Evaluation of Seven Different Commercially Available Real-Time PCR Assays for Detection of Shiga Toxin 1 and 2 Gene Subtypes

Abstract

Following the recent outbreak of Shiga toxin-producing Escherichia coli (STEC) O104:H4 infection in Germany, the demand for fast detection of STEC has again increased. Various real-time PCR-based methods enabling detection of Shiga toxin genes (stx) have been developed and can be used for applications in food microbiology. The present study was conducted to evaluate the reliability of seven commercially available real-time PCR systems for detection of stx1 and stx2 subtypes. For this purpose, pure cultures of 18 STEC strains harboring all known stx1 and/or stx2 subtypes were tested. Only one of the seven real-time PCR systems detected all known stx1 and stx2 subtypes. Six systems failed to detect the stx2f subtype. One system missed stx2 subtypes reported in association with severe human disease. Because the presence of certain stx genes (subtypes) is considered an important indicator of STEC virulence, systems differentiating between the stx1 and stx2 gene groups provide added value. Reliable and fast detection of stx genes is of major importance for both diagnostic laboratories and the food industry.

Evaluation of a Loop-Mediated Isothermal Amplification Suite for the Rapid, Reliable, and Robust Detection of Shiga Toxin-Producing Escherichia coli in Produce

“…Other studies also reported the failure of some PCR/qPCR assays to detect genetically distant stx subtypes, e.g., stx 2b , stx 2f , and stx 2g (40,45). A recent evaluation of seven commercially available qPCR systems for stx subtypes also returned variable results (41). Therefore, careful selection and evaluation of primers/ probes are critical in developing these NAATs.…”

“…Few studies have closely examined the ability of PCR and qPCR to detect STEC stx or eae subtypes (39)(40)(41), and to our knowledge, only one recent study explored the ability of LAMP to detect eae variants (42). There are currently 3 stx 1 subtypes (stx 1a , stx 1c , and stx 1d ), 7 stx 2 subtypes (a through g), and about 30 eae subtypes (␣, ␤, ␥, ε, etc.)…”

Evaluation of Enzyme Immunoassays and Real-Time PCR for Detecting Shiga Toxin-Producing Escherichia coli in Southern Alberta, Canada

“…The Centers for Disease Control and Prevention guidelines from October 2009 recommend simultaneous culture of stool samples and detection of Shiga toxins and/or their genes for all STEC isolates (31). Amplification and enzyme immunoassay (EIA) kits for STEC detection are commercially available (30,(32)(33)(34).…”

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

“…In contrast, the T m value of stx2f (81.729 C) was lower than those of stx2a, stx2b, stx2c, stx2d, and stx2e, and the T m value of stx2g (83.649 C) was slightly higher than those of stx2a, stx2b, stx2c, stx2d, and stx2e. Several real-time PCR assays for detection of stx1 and stx2 have been developed (8)(9)(10)(11), and real-time PCR assays using TaqMan probes, as reported by Derzelle et al (12), have been shown to detect all known stx2 subtypes. Nonetheless, real-time PCR with TaqMan probes is expensive as compared to that based on SYBR Green I (31).…”

“…Furthermore, the stx2 gene of enterohemorrhagic E. coli (EHEC) has recently been classified into 7 subtypes (stx2a, stx2b, stx2c, stx2d, stx2e, stx2f, and stx2g) (7). Although real-time PCR assays have been developed to detect these 7 stx2 subtypes (8)(9)(10)(11)(12), detection of these subtypes by RFBS24 has not yet been evaluated.…”