猪链球菌和猪多杀性巴氏杆菌多重实时荧光PCR方法的建立

Establishment of a multiplex real-time fluorescent PCR method for simultaneous detection of Streptococcus suis and Pasteurella multocida

摘要:

【背景】猪链球菌(Streptococcus suis，SS)和猪多杀性巴氏杆菌(Pasteurella multocida，Pm)都是能引起宿主致病的人畜共患病原菌，常出现混合感染，临床诊断上易与猪瘟、猪丹毒等混淆。【目的】快速、有效鉴别猪链球菌病和猪多杀性巴氏杆菌病，建立一种能同时检测2种病原的多重实时荧光定量PCR检测方法。【方法】基于猪链球菌的gdh基因和猪多杀性巴氏杆菌的plpE基因，设计2对特异引物及TaqMan探针，以细菌16S rRNA基因设计通用引物及探针，通过对反应条件优化，建立了一种能同时检测猪链球菌和猪多杀性巴氏杆菌的多重实时荧光定量PCR检测方法。【结果】该方法能够特异性地检测猪链球菌和猪多杀性巴氏杆菌，与细菌分离后的测序结果验证完全一致。此方法对重组质粒标准品的最低检出浓度分别为4.53×10²copies/μL和3.97×10²copies/μL。重复性试验结果显示，该方法的组内和组间变异系数均小于3%。【结论】本实验所建立的方法准确、简便、可靠，能够用于2种病原菌的同时检测，为猪链球菌病和猪多杀性巴氏杆菌病的防治提供了有效的检测工具，具有重要的流行病学意义和临床应用价值。

关键词:猪链球菌(SS);猪多杀性巴氏杆菌(Pm);多重实时荧光定量PCR

Abstract:

[Background] Streptococcus suis (SS) and Pasteurella multocida (Pm) are zoonotic pathogens that can cause severe diseases in humans, which are easily confused with swine fever and swine erysipelas in clinical diagnosis because of mixed infections. [Objective] To establish a multiplex real-time fluorescent quantitative PCR assay for simultaneous detection of SS and Pm, so as to realize early diagnosis and treatment. [Methods] Two pairs of specific primers and TaqMan were designed based on the gdh gene of SS and the plpE gene of Pm. The universal primers and probe were designed based on the sequence of bacterial 16S rRNA gene. After optimization of the reaction conditions, a multiplex real-time fluorescence quantitative PCR method was established for the simultaneous detection of SS and Pm.[Results] This method can specifically detect SS and Pm, and the results was entirely consistent with the sequencing results after isolation of bacteria. The lower limits of detection for recombinant plasmid standards were 4.53×10² copies/μL and 3.97×10² copies/μL, respectively. The results of repeated experiments showed that the intra- and inter-group coefficients of variation were less than 3%. [Conclusion] The method established in this study was simple, accurate, and reliable. It can be used for the simultaneous detection of SS and Pm and provides an effective detection tool for the prevention and treatment of the diseases caused by SS and Pm.

Key words:Streptococcus suis;Pasteurella multocida;multiplex real-time fluorescence quantitative PCR