Quantitative PCR Assays for the Strain-Specific Identification and Enumeration of Probiotic Strain Lacticaseibacillus rhamnosus X253

Abstract

Probiotics are universally recognized for their health benefits, despite the fact that their effects depend on the strain. Identification and enumeration of probiotic strains are required prior to evaluating their effectiveness. Lacticaseibacillus rhamnosus X253 is a potential probiotic strain with antioxidant capacity. Comparative genomics and single nucleotide polymorphisms (SNPs) were used to identify a strain-specific locus within the holA gene for strain X253 that was distinct in 30 different L. rhamnosus strains. Using quantitative PCR, the primers and probe designed for the locus were able to distinguish L. rhamnosus X253 from the other 20 probiotic strains. The chosen locus remained stable over 19 generations. The sensitivity of the assay was 0.2 pg genomic DNA of L. rhamnosus X253, or 103 cfu/mL bacteria of this strain. In terms of repeatability and reproducibility, relative standard deviations (RSD) were less than 1% and 3%, respectively. Additionally, this assay achieved accurate enumerations of L. rhamnosus X253 in spiked milk and complex powder samples. The strain-specific assay could be used for quality control and compliance assessment of dairy products.

A novel repeat sequence-based PCR (rep-PCR) using specific repeat sequences of Mycobacterium intracellulare as a DNA fingerprinting

“…This reflects a shift in the position of the repetitive sequences in the genomes of the three strains, and the actual PCR also produced varied electrophoretic patterns depending on the strain. Third, since the reproducibility tests for samples before and after subculturing can reflect that the PCR is not the non-specific response (Cangelosi et al, 2004;Zhao et al, 2022), it was confirmed using samples before and after In this study, designed rep-PCR showed 7 identical groups and statistical correlation with the VNTR profiles (Cramer's V value of 0.814) (Figure 4). There were two sets of serial isolated M. intracellulare strains; the first set (KBN12P06800 in group 7 and KBN12P06801 in group 7 from a patient A) showed same rep-PCR group and shared on 22/23 VNTR loci (96%) (Figure 4).…”

“…This reflects a shift in the position of the repetitive sequences in the genomes of the three strains, and the actual PCR also produced varied electrophoretic patterns depending on the strain. Third, since the reproducibility tests for samples before and after subculturing can reflect that the PCR is not the non-specific response ( Cangelosi et al, 2004 ; Zhao et al, 2022 ), it was confirmed using samples before and after subculturing that designed rep-PCR has a greater level of reproducibility (Pearson similarity 95–98%) than previous published rep-PCR ( Figure 2 ).…”

“…In the MAC-PD, a recurrence caused by strains having different genotypes are frequent ( Koh et al, 2017 ; Akoglu, 2018 ; Heidari et al, 2018 ; Gautam, 2022 ; Wang et al, 2022 ), requiring a more advanced approach than naïve species identification. Various studies on the efficacy of NTM treatment have employed epidemiological approaches of the intraspecies classification level using ( Englund, 2003 ; Bussone et al, 2012 ; van der Zanden et al, 2012 ; San Millán et al, 2013 ; Thomson et al, 2013 , 2014 ; Koh et al, 2017 ; Heidari et al, 2018 ; Karauz et al, 2018 ; van Weezep et al, 2019 ; Gautam, 2022 ; Zhao et al, 2022 ). Since WGS, PFGE, and VNTR require a specialized technique, labor-intensive or time-consuming approaches, several clinical studies used rep-PCR ( Cangelosi et al, 2004 ; Jagielski et al, 2016 ; Jhun et al, 2018a , b ; Shin et al, 2020a , b ; Furuuchi et al, 2022 ) for diagnosis of reinfection/relapse, recurrent after latency, evaluation of antibiotics therapy in recurrent MAC-PD, and MAC bacteremia follow-up study.…”