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A Validated Real-Time PCR Method for the Specific Identification of Probiotic Strain Lactobacillus rhamnosus GG (ATCC
Hanan R Shehata1,2, Steven G Newmaster1
1NHP Research Alliance, College of Biological Sciences, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
Background:
Strain Lactobacillus rhamnosus GG is one of the best-studied and most widely used probiotic strains, with various health benefits. Because probiotic health benefits and safety are strain specific, the availability of a reliable assay for specific identification of Lactobacillus rhamnosus GG is vital to ensure probiotic efficacy.
Objective:
To design and validate a probe-based real-time PCR assay for specific identification of strain Lactobacillus rhamnosus GG.
Method:
Rapid Annotation using Subsystem Technology (RAST) was used to find a unique sequence region in the genome of Lactobacillus rhamnosus GG. A probe-based assay was designed and evaluated for specificity, sensitivity, efficiency, repeatability, and reproducibility.
Results:
RAST identified a unique gene coding for a hypothetical protein in the genome of Lactobacillus rhamnosus GG. The assay successfully amplified all 22 target samples and did not amplify any of the 28 non-target strains, achieving 100% true positive and 0% false positive results. The Limit of Detection (LOD) was determined to be 0.001 ng. Reaction efficiency values, from three dilution series, were 96.4%, 93.3%, and 96.8% with R square values of 0.9974, 0.9981, and 0.9998, respectively. Relative standard deviation (RSD, %) of repeatability was below 1% and RSD of reproducibility was below 4%.
Conclusions:
This Lactobacillus rhamnosus GG specific assay proved to be specific, sensitive, efficient, and reproducible. Since the assay was evaluated on two real-time PCR platforms, including a portable one, the assay can be used for onsite testing throughout the supply chain.
Highlights:
The availability of validated and reliable assays for strain-specific identification plays a vital role in achieving compliance in probiotic products.
