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

Abstract

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.

Keywords: bacterial detection; probiotics; qPCR; sepsis.

Fig 1

Mauve alignment of L. plantarum ATCC 202195 gene FEE41_08190 to the 10 most homologous sequences in NCBI. The relative height and color of the bar at the top of the figure indicate the percent identity across all 11 sequences, with green indicating high homology, yellow indicating homology in some sequences, and red indicating no homology. The black lines indicate unique base pairs. The region of the gene that is amplified using the primers and probe designed in this study is outlined with a black box and magnified. Unique base pairs are shown in bold, and the SNP in the probe sequence is highlighted in red.

Fig 2

Standard curve of the qPCR assay for L. plantarum ATCC 202195. Cycle quantification (Cq) numbers were plotted against the log₁₀ starting quantity cell numbers from six independent experiments, each including three replicates per standard. The gray shaded area represents the 95% CI of the slope of the standard curve.

Fig 3

Limit of detection of the qPCR assay for L. plantarum ATCC 202195. Cycle quantification (Cq) numbers were plotted against the log₁₀ starting quantity cell numbers from two sets of qPCR experiments, which are outlined in Table 3. The reaction efficiency was calculated to be 97.8% using the following equation, 10^(–1/slope) – 1, where the slope was equal to −3.3758, as determined from a linear regression model. The gray shaded area represents the 95% CI of the slope of the standard curve.