Helicobacter pylori and other Helicobacter species DNA in human bile samples from patients with various hepato-biliary diseases

AIM： To investigate the presence of Helicobacter species by nested PCR of 16S rRNA genes followed by the presence of Flelicobacter pylori （H pylori） 16S rRNA, ureA, cagA genes in bile obtained at endoscopic retrograde cholangio-pancreatography （ERCP） from 60 Indian subjects. METHODS： Sixty bile samples were obtained from patients diagnosed with various hepato-biliary diseases and control subjects at ERCP. PCR analysis was carried out using primers for Helicobacter genus 16S rRNA gene and H pylori （16S rRNA, urea and cagA） genes. Gastric H pylori status was also assessed from biopsies obtained at endoscopy from patients with various hepatobiliary diseases and controls. The control group mainly consisted of subjects with gastric disorders. Sequencing analysis was performed to confirm that PCR products with 16S rRNA and cagA primers were derived from H pylori. RESULTS No Helicobacters were grown in culture from the bile samples. Helicobacter DNA was detected in bile of 96.7% and 6.6% of groups Ⅰ and Ⅱ respectively. Ten from group Ⅰ were positive for 16S rRNA and ureA and 9 were positive for cagA gene. In contrast of the 2 from the control, 1 amplified with 16S rRNA, ureA and cagA primers used. The sequences of the 16S rRNA genes and cagA were 99% similar to Helicobacter pylori. CONCLUSION： Helicobacters are associated with the pathogenesis of various hepato-biliary disorders.

Genomic selection of orange-spotted grouper(Epinephelus coioides)based on multiplex PCR enrichment capture sequencing

Orange spotted grouper(Epinephelus coioides)is an important mariculture fish,and genomic breeding of this grouper species has been hindered due to lack of efficient genotyping tools.Here,we developed a single nucleotide polymorphism(SNP)genotyping technology based on multiplex PCR enrichment capture sequencing,which mainly aims at target area for high-throughput sequencing,and 741 SNPs were designed for genomic selection(GS)of growth and ammonia tolerance traits at the same time.The multiplex PCR enrichment capture sequencing assay showed that the genotyping efficiency was more than 99%in the orange-spotted grouper and the predictive accuracy of body weight and ammonia tolerance traits was 82%and 96%,respectively.More importantly,the average identity of the sequences with these SNPs aligned to the genomes of giant grouper(E.lanceolatus)and brown-marbled grouper(E.fuscoguttatus)were both over 96%.Test data showed that the SNP genotyping efficiency was more than 94%in both giant grouper and brown-marbled grouper.In summary,these results indicated that the development of SNP loci and genotyping approach based on the multiple PCR enrichment capture sequencing are suitable for GS of growth and ammonia tolerance traits in various grouper species,and it would provide technical support for practical grouper breeding.

Selection of a DNA barcode for Nectriaceae from fungal whole-genomes

A DNA barcode is a short segment of sequence that is able to distinguish species. A barcode must ideally contain enough variation to distinguish every individual species and be easily obtained. Fungi of Nectriaceae are economically important and show high species diversity. To establish a standard DNA barcode for this group of fungi, the genomes of Neurospora crassa and 30 other filamentous fungi were compared. The expect value was treated as a criterion to recognize homologous sequences. Four candidate markers, Hsp90, AAC, CDC48, and EF3, were tested for their feasibility as barcodes in the identification of 34 well-established species belonging to 13 genera of Nectriaceae. Two hundred and fifteen sequences were analyzed. Intraand inter-specific variations and the success rate of PCR amplification and sequencing were considered as important criteria for estimation of the candidate markers. Ultimately, the partial EF3 gene met the requirements for a good DNA barcode: No overlap was found between the intra-and inter-specific pairwise distances. The smallest inter-specific distance of EF3 gene was 3.19%, while the largest intra-specific distance was 1.79%. In addition, there was a high success rate in PCR and sequencing for this gene (96.3%). CDC48 showed sufficiently high sequence variation among species, but the PCR and sequencing success rate was 84% using a single pair of primers. Although the Hsp90 and AAC genes had higher PCR and sequencing success rates (96.3% and 97.5%, respectively), overlapping occurred between the intraand inter-specific variations, which could lead to misidentification. Therefore, we propose the EF3 gene as a possible DNA barcode for the nectriaceous fungi.