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

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.

The power of DNA based methods in probiotic authentication.

Introduction: The global probiotic market is growing rapidly, and strict quality control measures are required to ensure probiotic product efficacy and safety. Quality assurance of probiotic products involve confirming the presence of specific probiotic strains, determining the viable cell counts, and confirming the absence of contaminant strains. Third-party evaluation of probiotic quality and label accuracy is recommended for probiotic manufacturers. Following this recommendation, multiple batches of a top selling multi-strain probiotic product were evaluated for label accuracy. Methods: A total of 55 samples (five multi-strain finished products and 50 single-strain raw ingredients) containing a total of 100 probiotic strains were evaluated using a combination of molecular methods including targeted PCR, non-targeted amplicon-based High Throughput Sequencing (HTS), and non-targeted Shotgun Metagenomic Sequencing (SMS). Results: Targeted testing using species-specific or strain-specific PCR methods confirmed the identity of all strains/species. While 40 strains were identified to strain level, 60 strains were identified to species level only due to lack of strain-specific identification methods. In amplicon based HTS, two variable regions of 16S rRNA gene were targeted. Based on V5-V8 region data, ~99% of total reads per sample corresponded to target species, and no undeclared species were detected. Based on V3-V4 region data, ~95%-97% of total reads per sample corresponded to target species, while ~2%-3% of reads matched undeclared species (Proteus species), however, attempts to culture Proteus confirmed that all batches were free from viable Proteus species. Reads from SMS assembled to the genomes of all 10 target strains in all five batches of the finished product. Discussion: While targeted methods enable quick and accurate identification of target taxa in probiotic products, non-targeted methods enable the identification of all species in a product including undeclared species, with the caveats of complexity, high cost, and long time to result.

Probiotic and postbiotic analytical methods: a perspective of available enumeration techniques.

Probiotics are the largest non-herbal/traditional dietary supplements category worldwide. To be effective, a probiotic strain must be delivered viable at an adequate dose proven to deliver a health benefit. The objective of this article is to provide an overview of the various technologies available for probiotic enumeration, including a general description of each technology, their advantages and limitations, and their potential for the future of the probiotics industry. The current "gold standard" for analytical quantification of probiotics in the probiotic industry is the Plate Count method (PC). PC measures the bacterial cell's ability to proliferate into detectable colonies, thus PC relies on cultivability as a measure of viability. Although viability has widely been measured by cultivability, there has been agreement that the definition of viability is not limited to cultivability. For example, bacterial cells may exist in a state known as viable but not culturable (VBNC) where the cells lose cultivability but can maintain some of the characteristics of viable cells as well as probiotic properties. This led to questioning the association between viability and cultivability and the accuracy of PC in enumerating all the viable cells in probiotic products. PC has always been an estimate of the number of viable cells and not a true cell count. Additionally, newer probiotic categories such as Next Generation Probiotics (NGPs) are difficult to culture in routine laboratories as NGPs are often strict anaerobes with extreme sensitivity to atmospheric oxygen. Thus, accurate quantification using culture-based techniques will be complicated. Another emerging category of biotics is postbiotics, which are inanimate microorganisms, also often referred to as tyndallized or heat-killed bacteria. Obviously, culture dependent methods are not suitable for these products, and alternative methods are needed for their quantification. Different methodologies provide a more complete picture of a heterogeneous bacterial population versus PC focusing exclusively on the eventual multiplication of the cells. Alternative culture-independent techniques including real-time PCR, digital PCR and flow cytometry are discussed. These methods can measure viability beyond cultivability (i.e., by measuring cellular enzymatic activity, membrane integrity or membrane potential), and depending on how they are designed they can achieve strain-specific enumeration.

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

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.

Enumeration of Probiotic Strain Lacticaseibacillus rhamnosus GG (ATCC 53103) Using Viability Real-time PCR.

