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【目的】通过构建猪链球菌(Streptococcus suis,SS)2型(SS2)和9型(SS9)双重快速检测方法，为监测SS的流行和传播提供技术支持。【方法】分别以SS2特有荚膜多糖糖编码基因(capsular polysaccharide 2J gene, cps2J)和SS9特有荚膜多糖糖编码基因(capsular polysaccharide 9H gene, cps9H)为靶标设计引物和探针，对反应体系和反应条件进行优化，建立基础型重组酶聚合酶等温扩增(Basic-recombinase polymerase amplification, Basic-RPA)快速检测技术，并对其特异性、灵敏度、重复性和临床应用效果进行评价。【结果】构建的SS2/9双重Basic-RPA检测方法引物为CPS2J212和CPS9H304，引物体积V(CPS2J212/μL)∶V(CPS9H304/μL)为2.4∶1.8，反应温度为39℃，反应时间为20 min。特异性结果表明，该方法与其他常见病原体无交叉反应，特异性良好。灵敏度结果显示，该方法针对SS2/9检测限达到1×10-3 mg·L-1，优于常规PCR的1×10-2 mg·L-1。重复性结果显示，该方法有良好的稳定性。用该方法对41例临床样品进行检测，其检出率为24.4%，与单重Basic-RPA方法检测结果一致，高于细菌分离方法的检出率12.2%，以及常规PCR方法的检出率19.5%。【结论】构建的SS2/9的双重Basic-RPA检测方法，特异性和稳定性好，灵敏度高，可应用于SS快速诊断和血清分型。
Abstract：【Objective】This research is conducted to develop a dual rapid detection method for Streptococcus suis serotype 2(SS2) and serotype 9(SS9), thereby providing technical support for monitoring the prevalence and spread of Streptococcus suis(SS). 【Method】Taking the Capsular Polysaccharide 2J gene(cps2J) specific to SS2 and the Capsular Polysaccharide 9H gene(cps9H) specific to SS9 as target respectively, specific primers and probes were designed to optimize the reaction system and conditions, and a rapid detection technology were established for basic recombinant enzyme polymerase isothermal amplification(Basic-RPA). Furthermore, the specificity, sensitivity, repeatability, and clinical applicability of this method were evaluated. 【Result】The primers for the established SS2/9 dual Basic-RPA detection method are CPS2J212 and CPS9H304, with a primer volume ratio of V(CPS2J212/μL) ∶ V(CPS9H304/μL) at 2. 4 ∶ 1. 8. The reaction is performed at 39 ℃ for 20 min. The specificity results show that this method had no cross-reaction with other common pathogens and had good specificity. The sensitivity results showed that the detection limit of this method for SS2/9 reached 1×10-3 mg·L-1, which is more sensitive than that of conventional PCR at 1×10-2 mg·L-1. Repeatability results showed the method has a good stability. The double Basic-RPA method was employed to analyze 41 clinical samples, yielding a detection rate of 24. 4%. This result was consistent with that obtained using the single Basic-RPA method. Furthermore, the detection rate achieved by the double Basic-RPA method was higher than those obtained by the bacterial isolation method(12. 2%) and conventional PCR method(19. 5%). 【Conclusion】The constructed dual Basic-RPA detection method of SS2/9 has good specificity and stability and high sensitivity, providing a new method for the rapid diagnosis and serotyping of SS and laying the foundation for the development of a more complex multiplex detection system.
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基本信息:
DOI：10.16445/j.cnki.1000-2340.20250828.002
中图分类号:S852.61
引用信息:
[1]张慧辉,王可心,李艳伟,等.猪链球菌2型和9型双重Basic-RPA检测方法的构建与应用[J].河南农业大学学报,2025,59(05):849-858.DOI:10.16445/j.cnki.1000-2340.20250828.002.
基金信息:
河南省高校科技创新人才项目（23HASTIT046）; 河南省优秀青年科学基金项目（232300421031）; 国家自然科学基金面上项目（32172876,32473070）