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首页> 美国卫生研究院文献>Journal of Clinical Microbiology >Development of a Multiplex PCR Assay for Rapid Molecular Serotyping of Haemophilus parasuis
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Development of a Multiplex PCR Assay for Rapid Molecular Serotyping of Haemophilus parasuis

机译：副猪嗜血杆菌快速分子血清分型的多重PCR检测方法的建立

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Haemophilus parasuis causes Glässer's disease and pneumonia in pigs. Indirect hemagglutination (IHA) is typically used to serotype this bacterium, distinguishing 15 serovars with some nontypeable isolates. The capsule loci of the 15 reference strains have been annotated, and significant genetic variation was identified between serovars, with the exception of serovars 5 and 12. A capsule locus and in silico serovar were identified for all but two nontypeable isolates in our collection of >200 isolates. Here, we describe the development of a multiplex PCR, based on variation within the capsule loci of the 15 serovars of H. parasuis, for rapid molecular serotyping. The multiplex PCR (mPCR) distinguished between all previously described serovars except 5 and 12, which were detected by the same pair of primers. The detection limit of the mPCR was 4.29 × 105 ng/μl bacterial genomic DNA, and high specificity was indicated by the absence of reactivity against closely related commensal Pasteurellaceae and other bacterial pathogens of pigs. A subset of 150 isolates from a previously sequenced H. parasuis collection was used to validate the mPCR with 100% accuracy compared to the in silico results. In addition, the two in silico-nontypeable isolates were typeable using the mPCR. A further 84 isolates were analyzed by mPCR and compared to the IHA serotyping results with 90% concordance (excluding those that were nontypeable by IHA). The mPCR was faster, more sensitive, and more specific than IHA, enabling the differentiation of 14 of the 15 serovars of H. parasuis.
机译：副猪嗜血杆菌引起猪格莱瑟氏病和肺炎。间接血细胞凝集（IHA）通常用于对该细菌进行血清分型，用一些不可分型的分离株区分出15个血清型。已对15个参考菌株的荚膜位点进行了注释，并发现血清型之间存在显着的遗传变异，但血清型5和12除外。在我们的> 200株。在这里，我们描述了基于副猪嗜血杆菌的15个血清型的胶囊基因座内变异的多重PCR的发展，用于快速分子血清分型。多重PCR（mPCR）区分了除5和12外的所有先前描述的血清型，它们通过同一对引物检测。 mPCR的检出限为4.29×10 5 ng /μl细菌基因组DNA，其高特异性是由于与猪的共生巴斯德氏菌和其他细菌病原体没有反应性。与计算机结果相比，先前测序的副猪嗜血杆菌的150个分离物的子集用于以100％的准确性验证mPCR。另外，使用mPCR可对两个计算机不可分型的分离株进行分型。通过mPCR分析了另外84个分离株，并与90％一致性的IHA血清分型结果进行了比较（不包括那些无法通过IHA分型的结果）。 mPCR比IHA更快，更灵敏且更具特异性，可区分副猪嗜血杆菌15种血清中的14种。

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