Probiotics are widely used in the food and medical fields for their functions in regulating the balance of the intestinal flora, promoting digestion, and enhancing human immunity [ 2 ]. Among the various probiotics,HN001 has unique effects in enhancing human immunity. Recent studies have shown that, in terms of modulating immunity in children,HN001 significantly reduces the cumulative prevalence of asthma in children at different ages, whereasHN019 does not [ 24 ]. The beneficial effects of probiotics are strain-specific and, in addition to strain characteristics, the efficacy of probiotic products depends on the number of live bacteria [ 11 ]. With the expansion of the probiotic industry, governments and industry stakeholders around the world have also stepped up their quality control oversight of commercial probiotic products. Several countries have also set limited standards for the number of live bacteria in probiotic products; for example, Italy, has established a feasible probiotic administration dose of 1 × 10CFU per day [ 25 ], and China requires probiotic health food products with a shelf life to have no fewer than 10CFU/mL of live bacteria per strain [ 26 ]. Currently, a major problem affecting the quality and safety of probiotic products is product mislabelling. The mislabelling of probiotic products may be reflected in the non-compliance of strains or the number of live bacteria [ 27 ]. Commercial probiotic products are often supplemented with a variety of active probiotics, and the diversity of probiotics and the stability of the live bacteria in these products pose challenges for quality monitoring. As a result, the mislabelling of commercial probiotic products may be unintentional. In addition to intentional mislabelling, the lack of strain-level probiotic live bacteria detection methods that are rapid, sensitive, and accurate creates difficulties for the regulatory enforcement of relevant authorities and the daily supervision of the probiotic industry. Therefore, there is an urgent need to develop an accurate quantitative method for the strain-level detection ofHN001 in commercial probiotic products.
L. rhamnosus
has been studied: Kim et al. [L. casei
,L. paracasei
, andL. rhamnosus
in a probiotic product via ring-mediated isothermal amplification, with a limit of detection as low as 103 CFU/mL; Wang et al. [L. rhamnosus
GG with a detection limit as low as 5 CFU/mL. However, none of these methods can achieve strain-levelL. rhamnosus
chenlv are exported all over the world and different industries with quality first. Our belief is to provide our customers with more and better high value-added products. Let's create a better future together.
HN001 detection, and it is also difficult to differentiate between dead and live bacteria in test samples. In contrast, in our study, anL. rhamnosus
HN001-specific single-copy gene fragment was used as the target gene. While achieving strain-level bacterial detection, the ddPCR quantification of the single-copy gene directly corresponded to the precise quantification ofL. rhamnosus
HN001. The detection system was optimised by treating the DNA to be detected with SD coupled with PMA using an orthogonal experimental design. Briefly, the SD-PMA-ddPCR established in this study made it easier for PMA to enter damaged cells through SD pretreatment, and PMA selectively penetrated the cell membrane of the dead bacteria to bind to the DNA and block its PCR amplification. ddPCR detected the unbound DNA of the live bacteria and realised the quantification of the live bacteria; thus, it differentiated between the dead/live bacteria. The theoretical detection limit of the SD-PMA-ddPCR method established in this study was as low as 4.2 × 10&#;10 ng/μL, and the detection limit of the simulated samples was as low as 1.42 × 105 CFU/g. For a practical applicability study of the samples, ten powder probiotic products were selected for testing. The number of probiotic species in the complex probiotic powders ranged from two to ten, with interference from other strains ofL. rhamnosus
. The results of the applicability study showed that the established SD-PMA-ddPCR method was able to rapidly and accurately detect the number of viable bacteria ofL. rhamnosus
HN001 in commercial probiotic powders, and the whole process of the assay took less than 1 h. To the best of our knowledge, the method established in the present study achieved the rapid quantitative detection of the viable bacteria ofL. rhamnosus
HN001 at the strain level for the first time. This method is a reliable tool for market supervision by relevant departments and daily monitoring in the probiotic industry, and it can effectively identify mislabelling in probiotic products labelled withL. rhamnosus
HN001, as well as safeguard their quality and safety.To solve the problem of probiotic characterisation and mislabelling, related research has gradually shifted from traditional culture methods to high-throughput sequencing. Traditional culture enumeration methods have also been used to achieve the enumeration of target probiotics in commercial products through the development of resuscitative and selective media [ 28 ]. High-throughput sequencing assesses the efficacy and potential safety risks of bacteria by sequencing the whole genome of a target strain, enabling the strain-level characterisation of the bacteria along with a gene-based functional annotation [ 29 ]. High-throughput sequencing based on metagenomics aims to evaluate the overall composition of the probiotics in complex probiotic products [ 30 ]. However, the above research methods have difficulties in balancing between rapid and accurate quantification, strain-level detection, and dead/live bacteria differentiation. As a result, molecular-biology-based assays have attracted researchers&#; attention. Among the probiotic assays developed based on molecular biology techniques, the rapid detection ofhas been studied: Kim et al. [ 31 ] achieved the rapid detection of, andin a probiotic product via ring-mediated isothermal amplification, with a limit of detection as low as 10CFU/mL; Wang et al. [ 32 ] used an ultrasensitive bacterial blotting electrochemical sensor to determineGG with a detection limit as low as 5 CFU/mL. However, none of these methods can achieve strain-levelHN001 detection, and it is also difficult to differentiate between dead and live bacteria in test samples. In contrast, in our study, anHN001-specific single-copy gene fragment was used as the target gene. While achieving strain-level bacterial detection, the ddPCR quantification of the single-copy gene directly corresponded to the precise quantification ofHN001. The detection system was optimised by treating the DNA to be detected with SD coupled with PMA using an orthogonal experimental design. Briefly, the SD-PMA-ddPCR established in this study made it easier for PMA to enter damaged cells through SD pretreatment, and PMA selectively penetrated the cell membrane of the dead bacteria to bind to the DNA and block its PCR amplification. ddPCR detected the unbound DNA of the live bacteria and realised the quantification of the live bacteria; thus, it differentiated between the dead/live bacteria. The theoretical detection limit of the SD-PMA-ddPCR method established in this study was as low as 4.2 × 10ng/μL, and the detection limit of the simulated samples was as low as 1.42 × 10CFU/g. For a practical applicability study of the samples, ten powder probiotic products were selected for testing. The number of probiotic species in the complex probiotic powders ranged from two to ten, with interference from other strains of. The results of the applicability study showed that the established SD-PMA-ddPCR method was able to rapidly and accurately detect the number of viable bacteria ofHN001 in commercial probiotic powders, and the whole process of the assay took less than 1 h. To the best of our knowledge, the method established in the present study achieved the rapid quantitative detection of the viable bacteria ofHN001 at the strain level for the first time. This method is a reliable tool for market supervision by relevant departments and daily monitoring in the probiotic industry, and it can effectively identify mislabelling in probiotic products labelled withHN001, as well as safeguard their quality and safety.