Establishment and application of an analytical in-house database (IHDB) for rapid discrimination of Bacillus subtilis group (BSG) using whole-cell MALDI-TOF MS technology.

Members of the Bacillus subtilis group (BSG) possess industrial applicability; unfortunately, B. subtilis and its phylogenetically closest species are indistinguishable from one another using 16S rDNA sequencing, physiological and biochemical tests. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a relatively novel technique for the fast and reliable identification of microorganisms. The aim of this study was to construct a unique analytical in-house database (IHDB) for BSG discrimination based on whole-cell protein fingerprinting using MALDI-TOF MS, as well as to discover biomarkers from the MS peaks to generate a classification model for further differentiation using the ClinProTools software. Type strains of 12 species (included five subspecies) of the BSG were used to build a main spectrum profile (MSP) to create an IHDB under the optimized parameters. The BSG isolates obtained from partial recA gene sequencing were used for IHDB validation. A total of 84 (100%) isolates were correctly identified to the species level and had high score values (mean score: 2.52). However, the IHDB had ambiguous identification at the subspecies level of Bacillus amyloliquefaciens. After implementation of the classification models, the strains could be clearly differentiated. We have successfully developed a rapid, accurate and cost-effective platform for the species- and subspecies-level discrimination of BSG based on the implementation of the IHDB and coupled with ClinProTools, which can be employed as an alternative technology to DNA sequencing and applied for efficient quality control of the microbial agent.

The dnaJ gene as a molecular discriminator to differentiate among species and strain within the Lactobacillus casei group.

Identifying Lactobacillus casei and its closely related taxa at the species and strain level using only phenotypic and genotypic (16S rDNA sequence homology analysis) techniques often yields inaccurate results. In this study, the dnaJ chaperone gene was investigated as a molecular target for inter- and intraspecies discrimination within the Lb. casei group as well as for the development of specific primers for species identification. The results showed that most of the examined strains could be clearly distinguished from closely related species based on the sequenced fragments. At the interspecies level, the dnaJ sequence similarities were 81.7%-85.5%. However, at the intraspecies level, the dnaJ sequence similarities were 96.2-100% and could be assigned to different haplotypes in Lactobacillus paracasei and Lactobacillus rhamnosus, respectively. Compared to the 16S rRNA gene, the dnaJ sequence showed greater variation at both the species and strain level. Thus, the dnaJ gene can be proposed as an alternative marker for the Lb. casei group that provides higher discriminatory power than the 16S rRNA gene. In addition, species-specific primers were developed and subsequently employed in two-plex minisequencing analysis and shown to be specific for Lb. paracasei and Lb. rhamnosus. Our data indicate that phylogenetic relationships in the Lb. casei group can be resolved using comparative sequence analysis of the dnaJ gene and that the Lb. paracasei and Lb. rhamnosus species can be simultaneously identified using a novel species-specific minisequencing assay.

Simultaneous discrimination of species and strains in Lactobacillus rhamnosus using species-specific PCR combined with multiplex mini-sequencing technology.

This study described the use of species-specific PCR in combination with SNaPshot mini-sequencing to achieve species identification and strain differentiation in Lactobacillus rhamnosus. To develop species-specific PCR and strain subtyping primers, the dnaJ gene was used as a target, and its corresponding sequences were analyzed both in Lb. rhamnosus and in a subset of its phylogenetically closest species. The results indicated that the species-specific primer pair was indeed specific for Lb. rhamnosus, and the mini-sequencing assay was able to unambiguously distinguish Lb. rhamnosus strains into different haplotypes. In conclusion, we have successfully developed a rapid, accurate and cost-effective assay for inter- and intraspecies discrimination of Lb. rhamnosus, which can be applied to achieve efficient quality control of probiotic products.

Cloning of a novel specific SCAR marker for species identification in Lactobacillus pentosus.

