Detection and absolute quantification of Lactiplantibacillus plantarum ATCC 202195 by quantitative real-time PCR

Abstract

ABSTRACT Lactiplantibacillus plantarum ATCC 202195 has probiotic properties that may be beneficial in early infancy. Since the effects of probiotics are often strain and dose specific, tools are needed to identify and enumerate L. plantarum ATCC 202195 with high sensitivity and specificity. Herein, a probe-based real-time quantitative PCR (qPCR) assay was developed to detect and enumerate L. plantarum ATCC 202195. The probe sequence contained a single nucleotide polymorphism within gene FEE41_08190 that was unique to L. plantarum ATCC 202195 at the time of the assay’s design. The linear dynamic range of the assay spanned five orders of magnitude with an R 2 that did not deviate from linearity and an amplification efficiency that ranged from 97% to 110% across six independent plates. At 95% confidence, the limit of detection in the assay was estimated at roughly 15 cells. No amplification was observed for reactions containing gDNA from seven other Lactobacillus species. This is the first report of a qPCR assay that detects and quantifies L. plantarum ATCC 202195. The assay is intended to be used as a resource for future clinical trials and commercial manufacturing settings. IMPORTANCE When administered for seven consecutive days shortly after birth, the probiotic bacterium Lactiplantibacillus plantarum ATCC 202195 plus fructooligosaccharide (FOS) was reported to reduce sepsis and lower respiratory tract infection events during early infancy in a randomized trial in India. Since probiotic effects are often strain specific, strain-level detection and quantification by routine molecular methods enables the monitoring of safety outcomes, such as probiotic-associated bacteremia, and allows for the quality of probiotic interventions to be assessed and monitored (i.e., verify strain identity and enumerate). Despite the potential clinical applications of L. plantarum ATCC 202195, an assay to detect and quantify this strain has not previously been described. Herein, we report the design of primer and probe sequences to detect L. plantarum ATCC 202195 and the development and optimization of a real-time PCR assay to detect and quantify the strain with high specificity and high sensitivity.