Establishment and Modification of Ninety-seven Pneumococcal Serotyping Assays Based on Quantitative Real-time Polymerase Chain Reaction / 生物医学与环境科学（英文）

OBJECTIVE@#To establish and modify quantitative real-time polymerase chain reaction (qPCR)-based serotyping assays to distinguish 97 pneumococcal serotypes.@*METHODS@#A database of capsular polysaccharide ( cps) loci sequences was generated, covering 97 pneumococcal serotypes. Bioinformatics analyses were performed to identify the cps loci structure and target genes related to different pneumococcal serotypes with specific SNPs. A total of 27 novel qPCR serotyping assay primers and probes were established based on qPCR, while 27 recombinant plasmids containing serotype-specific DNA sequence fragments were constructed as reference target sequences to examine the specificity and sensitivity of the qPCR assay. A panel of pneumococcal reference strains was employed to evaluate the capability of pneumococcal serotyping.@*RESULTS@#A total of 97 pneumococcal serotyping assays based on qPCR were established and modified, which included 64 serotypes previously reported as well as an additional 33 serotypes. Twenty-seven novel qPCR serotyping target sequences were implemented in the pneumococcal qPCR serotyping system. A total of 97 pneumococcal serotypes, which included 52 individual serotypes and 45 serotypes belonging to 20 serogroups, could not be identified as individual serotypes. The sensitivity of qPCR assays based on 27 target sequences was 1-100 copies/µL. The specificity of the qPCR assays was 100%, which were tested by a panel of 90 serotypes of the pneumococcal reference strains.@*CONCLUSION@#A total of 27 novel qPCR assays were established and modified to analyze 97 pneumococcal serotypes.

Validation of Reference Genes for Quantitative Real-Time PCR in Bovine PBMCs Transformed and Non-transformed by Theileria annulata

Theileria annulata is a tick-borne intracellular protozoan parasite that causes tropical theileriosis, a fatal bovine lymphoproliferative disease. The parasite predominantly invades bovine B lymphocytes and macrophages and induces host cell transformation by a mechanism that is not fully comprehended. Analysis of signaling pathways by quantitative real-time PCR (qPCR) could be a highly efficient means to understand this transformation mechanism. However, accurate analysis of qPCR data relies on selection of appropriate reference genes for normalization, yet few papers on T. annulata contain evidence of reference gene validation. We therefore used the geNorm and NormFinder programs to evaluate the stability of 5 candidate reference genes; 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ACTB (β-actin), PRKG1 (protein kinase cGMP-dependent, type I) and TATA box binding protein (TBP). The results showed that 18S rRNA was the reference gene most stably expressed in bovine PBMCs transformed and non-transformed with T. annulata, followed by GAPDH and TBP. While 18S rRNA and GAPDH were the best combination, these 2 genes were chosen as references to study signaling pathways involved in the transformation mechanism of T. annulata.