Identifying Lactobacillus species using only phenotypic and genotypic (16S rDNA sequence analysis) techniques yields inaccurate results. The objective of this study was to develop species-specific primers based on randomly amplified polymorphic DNA (RAPD) fingerprinting to distinguish species within the closely related Lactobacillus plantarum group. One of these primers, OPD-3, produced a species-specific band that was found only in the tested Lactobacillus pentosus. This specific fragment was isolated from agarose gel and ligated into a vector for DNA sequencing. A pair of primers, SpOPD3Lpen-F1/R1, that were highly specific sequence-characterized-amplified-regions (SCARs) were designed according to the nucleotide sequences of the specific RAPD marker. These primers were used for PCR analysis of the template DNA of the Lactobacillus strains, and a single 542 bp species-specific band was found only in L. pentosus. Using PCR, a novel species-specific primer pair is shown to rapidly, accurately and effectively distinguish L. pentosus from other species in the L. plantarum group of probiotic bacteria.

Utilization of elongation factor Tu gene (tuf) sequencing and species-specific PCR (SS-PCR) for the molecular identification of Acetobacter species complex.

The aim of this study was to use tuf gene as a molecular target for species discrimination in the Acetobacter genus, as well as to develop species-specific PCR method for direct species identification of Acetobacter aceti. The results showed that most Acetobacter species could be clearly distinguished, and the average sequence similarity for the tuf gene (89.5%) among type strains was significantly lower than that of the 16S rRNA gene sequence (98.0%). A pair of species-specific primers were designed and used to specifically identify A. aceti, but none of the other Acetobacter strains. Our data indicate that the phylogenetic relationships of most strains in the Acetobacter genus can be resolved using tuf gene sequencing, and the novel species-specific primer pair could be used to rapidly and accurately identify the species of A. aceti by the PCR based assay.

Molecular discrimination and identification of Acetobacter genus based on the partial heat shock protein 60 gene (hsp60) sequences.

BACKGROUND: To identify the Acetobacter species using phenotypic and genotypic (16S rDNA sequence analysis) technique alone is inaccurate. The aim of this study was to use the hsp60 gene as a target for species discrimination in the genus Acetobacter, as well as to develop species-specific polymerase chain reaction and mini-sequencing methods for species identification and differentiation. RESULTS: The average sequence similarity for the hsp60 gene (89.8%) among type strains was significantly less than that for the 16S rRNA gene (98.0%), and the most Acetobacter species could be clearly distinguished. In addition, a pair of species-specific primer was designed and used to specifically identify Acetobacter aceti, Acetobacter estunensis and Acetobacter oeni, but none of the other Acetobacter strains. Afterwards, two specific single-nucleotide polymorphism primers were designed and used to direct differentiate the strains belonging to the species A. aceti by mini-sequencing assay. CONCLUSION: The phylogenetic relationships in the Acetobacter genus can be resolved by using hsp60 gene sequencing, and the species of A. aceti can be differentiated using novel species-specific PCR combined with the mini-sequencing technology.

Use of novel species-specific PCR primers targeted to DNA gyrase subunit B (gyrB) gene for species identification of the Cronobacter sakazakii and Cronobacter dublinensis.

Cronobacter sakazakii and its phylogenetically closest species are considered to be an opportunistic pathogens associated with food-borne disease in neonates and infants. Neither phenotypic nor genotypic (16S ribosomal DNA sequence analysis) techniques can provide sufficient resolutions for accurately and rapidly identification of these species. The objective of this study was to develop species-specific PCR based on the gyrB gene sequence for direct species identification of the C. sakazakii and Cronobacter dublinensis within the C. sakazakii group. Two pair of species-specific primers were designed and used to specifically identify C. sakazakii and C. dublinensis, but none of the other C. sakazakii group strains. Our data indicate that the novel species-specific primers could be used to rapidly and accurately identify the species of C. sakazakii and C. dublinensis from C. sakazakii group by the PCR based assays.

Species identification of Wickerhamomyces anomalus and related taxa using ß-tubulin (ß-tub) DNA barcode marker.

Wickerhamomyces anomalus is used in food and feed processing, although the species has been reported as an opportunistic human pathogen, predominantly in neonates. Neither phenotypic nor the most frequently applied genotypic marker (D1/D2 LSU ribosomal DNA) provide sufficient resolution for accurate identification of this yeast. In this study, the ß-tubulin gene was used for species identification by direct DNA sequencing and as marker in a species-specific PCR assay. The results showed that all examined W. anomalus strains were clearly distinguished from the closely related species by comparative sequence analysis of the ß-tubulin gene. In addition, the species-specific primers were also developed based on the ß-tubulin gene, which was employed for polymerase chain reaction with the template DNA of Wickerhamomyces strains. A single 218 bp species-specific band was found only in W. anomalus. Our data indicate that the phylogenetic relationships between these strains are easily resolved by sequencing of the ß-tubulin gene and combined with species-specific PCR assay.

