA genes from many Y. enterocolitica strains (6), we designed TaqMan probes and primers (Table 1).An experiment using the entire reaction system was performed using a 20-l volume containing 10 l premix (TaKaRa; China), 7.2 l ultrapure distilled water, 0.2 l ROXII, and 0.2 l (100 nmol/liter) of each primer and probe. A two-step method was adopted. The cycling conditions for the use of a Rotor-Gene Q system consisted of 1 cycle of initial denaturation at 95°C for 10 s followed by 40 cycles of melting at 95°C for 5 s and elongation at 60°C for 30 s. And for the use of a ABI 7500 Fast system, cycling was performed using one initial denaturation at 95°C for 20 s followed by 40 cycles of melting at 95°C for 3 s and elongation at 60°C for 30 s.A total of 168 pathogenic Y. enterocolitica strains (3 ail sequence patterns and 8 foxA sequence patterns) and a total of 41 nonpathogenic Y. enterocolitica strains (13 foxA sequence patterns) were used to assess the sensitivity and specificity of the method. Most of these strains were isolated from animals, mainly swine and mice, in China. Furthermore, 258 non-Y. enterocolitica strains were used to test the specificity of the two detection systems. Most of the strains were Gram-negative bacteria of various genera. All the strains were isolated from patients and identified by using a Vitek Compact 2 biochemical identification instrument (bioMérieux). The results showed that both the ail and foxA gene detection systems have 100% specificity.Standard curves and sensitivity data were obtained by amplifying standard plasmid that had been serially diluted 10-fold. In parallel, we detected the sensitivity of conventional PCR. The results suggested that the slope was Ϫ3.09 and the R 2 was 0.99 for the ail system and that the slope was Ϫ3.16 and the R 2 was 0.99 for the foxA system. The detection limit was 10 2 copies/l for both detection systems. This represented sensitivity 10 times greater than that of the conventional PCR detection.To exclude false-negative results caused by potential inhibitors, we used the internal amplification control (IAC) developed by Fricker et al. (7). A total of 15 pathogenic Y. enterocolitica strains were used to test the IAC with the ail and foxA detection systems. When the ail and foxA detection systems were mixed with the IAC, both of...

Detection of 12 Common Food-Borne Bacterial Pathogens by TaqMan Real-Time PCR Using a Single Set of Reaction Conditions

“…Although TaqMan probes are more costly than SYBR green, the use of the sequence specific probes ensures high sensitivity, and specificity (Postollec et al, 2011; Law et al, 2014). Some target genes selected in the present study, as well as functional genes related to virulence or metabolism, have showed good specificity toward C. jejuni, Shigella spp., and Y. enterocolitica , when compared to standard methods (Wiemer et al, 2011; Wang et al, 2014; Van Lint et al, 2015). Three specific genes, namely, rfbE from E. coli O157:H7, hilA from S. enterica and prfA from L. monocytogenes were selected in our study to design primers and probes while fliC, invA , and hlyA were used in previous studies (Zhou et al, 2017).…”

“…The virulence genes have been used as target genes for nucleic acid-based assays (Finlay and Falkow, 1997; Wiemer et al, 2011; Van Lint et al, 2015). Namely, flic, hlyA, rfbE were selected from E. coli O157:H7, invA, staG from Salmonella spp., ail, nuc from Staphylococcus aureus and foxA genes from Yersinia enterocolitica to design primers and probes in real-time TaqMan PCR assay (Ranjbar et al, 2014b; Wang et al, 2014; Ding et al, 2017; Zhou et al, 2017). Additionally, a simultaneous quantifying detection of V. parahaemolyticus and L. monocytogenes by TaqMan-based real-time PCR using primers and probes that target tlh and hlyA genes, respectively, was developed (Zhang et al, 2015).…”

High-Throughput 16S rRNA Sequencing to Assess Potentially Active Bacteria and Foodborne Pathogens: A Case Example in Ready-to-Eat Food

“…Real-time PCR of gene markers of Y. enterocolitica pathogenic strains was performed as previously described [29] with the exception that SYBR Green I dye was used instead of Taqman probes for monitoring the amplification.…”

Identification of Atypical Enteropathogenic Escherichia coli O98 from Golden Snub-Nosed Monkeys with Diarrhea in China

“…and Yersinia detection, the fecal samples were mixed with phosphate-buffered saline and centrifuged at 500 g for 10 min, and the supernatant was used to extract the DNA using a TianGen DNA extraction kit (TianGen, Beijing, China) according to the manufacturer’s instructions. The DNA was subjected to qPCR with the primers and reaction conditions described in previous reports ( 34 , 35 ).…”