Real-time polymerase chain reaction methods for strain specific identification and enumeration of strain Lacticaseibacillus paracasei 8700:2

Abstract

Introduction: Reliable and accurate methods for probiotic identification and enumeration, at the strain level plays a major role in confirming product efficacy since probiotic health benefits are strain-specific and dose-dependent. In this study, real-time PCR methods were developed for strain specific identification and enumeration of L. paracasei 8700:2, a probiotic strain that plays a role in fighting the common cold.

Methods: The assay was designed to target a unique region in L. paracasei 8700:2 genome sequence to achieve strain level specificity. The identification assay was evaluated for specificity and sensitivity. The enumeration viability real-time PCR (v-qPCR) method was first optimized for the viability treatment, then the method was evaluated for efficiency, limit of quantification, precision, and its performance was compared to plate count (PC) and viability droplet digital PCR (v-ddPCR) methods.

Results: The identification method proved to be strain specific and highly sensitive with a limit of detection of 0.5 pg of DNA. The optimal viability dye (PMAxx) concentration was 50 μM. The method was efficient (> 90% with R ² values > 0.99), with a linear dynamic range between 6*10² and 6*10⁵ copies. The method was highly precise with a relative standard deviation below 5%. The Pearson correlation coefficient (r) was 0.707 for PC and v-qPCR methods, and 0.922 for v-qPCR and v-ddPCR. Bland-Altman method comparison showed that v-qPCR always gave higher values compared to PC method (relative difference ranging from 119% to 184%) and showed no consistent trend (relative difference ranging from -20% to 22%) when comparing v-qPCR and v-ddPCR methods.

Discussion: The difference between PC and v-PCR methods can potentially be attributed to the proportion of cells that exist in a viable but non culturable (VBNC) state, which can be count by v-PCR but not with PC. The developed v-qPCR method was confirmed to be strain specific, sensitive, efficient, with low variance, able to count VBNC cells, and has shorter time to results compared to plate count methods. Thus, the identification and enumeration methods developed for L. paracasei 8700:2 will be of great importance to achieve high quality and efficacious probiotic products.

Keywords: Lactobacillus paracasei 8700:2; PMAxx; probe-based assay; probiotics; real-time PCR; strain specific PCR assay; viability PCR; viable but non-culturable.

Figure 1

Evaluating the analytical sensitivity of Lacticaseibacillus paracasei 8700:2 strain-specific identification assay. Three DNA dilution series were prepared from starting concentrations of 10 ng/μl (Dilution series 1), 5 ng/μl (Dilution series 2), and 2 ng/μl (Dilution series 3). Dilution series consisted of five points each (10-fold) and each dilution was tested in triplicate to establish standard curves. The limit of detection was determined to be 0.5 pg. of DNA, corresponding to 150 target copies.

Figure 2

Optimization of viability treatment of L. paracasei 8700:2 strain-specific quantitative assay. (A) Final concentrations of PMAxx of 0, 25, 50, 100, and 150 μM were evaluated. PMAxx at concentrations of 25, 50, 100, and 150 μM resulted in significant shifts in Cq values from heat killed samples. PMAxx at a final concentration of 50 μM was selected as a potential optimal concentration. (B) Confirming 50 μM as an efficient PMAxx concentration using five mono-strain reference samples.

Figure 3

Determination of linear dynamic range, and reaction efficiency of L. paracasei 8700:2 strain-specific quantitative assay. Three independent serial dilution series were prepared from L. paracasei 8700:2 reference samples. The linear dynamic range was established between 6*10² to 6*10⁵ genomes. Reaction efficiency was 91.5, 93.7, and 90.9%. A vertical line at a Cq value of 33 cycles refers to reaction cut-off.

Figure 4

Evaluating precision of L. paracasei 8700:2 strain-specific quantitative assay. Two samples were tested at four dilutions to determine assay precision. The relative standard deviation (RSD%) ranged from 0.02 to 3.99% and from 0.18 to 4.23% for repeatability and reproducibility, respectively.

Figure 5

Correlation of L. paracasei 8700:2 viability real-time PCR method to plate count and viability droplet digital PCR methods. (A) Correlation between cell counts determined using viability real-time PCR (v-qPCR) and plate count (PC) method (Pearson r = 0.707). (B) Correlation between cell counts determined using viability real-time PCR (v-qPCR) and viability droplet digital PCR (v-ddPCR; Pearson r = 0.922).

Figure 6

Method agreement analysis for L. paracasei 8700:2 viability real-time PCR method with plate count and viability droplet digital PCR methods. (A) Bland–Altman method comparison (difference versus average analysis) for viability real-time PCR (v-qPCR) method and plate count (PC) method (Relative difference ranging from 119 to 184%). (B) Bland–Altman method comparison (difference versus average analysis) for viability real-time PCR (v-qPCR) method and viability droplet digital PCR (v-ddPCR) method (Relative difference ranging from −20 to 22%).

Figure 7

Assessing the ability of L. paracasei 8700:2 viability real-time PCR method to detect cell death following storage. Storage for 1 year resulted in 2.2-to-4.4-fold reduction in cell counts using plate count (PC) method and 1.5 to 2.4 fold reduction in cell counts using viability real-time PCR (v-qPCR).