基于单基因的实时荧光定量反转录-聚合酶链反应定性检测猪霍乱沙门菌

Detection of Salmonella Choleraesuis with single gene-based real time fluorescent quantitative reverse transcription polymerase chain reaction

目的建立基于单基因的猪霍乱沙门菌的实时荧光定量反转录-聚合酶链反应(rRT-PCR)检测方法。方法通过对猪霍乱沙门菌基因组及其他血清型沙门菌基因组、人类基因组的生物信息学比对及普通PCR检测分析,获得猪霍乱沙门菌特有而其他细菌及人类基因组中不存在的特异基因SC1242,针对该基因设计特异性引物,通过优化反应条件,建立检测该靶基因的rRT-PCR方法,利用123株多种血清型沙门菌及非沙门菌菌株RNA评价该方法的特异性及敏感性。同时对猪霍乱沙门菌全血模拟标本进行敏感性检测。结果利用该方法检测20株猪霍乱沙门菌均为阳性,其余菌株均扩增阴性。对纯菌RNA检测中,rRT-PCR的最低检测限度为50 fg/反应,约为96个拷贝/反应。在以全血模拟样品提取核酸为模板的检测中,敏感性达90 cfu/ml。结论 rRT-PCR方法检测猪霍乱沙门菌特异性好、灵敏度高,对猪霍乱沙门菌的早期诊断具有潜在应用价值。

Objective To establish a single gene-based assay of real time fluorescent quantitative reverse transcription polymerase chain reaction（ rRT-PCR） assay to detect Salmonella（ S.） Choleraesuis. Methods To obtain the specific genes of S. Choleraesuis,two screening rounds were performed. Firstly,the genes shared by sequenced S. Choleraesuis strains were identified and aligned with the genome sequences of other Salmonella serotypes in Gen Bank,and with the human genome. The alignments less than 50% of the gene size were ignored. In the second round,123 Salmonella strains covering 24 Salmonella serotypes were used to examine the specificity of these genes by PCR. The result specific gene,SC1242,was used as the target to develop the rRT-PCR assay. The specificity and detection limit of the rRT-PCR assay was evaluated by using pure cultured strain and S. Choleraesuis simulated blood specimens. Results Twenty S. Choleraesuis isolates were amplified to be positive with established rRT-PCR assay,other 103 isolates were amplified to be negative. For purified total RNA from the pure cultured isolates,the detection limit of the assay was 50 fg / μl per reaction,equal to 96 molecular copies per reaction. The sensitivity was90 cfu / ml in the extraction of nucleotide from the simulated blood. Conclusion The established rRT-PCR assay for detecting S. Choleraesuis with high sensitivity and specificity would be suitable for the rapid detection of S. Choleraesuis.