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Lavers are typically consumed in dried or seasoned forms. However, commercially processed lavers can lead to seafood fraud because it is impossible to authenticate the original species based on morphological characteristics alone. In this study, we developed a capillary electrophoresis-based multiplex polymerase chain reaction (PCR) to authenticate six different laver species. The species-specific primer sets to target the chloroplast rbcL or rbcS genes were newly designed. We successfully established both singleplex and multiplex conditions, which resulted in specific amplicons for each species (N. dentata, 274 bp; N. yezoensis, 211 bp; N. seriata, 195 bp; N. tenera, 169 bp; N. haitanensis, 127 bp; P. suborbiculata, 117 bp). Moreover, the assays were sensitive enough to detect DNA ranging from 10 to 0.1 pg of DNA. The optimized capillary electrophoresis-based multiplex PCR was successfully applied to 40 commercial laver products. In addition to detecting the laver species as stated on the commercial label, the assay discovered cases where less expensive species were mixed in. With its advantageous properties, such as short amplicon size, high specificity, and superior sensitivity, this assay could be used for the authentication of the six laver species. [ABSTRACT FROM AUTHOR]

Granular ark (Tegillarca granosa), broughton's ribbed ark (Anadara broughtonii), and half-crenate ark (Anadara kagoshimensis) are important fishery resources throughout Asia; granular ark exhibiting a higher economic value due to its rarity. However, due to the similar morphological characteristics of the three species, the less valuable species could be exploited for food fraud. In this study, we developed a rapid on-site identification method based on a microfluidic chip for the detection of the three ark shell species. We designed new species-specific primers, targeting the genes encoding mitochondrial cytochrome b or cytochrome c oxidase I, for the identification of the three ark shells and estimated their specificity against 17 species, which amplified only the target species. The sensitivity of each primer was 0.001 ng. In addition, this method was further improved to develop a direct ultrafast polymerase chain reaction (PCR) for on-site food monitoring, which would allow for completing the entire procedure (from sampling to obtaining the results) within 25 min without DNA extraction. Our direct, ultrafast PCR was successfully applied to differentiate the three species from 29 commercial products. Therefore, this assay could be used as a rapid and cost-effective approach for the on-site identification of ark shells in commercial food products. [ABSTRACT FROM AUTHOR]

Background: Lactobacillus species are used as probiotics and play an important role in fermented food production. However, use of 16S rRNA gene sequences as standard markers for the differentiation of Lactobacillus species offers a very limited scope, as several species of Lactobacillus share similar 16S rRNA gene sequences. In this study, we developed a rapid and accurate method based on comparative genomic analysis for the identification of 37 Lactobacillus species that are commonly used in probiotics and fermented foods. Results: To select species-specific sequences or genes, a total of 180 Lactobacillus genome sequences were compared using Python scripts. In 14 out of 37 species, species-specific sequences could not be found due to the similarity of the 16S–23S rRNA gene. Selected unique genes were obtained using comparative genomic analysis and all genes were confirmed to be specific for 52,478,804 genomes via in silico analysis; they were found not to be strain-specific, but to exist in all strains of the same species. Species-specific primer pairs were designed from the selected 16S–23S rRNA gene sequences or unique genes of species. The specificity of the species-specific primer pairs was confirmed using reference strains, and the accuracy and efficiency of the polymerase chain reaction (PCR) with the standard curve were confirmed. The PCR method developed in this study is able to accurately differentiate species that were not distinguishable using the 16S rRNA gene alone. This PCR assays were designed to detect and identify 37 Lactobacillus species. The developed method was then applied in the monitoring of 19 probiotics and 12 dairy products. The applied tests confirmed that the species detected in 17 products matched those indicated on their labels, whereas the remaining products contained species other than those appearing on the label. Conclusions: The method developed in this study is able to rapidly and accurately distinguish different species of Lactobacillus, and can be used to monitor specific Lactobacillus species in foods such as probiotics and dairy products. [ABSTRACT FROM AUTHOR]

