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Updated: Jun 4, 2026
Detection and Genogrouping of Noroviruses from Children's Stools By Taqman One-step RT-PCR
Published on: July 22, 2012
O serogroup specific real time PCR assays for the detection and identification of nine clinically relevant non-O157
Andrew Lin1, Omar Sultan, Henry K Lau
1U.S. Food and Drug Administration, San Francisco District Laboratory, 1431 Harbor Bay Parkway, Alameda, CA 94502, USA. andrew.lin@fda.hhs.gov
Abstract:
TaqMan™ real time PCR assays were designed for each of the non-O157 STEC O serogroups most commonly associated with human illness: O26, O45, O91, O103, O111, O113, O121, O128, and O145. The nine RT-PCR assays can be run as single assays when a known pathogen is of concern, or multiplexed in three reactions, to quickly screen for the most clinically relevant O serogroups. All assays included an internal amplification control constructed from the green fluorescent protein gene as an indicator of PCR inhibition. Of 103 strains tested, the inclusive tests accurately identified the O serogroup for 101 strains. The exclusive tests for each assay yielded no false positives for over 120 Escherichia coli strains and 23 non-E. coli bacteria tested. Furthermore, the RT-PCR assays were tested by inoculating four food matrices, milk, apple juice, lettuce, and ground beef, at ≤30 CFU/25 g or mL. Following a 24h selective enrichment, the RT-PCR assays detected STECs in all foods except for one ground beef sample inoculated with O111, and all apple juice samples inoculated with O113. The assays could also detect each O serogroup in human stool specimens inoculated with STECs at 1000 CFU/0.5 g of stool following 24 h enrichment.
Insights
New real-time PCR (RT-PCR) assays accurately detect common non-O157 Shiga toxin-producing E. coli (STEC) serogroups in food and clinical samples, improving pathogen identification.
Area of Science:
- Microbiology
- Molecular Biology
- Food Safety
Background:
- Shiga toxin-producing Escherichia coli (STEC) non-O157 serogroups are significant causes of human illness.
- Accurate and rapid detection methods are crucial for public health and food safety.
Purpose of the Study:
- To develop and validate novel TaqMan™ real-time PCR (RT-PCR) assays for the rapid identification of common non-O157 STEC O serogroups.
- To assess the performance of these assays in various food matrices and clinical specimens.
Main Methods:
- Designed nine specific RT-PCR assays targeting O serogroups O26, O45, O91, O103, O111, O113, O121, O128, and O145.
- Incorporated an internal amplification control using the green fluorescent protein gene to monitor PCR inhibition.
- Validated assays using 103 STEC strains, over 120 E. coli strains, and 23 non-E. coli bacteria, and tested them in spiked food samples (milk, apple juice, lettuce, ground beef) and human stool specimens after enrichment.
Main Results:
- The inclusive tests correctly identified the O serogroup for 101 out of 103 STEC strains.
- Exclusive tests showed no false positives with other E. coli or non-E. coli bacteria.
- RT-PCR assays successfully detected STECs in spiked food samples, with minor exceptions in ground beef (O111) and apple juice (O113).
- Detection was also successful in inoculated human stool specimens following enrichment.
Conclusions:
- The developed RT-PCR assays offer a sensitive and specific method for detecting clinically relevant non-O157 STEC serogroups.
- These assays can be used individually or multiplexed for efficient screening in food safety and clinical diagnostics.
- The assays demonstrate potential for rapid STEC identification in diverse sample types, aiding in outbreak response and surveillance.
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