Establishment and evaluation of multiplex PCR for detection of main pathogenic bacteria of endometritis in Tibetan sheep / 生物工程学报

A multiplex PCR method was developed to detect the main pathogens of Qinghai Tibetan sheep endometritis. First, the genomes of five standard bacterial strains were extracted and specific primers were selected; the multiplex PCR method was established by using the genome of the standard strain as a template. The samples were collected by sterile cotton swab from Tibetan sheep uterus, and then placed in LB medium and numbered. After 48 h, the genomes of cultured bacteria were extracted and detected by single PCR method, then the positive samples were recorded. The positive samples detected by single PCR were selected for multiplex PCR detection and recorded again. The coincidence rate between these two methods was calculated to measure the accuracy of multiplex PCR. In order to identify the species of the pathogen, 30 positive samples verified by single and multiplex PCR were randomly selected for bacterial isolation and identification. In the 600 samples, the infected ratio of Streptococcus agalactiae (GBS) was 47.33%, Escherichia coli 34.83%, Staphylococcus aureus 6.5%, Salmonella and Trueperella pyogenes were negatively detected. Among the positive samples detected by multiplex PCR, the positive ratio of GBS was 45.50%, E. coli 33.50%, S. aureus 6.5%. Comparison of two detection results, Multiplex PCR detection coincidence rate is more than 95%. The isolated pathogens were identified as E. coli, GBS and S. aureus, which was consistent with the results of two methods. The multiplex PCR method was successfully established and the main pathogens of endometritis in Qinghai Tibetan sheep were GBS, E. coli and S. aureus.

Transcriptional profiling of two different physiological states of the yak mammary gland using RNA sequencing.

Yak milk is superior to common cow milk in nutrients including protein, fat and calories. However, the milk yield of the yak is very much lower compared with other dairy bovines. To understand the molecular mechanisms of lactogenesis, lactation and mammary gland development, mammary tissue samples were taken from five yaks during a dry period (DP, n = 3) and lactation period (LP, n = 2). Two types of cDNA sequence libraries that reflected the different physiological states of the mammary gland were constructed using RNA sequencing technology. After removing reads containing adapters, reads containing poly-N and low-quality reads from the raw data, 45,423,478 to 53,274,976 clean reads were obtained from these libraries. A total of 74.72% to 80.65% of the high-quality sequence reads were uniquely aligned to the BosGru v2.0 yak reference genome. Using the DESeq R package, 360 differentially expressed genes were detected between the two groups when the adjusted P value (padj < 0.05) was used as the cutoff value; this included 192 upregulated and 168 downregulated genes in the yak mammary gland tissue of the DP compared to the LP. A gene ontology analysis revealed that the most enriched GO terms were protein binding, multi-organism process, immune system and others. KEGG pathway analysis indicated that the differentially expressed genes were mostly enriched in Hippo signaling, insulin signaling, steroid biosynthesis and others. The analysis of the up- and downregulated genes provides important insights into the molecular events involved in lactogenesis, lactation and mammary gland development and will guide further research to enhance milk yield and optimize the constituents of yak milk.