Probiotic health benefits are strain specific and are dose dependent. Hence, administering the correct strains, at the recommended doses is essential to achieve probiotic health benefits. Reliable methods are needed to facilitate probiotic strain identification and enumeration. Plate count methods are the most commonly used methods for probiotic enumeration. However, these methods are time-consuming, laborious, highly variable, and non-specific. Here, we developed a real-time PCR method for enumeration of a commonly used strain, Lacticaseibacillus rhamnosus GG. The method utilizes PMAxx as a viability dye to enumerate viable cells only. Optimization of viability treatment showed that PMAxx at a final concentration of 50 µM was effective in inactivating DNA from dead cells, and that bead beating for 5 min at 3000 rpm was effective in liberating DNA. The assay demonstrated high efficiency between 93 and 102%, with R2 values > 0.99. The assay showed high precision with relative standard deviation (RSD%) below 2.3%. Assay performance was compared to a plate count method in which there was a strong correlation between both methods (Pearson r = 0.8443). This method offers a 10 × shorter time for results and a higher precision compared to plate count methods. Furthermore, this method enables specific enumeration of L. rhamnosus GG in multi-strain products, which is not possible to achieve using plate count methods. This novel method facilitates faster and more accurate enumeration of L. rhamnosus GG as a raw ingredient as well as in finished products which enables better quality assurance and efficacy of probiotics for consumers.

Niche specificity and functional diversity of the bacterial communities associated with Ginkgo biloba and Panax quinquefolius.

Plant-associated bacteria can establish mutualistic relationships with plants to support plant health. Plant tissues represent heterogeneous niches with distinct characteristics and may thus host distinct microbial populations. The objectives of this study are to investigate the bacterial communities associated with two medicinally and commercially important plant species; Ginkgo biloba and Panax quinquefolius using high Throughput Sequencing (HTS) of 16S rRNA gene, and to evaluate the extent of heterogeneity in bacterial communities associated with different plant niches. Alpha diversity showed that number of operational taxonomic units (OTUs) varied significantly by tissue type. Beta diversity revealed that the composition of bacterial communities varied between tissue types. In Ginkgo biloba and Panax quinquefolius, 13% and 49% of OTUs, respectively, were ubiquitous in leaf, stem and root. Proteobacteria, Bacteroidetes, Actinobacteria and Acidobacteria were the most abundant phyla in Ginkgo biloba while Proteobacteria, Bacteroidetes, Actinobacteria, Plantomycetes and Acidobacteria were the most abundant phyla in Panax quinquefolius. Functional prediction of these bacterial communities using MicrobiomeAnalyst revealed 5843 and 6251 KEGG orthologs in Ginkgo biloba and Panax quinquefolius, respectively. A number of these KEGG pathways were predicted at significantly different levels between tissues. These findings demonstrate the heterogeneity, niche specificity and functional diversity of plant-associated bacteria.

Real-Time PCR Assays for the Specific Identification of Probiotic Strains Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC (NCIMB 30242).

The broad spectrum of health benefits attributed to probiotics has contributed to a rapid increase in the value of the probiotic market. Probiotic health benefits can be strain specific. Thus, strain-level identification of probiotic strains is of paramount importance to ensure probiotic efficacy. Both Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC (NCIMB 30242) strains have clinically proven health benefits; however, no assays were developed to enable strain-level identification of either of these strains. The objective of this study is to develop strain-specific PCR-based methods for Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC strains, and to validate these assays according to the guidelines for validating qualitative real-time PCR assays. Using RAST (Rapid Annotation using Subsystem Technology), unique sequence regions were identified in the genome sequences of both strains. Probe-based assays were designed and validated for specificity, sensitivity, efficiency, repeatability, and reproducibility. Both assays were specific to target strain with 100% true positive and 0% false positive rates. Reaction efficiency for both assays was in the range of 90 to 108% with R square values > 0.99. Repeatability and reproducibility were evaluated using five samples at three DNA concentrations each and relative standard deviation was < 4% for repeatability and < 8% for reproducibility. Both of the assays developed and validated in this study for the specific identification of Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC strains are specific, sensitive, and precise. These assays can be applied to evaluate and ensure compliance in probiotic products.

Locked Nucleic Acid Hydrolysis Probes for the Specific Identification of Probiotic Strains Bifidobacterium animalis subsp. lactis DSM 15954 and Bi-07™.

