Real-time PCR methods for identification and stability monitoring of Bifidobacterium longum subsp. longum UABl-14 during shelf life

Abstract

Bifidobacterium longum subsp. longum UABl-14^™ is an important probiotic strain that was found to support digestive health. Here we present the development and validation of real-time PCR methods for strain-specific identification and enumeration of this important strain. The identification method was evaluated for specificity using 22 target samples and 30 non-target samples. All target samples successfully amplified, while no amplification was observed from any non-target samples including other B. longum strains. The identification method was evaluated for sensitivity using three DNA dilution series and the limit of detection was 2 pg. of DNA. Coupled with a viability dye, the method was further validated for quantitative use to enumerate viable cells of UABl-14. The viability dye treatment (PMAxx) was optimized, and a final concentration of 50 μM was found as an effective concentration to inactivate DNA in dead cells from reacting in PCR. The reaction efficiency, linear dynamic range, repeatability, and reproducibility were also evaluated. The reaction efficiency was determined to be 97.2, 95.2, and 95.0% with R² values of 99%, in three replicates. The linear dynamic range was 1.3 × 10² to 1.3 × 10⁵ genomes. The relative standard deviation (RSD%) for repeatability ranged from 0.03 to 2.80, and for reproducibility ranged from 0.04 to 2.18. The ability of the validated enumeration method to monitor cell counts during shelf life was evaluated by determining the viable counts and total counts of strain UABl-14 in 18 multi-strain finished products. The viable counts were lower than label claims in seven products tested post-expiration and were higher than label claims in products tested pre-expiration, with a slight decrease in viable counts below label claim in three samples that were tested 2-3 months pre-expiration. Interestingly, the total counts of strain UABl-14 were consistently higher than label claims in all 18 products. Thus, the method enables strain-specific stability monitoring in finished products during shelf life, which can be difficult or impossible to achieve using the standard plate count method. The validated methods allow for simultaneous and cost-effective identification and enumeration of strain UABl-14 and represent an advancement in the quality control and quality assurance of probiotics.

Keywords: Bifidobacterium longum subsp. longum UABl-14; PMAxx; probe-based assay; probiotics; real-time PCR; strain specific PCR assay; viability PCR; viable but non culturable.

Figure 1

Evaluating the sensitivity of UABl-14 strain-specific assay. Three 10-fold dilution series of DNA were used to establish standard curves. The LOD was 0.002 ng of DNA or 755 copies.

Figure 2

Optimization of viability pre-treatments of UABl-14 strain-specific assay. (A) Agarose gel electrophoresis to examine the integrity of the DNA from non-heated and heat-killed cells. M is E-Gel^™ 1 Kb Plus DNA ladder. Samples 1–3 are the DNA from samples T-1, T-2, and T-3 (non-heated) and samples 4–6 are the DNA from samples T-1, T-2, and T-3 (heat-killed). (B) PMAxx viability dye treatments at 0 μM, 50 μM, 100 μM, and 150 μM were evaluated. PMAxx at 50 μM was used as an effective concentration in inactivating DNA from dead cells.

Figure 3

Evaluating the reaction efficiency and precision of UABl-14 strain-specific assay. Reaction efficiency of the UABl-14 strain-specific assay was determined to be 97.2, 95.2, and 95% with R² values of 99% and p value of 0.0004, 0.0005, and 0.0005 in three replicates.

Figure 4

Evaluating the precision of UABl-14 strain-specific assay. Repeatability and reproducibility were evaluated using 3 samples tested at five dilutions. The RSD% for repeatability ranged from 0.71 to 2.36, 0.03 to 1.51, and 0.43 to 2.80, and RSD% for reproducibility ranged from 0.06 to 0.61, 0.10 to 1.20, and 0.04 to 2.18 for the 3 samples.

Figure 5

Assessing the ability of UABl-14 strain-specific assay in monitoring strain stability in 18 multi-strain finished products during shelf life. The viable counts were lower than label claims in all 7 products tested post expiration dates and were higher than label claims in products tested pre-expiration dates, with the exception of samples that were within 3 months of expiration.

Figure 6

Total counts (viable and dead) and viable counts of strain UABl-14 versus label counts in 18 multi-strain finished products during shelf life. Unlike the viable counts of strain UABl-14, the total counts of strain UABl-14 were consistently higher than label claims in all 18 products.