The 6 best PCR methods for detecting STEC

Detection of Shiga toxin-producing E. coli is arguably the most complex microbiological analysis at the moment (in food microbiology!).

After explaining what STEC is, why they are complicated to detect, and how to choose the most suitable method for your lab, here is the final step: A selection of 6 reliable PCR methods to detect STEC.

To make this selection, we asked the SuperMicrobiologists community about the methods they use for detecting STEC. We then contacted the representatives of these brands to learn more about their real-time PCR technology.

In this article, we aim to highlight the advantages of each of these technologies to help you in your selection.

Now, all that’s left is for you to try them… and give us your feedback ;-).

Content :

• Assurance GDS by Merck
• IQ-Check by Bio-Rad
• dd-Check STEC Solution by Bio-Rad
• BACGene by Gold Standard Diagnostics

Assurance GDS MPX by Merck*

Merck offers 3 kits for the detection of the Top 7 STEC :

• Assurance GDS MPX for Top 7 STEC : For the screening. It detects stx1, stx2 and eae and O157H7. It utilizes an IMS approach prior to the PCR to capture the organisms belonging to the top 7 O-serogroup.
  
• Assurance GDS MPX ID for the Top 7 STEC : For the confirmation. It detects O103, O111, O121, O145, O26 and O45 serogroups.
  
• Assurance GDS EHEC ID : For the detection of O157:H7 serotype.

Validation: The Assurance GDS MPX Top 7 STEC is a certified method by AOAC and AFNOR 

AOAC validated matrices:

• Raw beef (375g)
• Stainless steel surface
• Spinach and leafy greens (25-375g)

AFNOR validated matrices:

• Raw meat (25 and 375g)
• Raw milk product (25g)

Strengths of the Assurance MPX for Top 7 STEC

• Reduction in False Positives: The IMS step captures only live cells, reducing the number of unconfirmed presumptive positives.
  
• Simple and Reliable Culture Confirmation: Using immuno-capture for PCR results eliminates biases from sample retesting.

*The Life Science business of Merck operates as MilliporeSigma in the U.S. and Canada.

iQ-Check STEC by Bio-Rad

Bio-Rad offers 2 kits for the detection of the Top 7 STEC:

• iQ-Check STEC VirX: For the detection of stx1-stx2 and eae genes.
  
• iQ-Check STEC SerO: For the detection of serotypes O26, O111, O45, O145, O121, O103, and E. coli O157

Validation : The iQ-Check STEC VirX method is AOAC and MicroVal certified.

AOAC Validated Matrices:

• Raw beef (375g)
• fresh spinach (375g)
• Cannabis (5 to 25g)
• All purpose flours (375g)

MicroVal Validation Scope 

• Detection of stx and eae genes
• Broad range of food, up to 375 g

Strengths of the IQ-Check STEC method

• Fast Results: 10 hours for raw meat and 15 hours for other samples.
   
• Validated on current problematic matrices: Flours, cannabis and vegetables.
  .
• Automation: Fully automated solution with iQ-Check Prep.

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dd-Check STEC Solution by Bio-Rad

The Bio-Rad dd-Check STEC solution allows co-localization of stx and eae virulence genes within a single bacteria.

The ddPCR technology generates thousands of uniform nanoliter-sized droplets in which there is only 1 bacteria.

Then a RT-PCR cycle is run in each single droplet. If the stx and eae genes are located on the same bacteria, then it is a true positive.

This technology reduces the percentage of false positives (when stx and eae are present in the same sample, but coming from 2 different bacteria). 

It provides a confirmation response in approximately 6 hours.

Validation : The dd-check STEC Solution is validated by the AOAC Performance Tested Methods^SM (PTM) Program.

AOAC Validated Matrices:

• Raw beef trim
• Fresh spinach
• Carcass sampling cloths

Strengths of the dd-Check STEC Solution

• Fewer “STEC presumptive positives” (false positives) save time by reducing unnecessary confirmations.
  
• Faster screening and confirmation times help products reach the market sooner.

BACGene by Gold Standard Diagnostics

Gold Standard Diagnostics offers 4 kits for the detection of the Top 7 STEC : 

• BACGene Mplex STEC Screen : for the detection of stx 1, stx 2 and eae genes.
  
• BACGene Mplex STEC SEROtype1 : for the detection of O111, O103, O26 (in one multiplex reaction).
  
• BACGene Mplex STEC SEROtype2 : for the detection of O145, O121 and O45 (in one multiplex reaction).
  
• BACGene E.coli O157:H7.

Kits can be used with 2 differents RT-PCR platforms : Agilent AriaMx^TM and Bio-Rad CFX96 Touch^TM Deep Weel

Validation : The BACGene STEC method from GSD is AOAC PTM^SM certified

AOAC Validated Matrices :

• Raw beef (375g)
• Romaine lettuce (375g)
• Flour (25g)
• Surface

Strengths of the BACGene STEC Top7 method

• Low sample dilution : As low as 1:3 for 375 g of beef trim and ground beef.
  
• Time-effective : Only 10 hours enrichment time for beef trim and ground beef.
  
• Optional use of certified free DNA-removal treatment to reduce risk of unconfirmed PCR positives.

More than 100,000 tests are performed every year using BACGene STEC kits.

The other methods mentioned by the SuperMicrobiologists are: Hygiena, Thermo and bioMerieux.

Why are some brands “better” presented than others?

Following the survey among the SuperMicrobiologists, we contacted all the favored brands to offer them sponsorship of this article.

This sponsorship allows us to offer all our content for free.

The brands “better” presented are those that agreed to support SuperMicrobiologists by sponsoring the article. We thank them.

So, if you need a PCR method for your STEC, start by testing these brands!

What about you ? Which method did you choose for detecting STEC? Come share your experience in the comments.