猪链球菌血清4型毒力株鉴定多重PCR方法的建立

A multiplex PCR method for identification of virulent strains of Streptococcus suis serotype 4

摘要:

猪链球菌(Streptococcus suis)不仅是猪的重要致病菌，还是一种人兽共患病原体。该菌血清型众多，其中血清4型(SS4)属于可感染人类的血清型，因其潜在的高致病性，对公共卫生构成威胁。目的 开发一种基于SS4毒力株特有毒力相关基因的多重PCR方法，以实现SS4毒力株的精准鉴定。方法 基于前期研究，选择sly、igdE、tran和sao这4个SS4毒力株特有的靶基因，结合SS4血清型特异基因wzy作为内参基因，设计了一套五重PCR方法。通过优化多重PCR扩增体系，进行了特异性和敏感性试验，并应用该方法检测新分离的SS4菌株。同时，结合斑马鱼毒力试验验证该方法的准确性。结果 该多重PCR方法能特异性扩增靶基因，有效区分SS4毒力株与弱毒株。其敏感性高，最低可检测到4.1×10² CFU的菌落数或12.5 pg的基因组DNA。特异性验证表明，该方法能准确辨识SS4毒力株。利用该方法检测临床分离的6株SS4菌株：3株菌株被鉴定为毒力株，对斑马鱼的致病性较高，死亡率为60.00%-86.67%；3株被鉴定为弱毒株的菌株对斑马鱼的致病性低，死亡率为0-6.67%。结论 本研究开发了一种基于猪链球菌毒力相关基因的多重PCR方法，能够准确、灵敏地鉴定SS4毒力株，为猪链球菌病的早期诊断和有效防控提供了技术支撑。

Abstract:

Streptococcus suis is a major pathogen in pigs and also a zoonotic pathogen. This bacterium has numerous serotypes, among which S. suis serotype 4 (SS4) is known to infect humans and poses a threat to public health due to its potential high pathogenicity.Objective To develop a multiplex PCR method based on specific virulence-associated genes of SS4 virulent strains to achieve precise identification of such strains.Methods Based on previous research results, four target genes specific to SS4 virulent strains—sly, igdE, tran, and sao—were selected, and the SS4 serotype-specific gene wzy was used as an internal reference gene to design a pentaplex PCR method. After optimization of the multiplex PCR amplification system, specificity and sensitivity tests were conducted. This method was then employed to detect newly isolated SS4 strains. Additionally, zebrafish virulence assays were performed to validate the accuracy of this method.Results The multiplex PCR method specifically amplified the target genes, effectively distinguishing SS4 virulent strains from lowly virulent strains. The method exhibited high sensitivity, with a minimum detection limit of 4.1×10² CFU or 12.5 pg of genomic DNA. Specificity validation confirmed that this method accurately identified SS4 virulent strains. This method was then employed to examine six clinically isolated SS4 strains. Three strains were identified as virulent, showing high pathogenicity in zebrafish and causing a mortality rate of 60.00%-86.67%. The other three strains were identified as lowly virulent strains, exhibiting low pathogenicity in zebrafish and causing a mortality rate of 0-6.67%.Conclusion A multiplex PCR method based on virulence-associated genes of S. suis was successfully developed, enabling accurate and sensitive identification of SS4 virulent strains. This method provides technical support for the early diagnosis and effective prevention and control of S. suis infections.