| 摘要: |
| 【目的】 建立致病性猪链球菌种及其1、2、7型的快速多重PCR检测方法，并进行初步的临床应用。【方法】 根据猪链球菌种特异的gdh基因序列及其1（14）、2（1/2）、7型特异的cps1I、cps2H、cps7H基因序列，分别设计4对引物，通过对单个基因PCR和多重PCR扩增条件、反应体系的优化，建立了快速检测猪链球菌种及其1（14）、2（1/2）、7型的4重PCR方法。利用保存的猪链球菌不同血清型菌株和其他相关标准菌株作为参考菌株,对建立的多重PCR方法进行特异性和敏感性试验。用所建立的4重PCR方法对河南省不同地市的39份猪扁桃体样品进行检测，并选取部分样品的PCR产物进行测序验证。【结果】 所建立的4重PCR方法特异、敏感，对猪链球菌2 型的最低检出水平为2.52 ×103 CFU/mL。临床检测及测序结果显示，该多重PCR准确性较高。【结论】 建立的4重PCR方法可用于猪链球菌主要致病血清型的快速检测。 |
| 关键词: 猪链球菌 种及血清1、2、7型 多重PCR 鉴别 |
| DOI： |
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| 基金项目:河南省重点科技攻关项目（072102130009）；国家科技部支撑计划项目（2006BAK02A21） |
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| Development and application of multiplex PCR assay for rapid detection of Streptococcus suis species and its main pathogenic serotypes 1,2 and 7 |
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| Abstract: |
| 【Objective】The study was done to develop rapid and specific multiplex PCR assay for detection of Streptococcus suis(S.suis) and its main pathogenic serotypes 1(and 14),2(and 1/2) and 7.【Method】Four pairs of primers were designed in this multiplex PCR assay,which was based on the sequences of the species specific gene coding for glutamate hydrogenase (gdh) of S.suis and serotypes specific genes of cps1I,cps2H and cps7H coding for the capsule of S.suisserotypes 1(and 14),2(and 1/2) and 7,respectively.By optimizing the single and multiplex PCR conditions and primers concentrations,a stable multiplex PCR assay was established.To evaluate the specificity,strains of other bacterial species related to S.suisor isolated from pigs were analyzed.The multiplex PCR assay was then applied to the detection of 39 tonsils,and several PCR products were sequenced to confirm the PCR assay.【Result】The PCR assay was rapid,exact,specific and sensitive.When genomic DNA of S.suis serotype 2 was used as template for PCR,the detection threshold of the test was 2.52 CFU per assay.【Conclusion】The multiplex PCR assay can be applied in rapid detection of S.suis and its main pathogenic serotypes from tonsils. |
| Key words: Streptococcus suis species,serotypes 1,2 and 7 multiplex PCR differentiation |
