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Title: Sensitive detection of Shiga toxin 2 and some of its Stx 2 variants in environmental samples by a novel immuno-PCR assay

Author
item He, Xiaohua
item QI, WENYUAN - Shanghai Academy Of Agricultural Sciences
item Quinones, Beatriz
item Patfield, Stephanie
item Cooley, Michael
item Mandrell, Robert

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/17/2011
Publication Date: 3/25/2011
Citation: He, X., Qi, W., Quinones, B., Mcmahon, S.A., Cooley, M.B., Mandrell, R.E. 2011. Sensitive detection of Shiga toxin 2 and some of its Stx 2 variants in environmental samples by a novel immuno-PCR assay. Applied and Environmental Microbiology. 77(1):3558-3564. doi:10.1128/AEM.02205-10.

Interpretive Summary: Shiga toxin producing Escherichia coli (STEC) in the environment has been reported frequently. However, detection of STEC in environmental samples remains difficult. We developed a novel and sensitive assay for the detection of Shiga toxin 2 (Stx2) and Stx2 variants, which are important the critical factor in human disease. The assay involves concentration of Stx2, an antibody linked to a DNA sequence that binds to the Stx, and PCR amplification of the DNA. The detection limit of the assay is 0.1 pg/mL with a quantification range of 10 to 100,000 pg/mL. This method is >1000-fold more sensitive than a conventional assay, and appears not to be affected by complex samples. The method was validated with complex samples by detection of Stx2 in all shown to be positive by real-time PCR and culture methods, demonstrating a 100% sensitivity and specificity. This assay is an effective screening method for evaluating the occurrence of STEC in the environment.

Technical Abstract: Shiga toxin producing Escherichia coli (STEC) in the environment has been reported frequently. However, robust detection of STEC in environmental samples remains difficult because the numbers of bacteria in samples often are below the detection threshold of the method. We developed a novel and sensitive immuno-polymerase chain reaction (IPCR) assay for the detection of Shiga toxin 2 (Stx2) and Stx2 variants. The assay involves immunocapture of Stx2 and real-time PCR amplification of the DNA marker linked to a detection antibody recognizing the Stx2A subunit. The detection limit of the assay is 0.1 pg/mL with a quantification range of 10 to 100,000 pg/mL. The IPCR method was more than 1000-fold more sensitivethan a conventional enzyme-linked immunosorbent assay (ELISA). The sensitivity of the IPCR for Stx2 was not affected by environmental sample matrices of soil, feral swine colon, or water from watersheds, but was inhibited slightly by cow feces. Application of the IPCR assay to 23 enriched cultures of soil, feral swine colon, watershed and feces samples collected from the environment revealed that the IPCR detected Stx2 in all 15 samples that were shown to be STEC-positive by real-time PCR and culture methods, demonstrating a 100% sensitivity and specificity. The modification of the IPCR we have described in this study will be a sensitive and specific screening method for evaluating the occurrence of STEC in the environment.