Probiotic health benefits are now well-recognized to be strain specific. Probiotic strain characterization and identification is thus important in clinical research and in the probiotic industry. This is becoming especially important with reports of probiotic products failing to meet the declared strain content, potentially compromising their efficacy. Availability of reliable identification methods is essential for strain authentication during discovery, evaluation and commercialization of a probiotic strain. This study aims to develop identification methods for strains Bifidobacterium animalis subsp. lactis DSM 15954 and Bi-07 (Bi-07™) based on real-time PCR, targeting single nucleotide polymorphisms (SNPs). The SNPs were targeted by PCR assays with locked nucleic acid (LNA) probes, which is a novel application in probiotic identification. The assays were then validated following the guidelines for validating qualitative real-time PCR assays. Each assay was evaluated for specificity against 22 non-target strains including closely related Bifidobacterium animalis subsp. lactis strains and were found to achieve 100% true positive and 0% false positive rates. To determine reaction sensitivity and efficiency, three standard curves were established for each strain. Reaction efficiency values were 86, 91, and 90% (R square values > 0.99), and 87, 84, and 86% (R square values > 0.98) for B. animalis subsp. lactis DSM 15954 and Bi-07 assays, respectively. The limit of detection (LOD) was 5.0 picograms and 0.5 picograms of DNA for DSM 15954 and Bi-07 assays, respectively. Each assay was evaluated for accuracy using five samples tested at three different DNA concentrations and both assays proved to be highly repeatable and reproducible. Standard deviation of Cq values between two replicates was always below 1.38 and below 1.68 for DSM 15954 and Bi-07 assays, respectively. The assays proved to be applicable to mono-strain and multi-strain samples as well as for samples in various matrices of foods or dietary supplement ingredients. Overall, the methods demonstrated high specificity, sensitivity, efficiency and precision and broad applicability to sample, matrix and machine types. These methods facilitate strain level identification of the highly monophyletic strains B. animalis subsp. lactis DSM 15954 and Bi-07 to ensure probiotic efficacy and provide a strategy to identify other closely related probiotics organisms.

Draft Genome Sequences of Lactobacillus delbrueckii subsp. bulgaricus Strains CBC-LB69 and CBC-LB8, Isolated from Homemade Dairy Foods in Bulgaria.

Here, we report the draft genome sequences of Lactobacillus delbrueckii subsp. bulgaricus strains CBC-LB69 and CBC-LB8. The strains were isolated from naturally processed, homemade dairy foods in Bulgaria. The two genome assemblies each resulted in 39 contigs with total lengths of 1,752,493 and 1,759,908 bp and GC contents of 49.80% and 49.90%, respectively.

Characterization of the microbiota of commercially traded finfish fillets.

The profile of human gut microbiota is known to be affected by diet and is linked to human health. Seafood is a highly consumed food and it accounts for a large proportion of food-borne illness. The objective of this study is to characterise the microbiota of fish fillets of various species sold in the Canadian market. We test 19 fish fillet samples from nine species in five fish families, ten of which were previously determined to be mislabeled as different species. The microbiota profiles were characterized using 16S rRNA gene high-throughput sequencing. Despite the complexities of the supply chain to produce these fillets, the major microbial groups were fairly consistent across samples. Significant differences in microbial taxa were observed between species, families, and based on labelling accuracy. Several putative spoilage and putative pathogenic taxa were identified. Studying food-associated microbiota can provide comprehensive information on food safety, authenticity, and traceability.

A Validated Real-Time PCR Method for the Specific Identification of Probiotic Strain Lactobacillus rhamnosus GG (ATCC 53103).