A novel non-synonymous SNP of the COLX gene and its association with duck reproductive traits.

Type X collagen (COLX) is a known marker of chondrocyte hypertrophy, which is exclusively expressed in hypertrophic chondrocytes and is reported to be involved in the process of mineralization. The purpose of this study was to investigate the relationship between COLX genotypes of single nucleotide polymorphism (SNP) and reproductive traits of Tsaiya ducks. A total of 336 Brown Tsaiya ducks from two lines, the control line (CL) with no selection and the selected line (SL), were employed for testing. We employed polymerase chain reaction -single strand conformation polymorphism and DNA sequencing to screen the polymorphisms of the COLX gene. One novel non-synonymous SNP in coding region (T74C: Val24Ala) of the COLX gene was found, and resulted in 3 genotypes TT, TC, and CC. The frequencies of genotype TT and allele T were high in both lines. Regarding egg weight at 40 weeks of age (EW40), based on SNP-trait association analysis, ducks with the CC genotype had a 4.09 and 4.15 g/egg lower EW40 as compared with ducks with the TT and TC genotypes in the CL, respectively (P < 0.05). In addition, significant positive dominance effect of 2.10 ± 1.05 g/egg for EW40 was detected (P = 0.0481). This finding indicated that selection for the genotype TT and TC ducks might contribute to an improved egg weight in the Tsaiya ducks. Further investigations on more duck populations with large sample sizes are needed to confirm.

Development of a novel PCR assay based on the gyrase B gene for species identification of Bacillus licheniformis.

Bacillus licheniformis is closely related to the Bacillus subtilis group, and could not be clearly identified using phenotypic and genotypic (16S rDNA sequence analysis) techniques alone. Some strains of this species are considered to be probiotic and are widely applied in the food and feed industry. The objective of this study was to develop species-specific PCR based on the gyrB gene sequence for direct species identification of the B. licheniformis within the B. subtilis group. A pair of species-specific primer was designed and used to specifically detect B. licheniformis, but none of the other B. subtilis group strains. Our data indicate that the novel species-specific primer could be used to rapidly and accurately identify the species of B. licheniformis from B. subtilis group by a PCR based assay.

The SNP genotypes of growth hormone gene associated with reproductive traits in Tsaiya ducks.

Our previous cDNA microarray study showed that the growth hormone (GH) gene may involve in the duck egg formation process. The purpose of this study was to investigate the relationship between GH genotypes of single nucleotide polymorphisms (SNPs) and reproductive traits of Tsaiya ducks. Primer pairs for the coding region in the GH were designed based on the duck genomic sequence. Polymorphisms were detected by polymerase chain reaction (PCR)-single strand polymorphism (SSCP) and were verified by DNA sequencing. Nineteen SNPs were identified in the duck GH gene, of which three coding SNPs (C3169T, C3700T and C5058G) were genotyped to investigate the associations with reproductive traits. The results showed that each SNP was associated with at least one duck fertility-related trait (p < 0.05). Haplotypes constructed on these three SNPs were associated with fertility rate (FR) and maximum duration of fertility (MDF) (p < 0.05). In particular, diplotype H1H1 was dominant for FR and MDF. This suggested that GH gene polymorphisms are associated with duck fertility-related traits. The SNPs in this gene may be used as potential markers for marker-assisted selection.

Discrimination of the Lactobacillus acidophilus group using sequencing, species-specific PCR and SNaPshot mini-sequencing technology based on the recA gene.