The closely related species, Lacticaseibacillus casei , L. paracasei , L. rhamnosus , L. chiayiensis , and L. zeae , are difficult to accurately discriminate by conventional identification methods. In this study, the bioTyper and in-house database of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was evaluated to discriminate five Lacticaseibacillus species. From the mass spectra of 130 isolates aligned with databases, 118 strains were correctly identified. On the other hand, databases could not accurately differentiate 12 isolates such as L. casei , L. rhamnosus and L. chiayiensis because the same colony was identified as two species with similar score. To overcome the database's limitations, the mass spectra were analyzed to discover species-specific protein peaks. The peaks at 6731 ± 1, 6849 ± 1, 7008 ± 1, 7376 ± 1, and 2593 ± 1 m/z were specifically found in the reference strains of L. casei , L. paracasei , L. rhamnosus , L. chiayiensis , and L. zeae , respectively. These peaks confirmed that the five peaks were consistently present in each species using 130 strains isolated from food samples. Our results demonstrate the high-resolution of MALDI-TOF MS technique for rapid and accurate classification of five species when used with databases coupled to specific peaks. • MALDI-TOF MS was applied to discriminate five Lacticaseibacillus species. • The bioTyper database yielded unreliable identification results for some isolates. • Specific protein peaks were able to overcome the limitations of the database. • This method can identify Lacticaseibacillus species as an alternative to PCR method. [ABSTRACT FROM AUTHOR]

Latilactobacillus sakei group comprises four closely related species, making it difficult to accurately distinguish them with standard markers such as the 16S rRNA gene. The objective of our study was to mine novel markers for PCR detection and discrimination of L. sakei group species and L. sakei subspecies by comparative pan-genomic analysis. A total of 63 genome sequences of L. sakei group species consisted of 119,899 coding genes, yielding 5741 pan-genomes, 831 core-genomes, 3347 accessory-genomes, and 1563 unique-genomes. The accessory-genome was compared to extract unique candidate genes common only to genomes of the same species. The candidate genes were then aligned with the other bacterial genomes to select marker genes present in all genomes of a given species, but not in the genomes of other species. We identified the arginine/ornithine antiporter, putative cell surface protein precursor, sodium:solute symporter, PRD domain protein, PTS sugar transporter subunit IIC, and phosphoenolpyruvate-dependent sugar phosphotransferase system EIIC as marker genes for L. sakei , L. sakei subsp. sakei , L. sakei subsp. carnosus , L. curvatus , L. graminis , and L. fuchuensis , respectively. Primer pairs were designed for each marker and showed 100% specificity for 48 lactic acid bacterial reference strains. The PCR method developed in this study was used to evaluate 106 strains isolated from fermented foods to demonstrate that the marker genes provided a viable alternative to the 16S rRNA gene. We also applied the method to the monitoring of kimchi samples to quantify L. sakei group species or subspecies. Our PCR method based on novel markers can rapidly identify L. sakei group with high accuracy and high throughput. • Novel markers for L. sakei group were identified by comparative pan-genomic analysis. • The marker genes accurately distinguish the four species and two subspecies. • Primer pairs designed for each marker gene showed 100% specificity in PCR. • The PCR method can qualify and quantify L. sakei group in fermented food. • The marker genes can serve as an alternative to the 16S rRNA gene sequence. [ABSTRACT FROM AUTHOR]

The Lactobacillus casei group, which includes the closely related species L. casei, L. paracasei , L. rhamnosus , and L. chiayiensis , has been under debate regarding its taxonomy because of the difficulty in distinguishing the species from each other. In the present study, we developed a novel real-time PCR assay for distinguishing the L. casei group species. The pan-genome, as determined by the genomes of 44 strains, comprised 6789 genes, comparative genomic analysis showed that L. casei group strains were classified by species. Based on these results, species-specific genes were identified, and primers were designed from those genes. Real-time PCR clearly distinguished each species of the L. casei group and specifically amplified only to the target species. The method was applied to 29 probiotic products, and the detected results and label claims were compared. Total 23 products were in accordance with the label claims, and the remaining products contained species different from those stated in the label claims. Our method can rapidly and accurately distinguish the L. casei group species in a single reaction. Hence, our assay can be applied to identify L. casei group species from food or environmental samples and to accurately determine the nomenclature of the species. • Specific genes of the L. casei group were identified using comparative genomics. • Novel primer pairs were designed to accurately distinguish L. casei group species. • A real-time PCR assay was developed to detect all the species in a single reaction. • The method was successfully applied to confirm L. casei group species in probiotic products. [ABSTRACT FROM AUTHOR]