BACKGROUND: Strain Lactobacillus rhamnosus GG is one of the best-studied and most widely used probiotic strains, with various health benefits. Because probiotic health benefits and safety are strain specific, the availability of a reliable assay for specific identification of Lactobacillus rhamnosus GG is vital to ensure probiotic efficacy. OBJECTIVE: To design and validate a probe-based real-time PCR assay for specific identification of strain Lactobacillus rhamnosus GG. METHOD: Rapid Annotation using Subsystem Technology (RAST) was used to find a unique sequence region in the genome of Lactobacillus rhamnosus GG. A probe-based assay was designed and evaluated for specificity, sensitivity, efficiency, repeatability, and reproducibility. RESULTS: RAST identified a unique gene coding for a hypothetical protein in the genome of Lactobacillus rhamnosus GG. The assay successfully amplified all 22 target samples and did not amplify any of the 28 non-target strains, achieving 100% true positive and 0% false positive results. The Limit of Detection (LOD) was determined to be 0.001 ng. Reaction efficiency values, from three dilution series, were 96.4%, 93.3%, and 96.8% with R square values of 0.9974, 0.9981, and 0.9998, respectively. Relative standard deviation (RSD, %) of repeatability was below 1% and RSD of reproducibility was below 4%. CONCLUSIONS: This Lactobacillus rhamnosus GG specific assay proved to be specific, sensitive, efficient, and reproducible. Since the assay was evaluated on two real-time PCR platforms, including a portable one, the assay can be used for onsite testing throughout the supply chain. HIGHLIGHTS: The availability of validated and reliable assays for strain-specific identification plays a vital role in achieving compliance in probiotic products.

Draft Genome Sequence of Lactobacillus rhamnosus Strain CBC-LR1, Isolated from Homemade Dairy Foods in Bulgaria.

Here, we report the draft genome sequence of Lactobacillus rhamnosus strain CBC-LR1, which was isolated from naturally processed, homemade dairy foods in Bulgaria. The genome was assembled in 29 contigs with a total length of 2,892,155 bp and a GC content of 46.7%. Genome annotation predicted 2,638 coding genes and 49 tRNA genes.

Draft Genome Sequence of Streptococcus thermophilus Strain CBC-S77, Isolated from Homemade Dairy Foods in Bulgaria.

Here, we report the draft genome sequence of Streptococcus thermophilus strain CBC-S77. The strain was originally isolated from naturally processed, homemade dairy foods in West Rhode Mountain, Bulgaria. The genome was assembled in 148 contigs with a total length of 1,707,130 bp, with 1,563 coding genes and a GC content of 39.11%.

Combined Targeted and Non-targeted PCR Based Methods Reveal High Levels of Compliance in Probiotic Products Sold as Dietary Supplements in United States and Canada.

Probiotics are defined as "live microorganisms that, when administered in adequate amounts, confer a health benefit on the host." The diverse health benefits have contributed to rapid increase in probiotic consumption and in the value of probiotic market, valued at USD 46 billion in 2019. For probiotics to be effective, the correct species/strains should be delivered viable in an adequate dose. The most commonly used methods for species/strain identification are DNA based methods including targeted and non-targeted methods (e.g., high-throughput sequencing, HTS). Using different DNA based methods, previous studies reported several cases of non-compliance in probiotic products. The objectives of this study are to evaluate levels of compliance in probiotic products (presence of all declared species/strains, absence of any contaminants or undeclared species, and meeting the declared minimum viable cell count) and to compare the performance of targeted and non-targeted methods in probiotic authentication. To the best of our knowledge, this is the largest study of its kind, testing 182 probiotic products, containing a total of 520 strains, collected from United States and Canada. Using species-specific assays, 11 species could not be detected in ten products. Missing species were Lactobacillus casei in seven products, Bifidobacterium longum and Bifidobacterium bifidum in one product, B. longum in one product while B. longum subsp. longum was mislabeled as B. longum subsp. infantis in another. Additionally, undeclared Bifidobacterium animalis subsp. lactis was detected in one product. Viable count was determined for 72 samples and was found to be lower than declared in five samples, including one product showing no viable cells. Overall, non-compliance was observed in 15 out of 182 products (8%). Additionally, undeclared species at relative abundance of ∼1-2% were found in 14 products using HTS, however, their presence could not be confirmed using species-specific assays. The results show that targeted PCR based methods enable species and strain level identification. The results also highlight the need to continue to develop strain-specific assays appropriate for use with multi-strain products. True strain-specific assays will enable strain authentication in both single-strain products and multi-strain products to ensure probiotic products meet the label claims and ensure probiotic efficacy.

Investigating appropriate molecular and chemical methods for ingredient identity testing of plant-based protein powder dietary supplements.