BACKGROUND: To clearly identify specific species and subspecies of the Lactobacillus acidophilus group using phenotypic and genotypic (16S rDNA sequence analysis) techniques alone is difficult. The aim of this study was to use the recA gene for species discrimination in the L. acidophilus group, as well as to develop a species-specific primer and single nucleotide polymorphism primer based on the recA gene sequence for species and subspecies identification. RESULTS: The average sequence similarity for the recA gene among type strains was 80.0%, and most members of the L. acidophilus group could be clearly distinguished. The species-specific primer was designed according to the recA gene sequencing, which was employed for polymerase chain reaction with the template DNA of Lactobacillus strains. A single 231-bp species-specific band was found only in L. delbrueckii. A SNaPshot mini-sequencing assay using recA as a target gene was also developed. The specificity of the mini-sequencing assay was evaluated using 31 strains of L. delbrueckii species and was able to unambiguously discriminate strains belonging to the subspecies L. delbrueckii subsp. bulgaricus. CONCLUSION: The phylogenetic relationships of most strains in the L. acidophilus group can be resolved using recA gene sequencing, and a novel method to identify the species and subspecies of the L. delbrueckii and L. delbrueckii subsp. bulgaricus was developed by species-specific polymerase chain reaction combined with SNaPshot mini-sequencing.

Application of the SNaPshot minisequencing assay to species identification in the Lactobacillus casei group.

This study used group-specific PCR combined with SNaPshot minisequencing for species identification within the Lactobacillus casei group. The L. casei group-specific PCR primer pair was designed using the rpoA gene sequence. A SNaPshot minisequencing assay using dnaK as a target gene was developed, and five SNP primers were designed by analysing the conserved regions of the dnaK sequences. The specificity of the minisequencing assay was evaluated using 63 strains of L. casei group species. The results showed that the group-specific PCR could assign Lactobacillus strains into the L. casei group, and the SNaPshot minisequencing assay was able to unambiguously and simultaneously discriminate strains belonging to the species L. casei, Lactobacillus paracasei, and Lactobacillus rhamnosus. In conclusion, we have successfully developed a rapid, accurate and cost-effective assay for species identification of members of the L. casei group.

Association of prolactin haplotypes with reproductive traits in Tsaiya ducks.

A previous cDNA microarray study showed that the prolactin (PRL) gene may be involved in the duck ovarian follicle development and egg formation process. The purpose of this study was to investigate the relationship between PRL genotypes of single nucleotide polymorphism (SNP) and reproductive traits of Tsaiya ducks. Primer pairs for the coding regions in the PRL were designed based on the duck genomic sequence database. Polymorphisms were detected by polymerase chain reaction (PCR)-single strand polymorphism (SSCP) and were verified by DNA sequencing. Six novel SNPs (T233C, T295C, G309T, C381A, G3941T and A3975C) were identified in the 1972 bp region of duck PRL gene, and all of them were located in non-coding regions. Single SNP-trait association analysis showed that each SNP was associated with at least one duck reproductive trait (P<0.05). Haplotype combinations constructed on these SNPs were associated with egg weight at 40 weeks of age (EW40), fertility rate (FR) and maximum duration of fertility (MDF) (P<0.0001). In particular, diplotype H1H2 had positive effect on EW40, whereas it had negative effect on FR and MDF (P<0.05). Positive effects of the diplotype H1H5 were observed for FR and MDF, but a negative effect was observed for EW40 (P<0.05). This suggested that the PRL gene plays an important role in the regulation of egg weight and fertility-related traits and could be a potential marker in a marker assisted selection program during duck balancing selection. Further investigations on more duck populations with large sample sizes are needed to confirm this finding.

Rapid identification of Lactobacillus plantarum group using the SNaPshot minisequencing assay.

This study used SNaPshot minisequencing for species identification within the Lactobacillus plantarum group. A SNaPshot minisequencing assay using dnaK as a target gene was developed, and five SNP primers were designed by analysing the conserved regions of the dnaK sequences. The specificity of the minisequencing assay was evaluated using 35 strains of L. plantarum group species. The results showed that the SNaPshot minisequencing assay was able to unambiguously and simultaneously discriminate strains belonging to the species L. plantarum subsp. plantarum, L. plantarum subsp. argentoratensis, Lactobacillus paraplantarum, Lactobacillus pentosus and Lactobacillus fabifermentans. In conclusion, a rapid, accurate and cost-effective assay was successfully developed for species identification of the members of the L. plantarum group.