Because of their beneficial effects, some enterococci species are used in probiotics, whereas some species are considered opportunistic pathogens implicated in human infections. High 16S rRNA sequence similarities among Enterococcus can impede their accurate classification. In this study, using pan-genome analysis, we developed an accurate method for the identification and quantification of six major Enterococcus species in different foods. Bioinformatics analysis revealed that some Enterococcus faecium genomes deposited in GenBank were misclassified. Six novel markers (FBPase_2 for Enterococcus durans , EcsB for Enterococcus faecalis , PurR for E. faecium , SDF for Enterococcus hirae , HTH_ARSR for Enterococcus lactis , and wxL for Enterococcus mundtii) were mined based on pan-genome analyses and used for quantitative polymerase chain reaction (qPCR). For all species, the detection limits in pure cultures and spiked food samples were 102 colony-forming units/ml (CFU/ml). This method facilitated the sensitive identification of six species in foods and the presence of nontarget bacteria. Moreover, qPCR efficiency was identified using 140 isolates and showed the same or higher resolution when compared with 16S rRNA sequencing. Our qPCR assay will help identify and quantify Enterococcus species in foods, thereby enabling a better understanding of ecological and safety aspects in food samples. • This study is the first to develop a qPCR method for six Enterococcus species. • This is the first report to identify misassigned E. faecium genomes in NCBI. • FBPase_2 , EcsB , PurR , SDF , HTH_ARSR , and WxL were identified as DNA markers. • Our DNA marker-based qPCR strategy showed high specificity and sensitivity. • This method can be used as an alternative to 16S rRNA sequencing. [ABSTRACT FROM AUTHOR]

The Lactobacillus acidophilus group consists of seven closely related species. Among these, Lb. acidophilus , Lb. gallinarum , and Lb. helveticus help maintain gut health and are used as a starter for fermented food. However, these species are difficult to differentiate using conventional methods due to the high similarity between the 16S rRNA and housekeeping genes. Thus, in this study, we selected biomarker genes to identify and discriminate the three species via pangenome analysis, and a multiplex SYBR Green real-time PCR that can be detected simultaneously in a single tube was developed. Pangenome analysis revealed three specific target genes: mucus-binding protein precursor to detect Lb. acidophilus , an amino acid ABC superfamily ATP binding cassette transporter carrier protein to detect Lb. gallinarum , and selenocysteine lyase to detect Lb. helveticus. The specificity was robustly verified using 26 Lb. acidophilus group strains and 62 other strains. The detection limits were 101 colony-forming units (CFU)/ml in pure culture. As per our findings, the developed method satisfactorily monitored Lb. acidophilus group species in probiotic and dairy products. This result suggests that real-time PCR based on specific targets provides a promising approach for the rapid, accurate, and sensitive identification of these three species. ● Pan-genome analysis revealed novel biomarker genes for three species. ● Biomarker genes can distinguish L. acidophilus , L. gallinarum , and L. helveticus. ● Multiplex SYBR Green real-time PCR showed high specificity and sensitivity. ● It could be applied to the rapid and accurate monitoring of three species in food. [ABSTRACT FROM AUTHOR]

[Display omitted] • This is the first study to develop a high-resolution PCR method for 25 species. • Pangenome analysis revealed molecular marker genes specific to each species. • The primer pairs showed 100% specificity and sensitivity in real-time PCR. • Some commercial products observed the absence of declared species on the label. • This assay was successfully applied to monitor commercial probiotic products. The health-promoting effects of probiotics are species-specific, and hence it is important to declare the correct information in products. However, some studies have identified issues related to the accuracy of labeling commercial probiotic products. In this study, we developed a high-resolution real-time PCR method based on pangenome analysis for a more affordable, rapid, and accurate identification of commercial probiotic products than sequencing methods. We selected 25 species or subspecies primarily used for probiotic strains and are closely related to them as targets. To extract molecular markers, 354 whole-genome sequences present in the target genomes but not in the pangenome of other genomes were compared, which resulted in the identification of molecular marker genes. The marker genes exhibited 100% specificity for 100 strains as assessed by the real-time PCR method. Fifty probiotic and dairy products were investigated to verify the information claimed on the label. Real-time PCR results showed that most products reflected the bacterial species declared in the label claim, whereas 12 products showed the presence of undeclared species or missing species. Our method for accurately verifying the labeling of probiotic products would be useful for quality control and safety. [ABSTRACT FROM AUTHOR]