猪链球菌2型实时荧光定量PCR检测方法的建立与应用

doi:10.16431/j.cnki.1671-7236.2016.12.004

《中国畜牧兽医》 ›› 2016, Vol. 43 ›› Issue (12): 3107-3113.doi: 10.16431/j.cnki.1671-7236.2016.12.004

猪链球菌2型实时荧光定量PCR检测方法的建立与应用

李建达^1,2, 于江², 张玉玉², 任素芳², 陈蕾², 郭立辉², 孙文博², 陈智², 王淞^1,2, 刘俊珍², 杜以军², 李俊², 杨灵芝³, 王金宝^1,2, 吴家强²

1. 青岛农业大学动物科技学院, 青岛 266109;
2. 山东省农业科学院畜牧兽医研究所, 济南 250100;
3. 山东滨州沃华生物工程有限公司, 滨州 256600

收稿日期:2016-05-18 出版日期:2016-12-20 发布日期:2016-12-22
通讯作者: 王金宝, 吴家强 E-mail:wangjb@saas.ac.cn;wujiaqiang2000@sina.com
作者简介:李建达(1992-),男,山东济南人,硕士生,研究方向:动物微生物与免疫学,E-mail:ljd4453@163.com;于江(1985-),女,山东莱州人,博士,研究方向:动物微生物与免疫学,E-mail:yujiang_2213@163.com
基金资助:
泰山学者特聘专家工程经费；山东省现代农业产业技术体系（SDAIT）；山东省农业科学院重大科技成果培育计划（2014CGPY04）；山东省农业重大应用技术创新项目；山东省自然科学基金（ZR2015YL078）；山东省农业科学院青年科研基金（2015YQN51）；山东省农业科学院农业科技创新工程项目（CXGC2016B14）

Establishment and Application of Quantitative Real-time PCR Method to Detect Streptococcus suis Serotype 2

LI Jian-da^1,2, YU Jiang², ZHANG Yu-yu², REN Su-fang², CHEN Lei², GUO Li-hui², SUN Wen-bo², CHEN Zhi², WANG Song^1,2, LIU Jun-zhen², DU Yi-jun², LI Jun², YANG Ling-zhi³, WANG Jin-bao^1,2, WU Jia-qiang²

1. College of Animal Science and Technology, Qingdao Agricultural University, Qingdao 266109, China;
2. Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences, Jinan 250100, China;
3. Wohua Biotech Co., Ltd., Binzhou 256600, China

Received:2016-05-18 Online:2016-12-20 Published:2016-12-22

摘要/Abstract

摘要：

试验根据猪链球菌2型荚膜多糖（CPS）抗原设计CPS2J基因特异性引物，建立猪链球菌2型实时荧光定量PCR检测方法。结果显示，该方法的标准曲线为y=-3.073x+36.87，r=0.995，熔解曲线只有单一的特异峰。敏感性试验显示，该方法可以检测出模板最低浓度为1.0×10¹拷贝/μL，是普通PCR的10倍；特异性试验显示，对猪链球菌2型具有良好的特异性，能够区分其他血清型猪链球菌和其他细菌；重复性试验变异系数为0.37%~0.63%，均低于2.5%。临床检测显示该方法的敏感性明显高于常规PCR方法和细菌分离的方法。以上结果表明，本研究建立的方法敏感性高、特异性强、重复性好，有利于对猪链球菌2型的快速检测。

关键词: 猪链球菌2型; 实时荧光定量PCR; 敏感性; 特异性; 重复性

Abstract:

In this study,a quantitative Real-time PCR method using the specific primers according to CPS2J gene was established to detect Streptococcus suis serotype 2. The result showed that the equation of standard curve was y=-3.073x+36.87,r=0.995,which demonstrated that the assay had good linear relationship.The melting curve analysis showed that there was only specific peak.Sensitivity test showed that the method could detect the template at the lowest concentration of 1.0×10¹ copies/μL,which was 10 times higher than the ordinary PCR.The specific tests showed that this method could able to detect Streptococcus suis serotype 2 specially and had no cross-reaction with other serotypes or other bacteria from swine. The CV of repeatability test was 0.37% to 0.63%,lower than 2.5%. The clinical diagnosis showed this assay was more sensitive than ordinary PCR and bacteria isolation. All the results showed that the established method was sensitive,specific and reproducible,which could be used for the rapid diagnosis and quantitative detection of Streptococcus suis serotype 2.

Key words: Streptococcus suis serotype 2; quantitative Real-time PCR; sensibility; specificity; repeatability

中图分类号:

S858.28

李建达, 于江, 张玉玉, 任素芳, 陈蕾, 郭立辉, 孙文博, 陈智, 王淞, 刘俊珍, 杜以军, 李俊, 杨灵芝, 王金宝, 吴家强. 猪链球菌2型实时荧光定量PCR检测方法的建立与应用[J]. 《中国畜牧兽医》, 2016, 43(12): 3107-3113.

LI Jian-da, YU Jiang, ZHANG Yu-yu, REN Su-fang, CHEN Lei, GUO Li-hui, SUN Wen-bo, CHEN Zhi, WANG Song, LIU Jun-zhen, DU Yi-jun, LI Jun, YANG Ling-zhi, WANG Jin-bao, WU Jia-qiang. Establishment and Application of Quantitative Real-time PCR Method to Detect Streptococcus suis Serotype 2[J]. , 2016, 43(12): 3107-3113.

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猪链球菌2型实时荧光定量PCR检测方法的建立与应用

Establishment and Application of Quantitative Real-time PCR Method to Detect Streptococcus suis Serotype 2

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