Multiplex real-time PCR assay for detection of Escherichia coli O157:H7 and screening for non-O157 Shiga toxin-producing E. coli

Abstract

BackgroundShiga toxin-producing Escherichia coli (STEC), including E. coli O157:H7, are responsible for numerous foodborne outbreaks annually worldwide. E. coli O157:H7, as well as pathogenic non-O157:H7 STECs, can cause life-threating complications, such as bloody diarrhea (hemolytic colitis) and hemolytic-uremic syndrome (HUS). Previously, we developed a real-time PCR assay to detect E. coli O157:H7 in foods by targeting a unique putative fimbriae protein Z3276. To extend the detection spectrum of the assay, we report a multiplex real-time PCR assay to specifically detect E. coli O157:H7 and screen for non-O157 STEC by targeting Z3276 and Shiga toxin genes (stx1 and stx2). Also, an internal amplification control (IAC) was incorporated into the assay to monitor the amplification efficiency.MethodsThe multiplex real-time PCR assay was developed using the Life Technology ABI 7500 System platform and the standard chemistry. The optimal amplification mixture of the assay contains 12.5 μl of 2 × Universal Master Mix (Life Technology), 200 nM forward and reverse primers, appropriate concentrations of four probes [(Z3276 (80 nM), stx1 (80 nM), stx2 (20 nM), and IAC (40 nM)], 2 μl of template DNA, and water (to make up to 25 μl in total volume). The amplification conditions of the assay were set as follows: activation of TaqMan at 95 °C for 10 min, then 40 cycles of denaturation at 95 °C for 10 s and annealing/extension at 60 °C for 60 s.ResultsThe multiplex assay was optimized for amplification conditions. The limit of detection (LOD) for the multiplex assay was determined to be 200 fg of bacterial DNA, which is equivalent to 40 CFU per reaction which is similar to the LOD generated in single targeted PCRs. Inclusivity and exclusivity determinants were performed with 196 bacterial strains. All E. coli O157:H7 (n = 135) were detected as positive and all STEC strains (n = 33) were positive for stx1, or stx2, or stx1 and stx2 (Table 1). No cross reactivity was detected with Salmonella enterica, Shigella strains, or any other pathogenic strains tested.ConclusionsA multiplex real-time PCR assay that can rapidly and simultaneously detect E. coli O157:H7 and screen for non-O157 STEC strains has been developed and assessed for efficacy. The inclusivity and exclusivity tests demonstrated high sensitivity and specificity of the multiplex real-time PCR assay. In addition, this multiplex assay was shown to be effective for the detection of E. coli O157:H7 from two common food matrices, beef and spinach, and may be applied for detection of E. coli O157:H7 and screening for non-O157 STEC strains from other food matrices as well.

Comparative genomics reveals signature regions used to develop a robust and sensitive multiplex TaqMan real‐time qPCR assay to detect the genus Dickeya and Dickeya dianthicola

“…Multiplexing for specific detection of multiple targets in a single reaction is time efficient, provides enhanced reliability and further validation of the outcomes (Li et al ). The probes labelled with internal ZEN quencher along with the traditional 3′ end quencher enhance quenching efficiency, provide less background noise and increase precision (Arif et al ).…”

“…The probes labelled with internal ZEN quencher along with the traditional 3′ end quencher enhance quenching efficiency, provide less background noise and increase precision (Arif et al ). Multiplexing the reaction with different primer and probe sets can adversely affect the sensitivity and can increase the background noise (Li et al ). However, the developed assay did not show any discrepancy in the detection limit when assays were performed individually as compared to the multiplex reaction; both detected 10 fg of genomic DNA.…”

Comparative genomics reveals signature regions used to develop a robust and sensitive multiplex TaqMan real-time qPCR assay to detect the genus Dickeya and Dickeya dianthicola

“…Multiplexing for specific detection of multiple targets in a single reaction is time efficient, provides enhanced reliability and further validation of the outcomes (Li et al 2017). The probes labelled with internal ZEN quencher along with the traditional 3'end quencher enhance quenching efficiency, provide less background noise and increase precision ).…”

“…The probes labelled with internal ZEN quencher along with the traditional 3'end quencher enhance quenching efficiency, provide less background noise and increase precision ). Multiplexing the reaction with different primer and probe sets can adversely affect the sensitivity and can increase the background noise (Li et al 2017). However, the developed assay did not show any discrepancy in the detection limit when assays were performed individually as compared to the multiplex reaction; both detected 10 fg of genomic DNA.…”

Multiplex polymerase chain reaction detection of Shiga toxin genes and antibiotic sensitivity of Escherichia coli O157:H7 isolated from beef meat in Quetta, Pakistan

“…To ensure food safety, rapid and reliable diagnostic tools are essential for detecting E. coli O157:H7 in different food products. Multiplex real-time polymerase chain reaction (PCR) has emerged as the most attractive alternative to culture-based and immunological-based methods for detecting E. coli O157:H7 (Jinneman, Yoshitomi, & Weagant, 2003;Li, Liu, & Wang, 2017;Tabashsum, Nazneen, Ahsan, Bari, & Yasmin, 2016;Van Giau, Nguyen, Nguyen, Le, & Nguyen, 2016;Zhou et al, 2017).…”