Plant-based protein powders are rapidly growing in popularity, and outdated quality assurance tools expose vulnerabilities to adulteration via different methods of "protein spiking". Adequate diagnostic tools are urgently needed to be able to authenticate protein source ingredients and screen for potential adulterants. We explored the application of three diagnostic tools for ingredient identification: targeted PCR with Sanger sequencing, NGS, and LC-MS/MS. We collected 33 samples of common commercial products from the plant-based protein powder market and sought to identify botanical components using the three technologies. We found success in detection with all approaches, with at least one main protein source being identified by at least one approach in all samples. The investigation uncovered challenges to data collection or result interpretation with each technology including but not limited to amplification biases with PCR technologies, potential influence of DNA degradation, and issues with protein solubility during isolation. Ultimately, each platform demonstrated utility along with certain caveats, which epitomized the importance of orthogonality of testing.

Re-visiting the occurrence of undeclared species in sausage products sold in Canada.

Meat and poultry are major protein sources for humans worldwide. Undeclared ingredients in processed meat products, like sausage, continue to be identified in retail products all over the world. In collaboration with the Canadian Food Inspection Agency, a previous study of products purchased in Canada showed 20% mislabelling rate in sausage meats when tested for beef, pork, chicken, turkey and horse using DNA barcoding and digital PCR. In a follow-up to this study, an additional 100 "single species" sausage products were collected from Canadian retail markets, one year after our earlier study, to determine the prevalence of undeclared meat species in sausage. A new hierarchy of complementary molecular methods was applied in this study, including the testing of new target species (sheep and goat), in addition to beef, pork, chicken, turkey and horse. First, all samples were tested using DNA barcoding using universal primers, which revealed that 97% of the samples contained the declared species, presumably as the predominant species. Second, all samples were tested using ddPCR assays specifically targeting beef, pork, chicken, and turkey, which revealed that five beef samples, three chicken samples and two turkey samples contained undeclared species. Additionally, ddPCR revealed the presence of undeclared sheep in five samples. Overall, using complementary molecular methods, 14% of the samples contained additional undeclared species. It was encouraging to find a reduced rate of mislabelling compared to the previous study, though it remains clear that meat mislabelling is still an issue affecting Canadian consumers. The results from this study can be used to support decision-making processes for future inspection and monitoring activities in order to control species substitution or adulteration to protect consumers.

Survey of mislabelling across finfish supply chain reveals mislabelling both outside and within Canada.

Seafood has become one of the most heavily traded food commodities in the era of globalization. International seafood supply chains are complex and contend with many difficulties in bringing an enormous variety of products to market. A major challenge involves accurately labelling products such that they comply with a diverse set of regulatory frameworks, ranging from country-of-origin through to the final point of consumer sale. Thanks to DNA barcoding, seafood mislabelling is now recognized as a global problem, with potentially negative impacts on human health, economy and the environment. Mislabelling can result from species misidentification, use of inappropriate common names, incomplete and/or out-dated regulatory frameworks, or through market substitution. While prior studies have focused primarily on retail and food service establishments, this study used barcoding to assess rates of finfish mislabelling at multiple points in the supply chain within Ontario, Canada. A total of 203 specimens from 12 key targeted species were collected from varied importers, registered processing plants and retailers in Southern Ontario and identified using DNA barcoding. Species identity of samples was used to assess conformity of labelling against the Canadian Food Inspection Agency's (CFIA) Fish List, which revealed an overall mislabelling rate of 32.3% among targeted species. The mislabelling rate was significantly different between samples collected from importers and retailers. Among the mislabelled samples were seven samples that originated from US and were properly labelled according to US Food and Drug Administration (FDA) Seafood List. This study evaluated the integrity of chain of custody documents and identified discrepancies in 43 samples (21.4%). Implementing seafood traceability throughout the supply chain and harmonizing labelling regulations between countries can help to ensure industry compliance in a globalized market, while sampling at multiple points in the supply chain can help to reveal causes.

Guidelines for Validation of Qualitative Real-Time PCR Methods for Molecular Diagnostic Identification of Probiotics.

