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PCR STEC Analysis

PRC STEC Analysis

ALS Life Sciences is INAB accredited and approved by the Irish Department of Agriculture, Food and the Marine for microbiological testing required for meat plants for USA trade to include the Shiga toxigenic E.coli detection method.

 

E. coli bacteria are normal flora in human and animal intestines and are usually harmless. However, Shiga toxin-producing Escherichia coli (STEC) are known to be highly pathogenic to humans. STECs are defined by the presence of the stx1 or stx2 (Shiga toxin genes) in their genome. The eae (intimin) gene is an additional virulence marker.

 

Real-time PCR detection of virulence genes in Shiga Toxin Producing E. coli (STEC) is required for testing minced meat/trimmings when product intended for grinding is planned to be produced for shipping to the US. If a positive sample is detected, then the 7 major serogroups (O157, O111, O26, O103, O145, O45 and O121) must then be tested for.

 

ALS can perform this analysis over a number of stages – being up to 2 PCR screens and in the event of a presumptive positive, a further series of confirmation steps which includes several microbial isolation steps, latex agglutination as well as 2 further PCR steps.

 

• PCR Screen 1: The iQ-Check STEC VirX kit, based on a multiplex real-time PCR system, allows the detection of the stx and eae virulence genes in one well, within a few hours after the end of the microbiological enrichment. A sample that is positive for both stx1/2 and eae is then tested with the iQ-Check STEC SerO real-time PCR kit

 

• PCR Screen 2: The iQ-Check STEC SerO kit, based on a multiplex real-time PCR system, allows the detection of these 7 major serogroups, including E. coli O157:H7, within a few hours after the iQ-Check STEC VirX result.

 

If a negative result is found after the first VirX PCR screen, no further steps are required. Where a presumptive is reported on the first PCR screen, analysis must be performed on the second SerO PCR screen.  If a negative result is reported at this stage, no further analysis is required.  Where a presumptive positive is reported on the second PCR screen, ALS need to proceed with isolation and identification of the serogroup(s) involved.  This is an extensive study, with several microbial isolation and PCR identification steps which takes about 3 days to complete. 

 

PCR generates many copies of target DNA. During the PCR reaction, several cycles of heating and cooling allow DNA denaturation, followed by primers annealing to the target region. The DNA polymerase then uses these primers and deoxynucleotide triphosphates (dNTPs) to extend the DNA, creating copies called amplicons. Specific probes are used to detect DNA by hybridizing to the amplicons. These probes are linked to a fluorophore which fluoresces only when hybridized to the target sequence. In the absence of target DNA, no fluorescence will be detected.  During each PCR cycle, the optical module measures this fluorescence, and the software plots the fluorescence intensity versus the number of cycles.