Field Trial Study on BAX® System Real-time PCR Assay for the Detection of Shigella in China FDA Testing Foods

Introduction: A scorpion probe-based real-time PCR assay followed by culture confirmation method was developed and validated in-house for the detection of Shigella species in various foods including raw and ready-to-eat meats, jelly and produce. The specificity of the assay for Shigella was demonstrated with a diverse range of Shigella species, related organisms, and unrelated genera. The assay was sensitive enough to detect less than 2 log colony-forming units (CFU)/ml of the target pathogen in both pure broth culture and food samples. Our in-house validation studies demonstrated BAX^® RT-Shigella assay performed as well as the three reference methods (FDA-BAM Chapter 6, ISO21567 and China National Standard GB4789.5-2010) for the detection of Shigella in 8-20 hour enrichment in the selected foods. 

Purpose: The objective of this study was to perform pre-commercial field trial study to detect Shigella in various local FDA testing foods by assay and reference culture method.

Methods: The 25 g of various food samples including ready-to-eat meats, poultry, sea foods, mushrooms, soy products, vegetable and fruit salads, granulated sugar, drinking water, and juice etc. were weighed and enriched in 225 ml of Shigella broth as adopted by China National Standard GB4789.5-2010. After 20 h enrichment in Shigella broth, samples were either lysed, detected by the test method, or following the reference methods for the detection of Shigella.

Results: For in-house validation study with spiking Shigella in similar foods, the test method showed 100% sensitivity and specificity, and Chi-square analysis indicates it performed equivalent to the reference methods. For the total 74 samples tested in the field trial studies, 6 samples inhibited PCR reaction, while all others were in agreement with the reference method. The PCR inhibition can be relieved by growing back the enrichments in BHI broth for additional 3 hours and test the lysate by the test method.  

Significance: Method comparison studies showed that the test method performed as well as the reference method for detecting Shigella in 20 hour enrichment in the testing foods. The test method allows for detection Shigella in 1 day instead of 2-3 days by culture method. It is rapid and convenient to screen large varieties of foods with complexity of background microflora.