关键词: 巴尔通体, 特异基因, 引物设计, 云平台, 荧光定量PCR

Abstract: Objective To establish a method of screening for Bartonella-specific genes based on the Microbial Data Analysis Cloud Platform, and to design universal primers and probes for detecting Bartonella spp. Methods On the Microbial Data Analysis Cloud Platform, the Special_Marker_Gene workflow was selected, the genome sequence of target bacteria was uploaded, and the parameters Align (coverage) and E (similarity) was set; when the operation was completed, a specific gene sequences list was obtained and validated by NCBI-BLAST. Real-time fluorescence quantitative PCR primers or probes were designed according to the screened fragments, and the experimental results were analyzed. Results Forty-nine Bartonella-specific genes were screened out, and primers and probes were designed by selecting gene sequences with high coverage and specificity after NCBI-BLAST validation. According to pre-experimental analysis, the Bar5 primers and TaqMan probe for the target sequence of the yfeC gene (ACS50472.1_79) in the genome of B. grahamii had a good detection effect:twelve Bartonella species could be amplified with good generality, and no fluorescent signals were found in the nucleic acid samples of other species or genera; the minimum detection limit was 3.47×10 copies/reaction; the within-group and between-group coefficients of variation were 0.02%-2.27% and 0.56%-0.83% respectively by repeatability analysis; at the same time, the standard curve revealed a good linear relationship, with a correlation coefficient (R²) of 1.00 and an amplification efficiency (E) of 101%. Conclusion It is simple and efficient to screen for Bartonella-specific genes using the Microbial Data Analysis Cloud Platform, which can be applied to quickly find alternative gene loci for bacterial identification and classification.

Key words: Bartonella, Specific gene, Primer design, Microbial Data Analysis Cloud Platform, Fluorescent quantitative PCR