Backgroud: Probiotics have been shown to benefit human health through several mechanisms, including their role in improving the health of our gastrointestinal tracts. The health benefits of probiotics are strain specific, and therefore it is critical to include the correct strains in probiotic products when claiming specific health benefits. Several studies have reported issues concerning the accuracy of labeling of commercial probiotic products, including inaccurate taxonomy, missing species, or undeclared species. Consequently, there is a growing need to develop and validate assays to reliably verify strain identity in commercial probiotic products. PCR-based methods are the most commonly used methods for food species ingredient diagnostics because they are simple, fast, sensitive, and can be validated. Objective: The aim of this paper is to set the guidelines for validating targeted qualitative real-time PCR assays to verify the presence of specific strains in a probiotic supplement. Methods and Results: Qualitative real-time PCR assays are validated to evaluate the assay performance in terms of specificity, sensitivity, repeatability, and reproducibility in detecting target strains. Conclusions and Highlights: Setting these guidelines will facilitate and streamline the validation process for qualitative real-time PCR-based assays for probiotic identity authentication in support of quality assurance systems.

An endophytic microbe from an unusual volcanic swamp corn seeks and inhabits root hair cells to extract rock phosphate.

In the animal microbiome, localization of microbes to specific cell types is well established, but there are few such examples within the plant microbiome which includes endophytes. Endophytes are non-pathogenic microbes that inhabit plants. Root hairs are single cells, equivalent to the nutrient-absorbing intestinal microvilli of animals, used by plants to increase the root surface area for nutrient extraction from soil including phosphorus (P). There has been significant interest in the microbiome of intestinal microvilli but less is known about the root hair microbiome. Here we describe a bacterial endophyte (3F11) from Zea nicaraguensis, a wild corn discovered in a Nicaraguan swamp above rock-P lava flowing from the San Cristobal volcano. Rock-P is insoluble and a major challenge for plants. Following seed coating and germination on insoluble-P, the endophyte colonized epidermal surfaces, ultimately colonizing root hairs intracellularly. The endophyte promoted root hair growth and secreted acids to solubilize rock-P for uptake by a larger root hair surface. The most interesting observation was that a seed-coated endophyte targeted and colonized a critical cell type, root hair cells, consistent with earlier studies. The endophyte maintained its targeting ability in two evolutionary divergent hosts, suggesting that the host recognition machinery is conserved.

Droplet digital polymerase chain reaction (ddPCR) assays integrated with an internal control for quantification of bovine, porcine, chicken and turkey species in food and feed.

Food adulteration and feed contamination are significant issues in the food/feed industry, especially for meat products. Reliable techniques are needed to monitor these issues. Droplet Digital PCR (ddPCR) assays were developed and evaluated for detection and quantification of bovine, porcine, chicken and turkey DNA in food and feed samples. The ddPCR methods were designed based on mitochondrial DNA sequences and integrated with an artificial recombinant plasmid DNA to control variabilities in PCR procedures. The specificity of the ddPCR assays was confirmed by testing both target species and additional 18 non-target species. Linear regression established a detection range between 79 and 33200 copies of the target molecule from 0.26 to 176 pg of fresh animal tissue DNA with a coefficient of determination (R2) of 0.997-0.999. The quantification ranges of the methods for testing fortified heat-processed food and feed samples were 0.05-3.0% (wt/wt) for the bovine and turkey targets, and 0.01-1.0% (wt/wt) for pork and chicken targets. Our methods demonstrated acceptable repeatability and reproducibility for the analytical process for food and feed samples. Internal validation of the PCR process was monitored using a control chart for 74 consecutive ddPCR runs for quantifying bovine DNA. A matrix effect was observed while establishing calibration curves with the matrix type under testing, and the inclusion of an internal control in DNA extraction provides a useful means to overcome this effect. DNA degradation caused by heating, sonication or Taq I restriction enzyme digestion was found to reduce ddPCR readings by as much as 4.5 fold. The results illustrated the applicability of the methods to quantify meat species in food and feed samples without the need for a standard curve, and to potentially support enforcement activities for food authentication and feed control. Standard reference materials matching typical manufacturing processes are needed for future validation of ddPCR assays for absolute quantification of meat species.