NHP Research Alliance, College of Biological Sciences, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
Department of Microbiology, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt.
J AOAC Int. 2020 Nov 1;103(6):1604-1609. doi: 10.1093/jaoacint/qsaa063.
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.
To design and validate a probe-based real-time PCR assay for specific identification of strain Lactobacillus rhamnosus GG.
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.
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%.
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.
The availability of validated and reliable assays for strain-specific identification plays a vital role in achieving compliance in probiotic products.
鼠李糖乳杆菌 GG 是研究最多、应用最广泛的益生菌菌株之一，具有多种健康益处。由于益生菌的健康益处和安全性取决于菌株，因此，能够可靠地鉴定鼠李糖乳杆菌 GG 的特定菌株的方法至关重要，这有助于确保益生菌的功效。
设计并验证一种基于探针的实时 PCR 方法，用于鉴定鼠李糖乳杆菌 GG 菌株。
使用快速分类系统（Rapid Annotation using Subsystem Technology，RAST）在鼠李糖乳杆菌 GG 基因组中寻找一个独特的序列区域。设计并评估了基于探针的方法的特异性、灵敏度、效率、重复性和重现性。
RAST 在鼠李糖乳杆菌 GG 基因组中识别出一个独特的基因，该基因编码一个假定的蛋白质。该检测方法成功地扩增了所有 22 个靶标样本，而未扩增任何 28 个非靶标菌株，获得了 100%的真阳性和 0%的假阳性结果。检测限（LOD）确定为 0.001ng。从三个稀释系列获得的反应效率值分别为 96.4%、93.3%和 96.8%，R 平方值分别为 0.9974、0.9981 和 0.9998。重复性的相对标准偏差（RSD，%）低于 1%，重现性的 RSD 低于 4%。
该鼠李糖乳杆菌 GG 特异性检测方法被证明具有特异性、灵敏度、高效性和重现性。由于该检测方法在两个实时 PCR 平台上进行了评估，包括一个便携式平台，因此该检测方法可用于整个供应链中的现场测试。
验证可靠的菌株特异性鉴定方法对于确保益生菌产品符合规定至关重要。
