河南农业大学学报2025，Vol.59Issue(5)：849-858,10.DOI:10.16445/j.cnki.1000-2340.20250828.002
猪链球菌2型和9型双重Basic-RPA检测方法的构建与应用
Establishment and application of dual Basic-RPA detection method for Streptococcus suis serotype 2 and serotype 9
摘要
Abstract
[Objective]This research is conducted to develop a dual rapid detection method for Strepto-coccus 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 con-ditions,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 consis-tent 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.关键词
猪链球菌/基础型重组酶聚合酶等温扩增/荚膜多糖/血清分型/双重检测Key words
Streptococcus suis/Basic-recombinase polymerase amplification(Basic-RPA)/capsule polysaccharides/serotype/dual detection分类
农业科技引用本文复制引用
张慧辉,王可心,李艳伟,魏小兵,王磊,胡建和,丁轲,刘明成,夏小静..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(5):849-858,10.基金项目
河南省高校科技创新人才项目(23HASTIT046) （23HASTIT046）
河南省优秀青年科学基金项目(232300421031) （232300421031）
国家自然科学基金面上项目(32172876,32473070) （32172876,32473070）
