猪链球菌纳米PCR检测方法的建立和应用

doi:10.7668/hbnxb.20192014

华北农学报 ›› 2021, Vol. 36 ›› Issue (4): 212-217. doi: 10.7668/hbnxb.20192014

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猪链球菌纳米PCR检测方法的建立和应用

张昆丽¹, 林本夫², 席振军¹, 杨冬霞¹, 卞志标¹, 宋帅¹, 蒋智勇¹, 蔡汝健¹, 李春玲¹

1. 广东省农业科学院动物卫生研究所, 广东省畜禽疫病防治研究重点实验室, 农业农村部兽用药物与诊断技术广东科学观测实验站, 广东广州 510640;
2. 广州市花都区动物卫生监督所, 广东广州 510800

收稿日期:2021-01-07 出版日期:2021-08-28
通讯作者: 李春玲(1963-),女,河南商丘人,研究员,博士,主要从事细菌病原学研究。
作者简介:张昆丽(1988-),女,云南玉溪人,博士,主要从事动物病原学与免疫学研究。
基金资助:
国家“十三五”重点计划专项课题（2016YFD0500709）；科技创新战略专项资金（高水平农科院建设）（R2019YJ-YB2005；R2020QD-047）；广州市科技计划项目（202102020385）

Establishment of a Nano PCR Assay for Detection of the Streptococcus suis

ZHANG Kunli¹, LIN Benfu², XI Zhenjun¹, YANG Dongxia¹, BIAN Zhibiao¹, SONG Shuai¹, JIANG Zhiyong¹, CAI Rujian¹, LI Chunling¹

1. Institute of Animal Health, Guangdong Academy of Agricultural Sciences, Key Laboratory of Livestock Disease Prevention of Guangdong Province, Scientific Observation and Experiment Station of Veterinary Drugs and Diagnostic Techniques of Guangdong Province, Ministry of Agriculture and Rural Affairs, P. R. China, Guangzhou 510640, China;
2. Guangzhou Huadu District Animal Health Supervision Institute, Guangzhou 510800, China

Received:2021-01-07 Published:2021-08-28

摘要/Abstract

摘要： 为了建立一种高效、灵敏的猪链球菌（SS）检测方法，针对SS的谷氨酸脱氢酶基因（gdh）合成特异性引物，通过优化退火温度与引物浓度，经特异性、敏感性、重复性以及临床样品检测，建立了SS的纳米PCR检测方法。特异性试验结果显示，该方法仅能从SS的基因组序列中扩增得到687 bp的特异性序列，与猪胸膜肺炎放线杆菌、副猪嗜血杆菌、猪大肠杆菌、沙门氏菌、葡萄球菌无交叉反应，且能同时识别1、2、7和9型4个目前流行的SS主要血清型，满足临床上多血清型SS感染检测的需要；敏感性试验结果显示，该方法对SS的最低检测下限为10 cfu/mL，与普通PCR方法相比，灵敏度提高了100倍；重复性试验表明，该方法检测效果稳定，重复性较好。用该方法和普通PCR对临床上收集的44份疑似SS感染样品进行检测，阳性率分别为72.7%（32/44），54.5%（24/44），表明该纳米PCR方法具有灵敏度高，特异性好等优点。综上，本研究建立的SS纳米PCR检测方法，能够准确、高效检测SS，为猪链球菌病的临床诊断和流行病学调查提供了技术支持。

关键词: 猪链球菌, 谷氨酸脱氢酶基因, 纳米PCR, 临床诊断

Abstract: This study was to develop an efficient and sensitive nano PCR for the detection of Streptococcus suis (SS). The specific primers were synthesized for the Glutamate dehydrogenase gene (gdh) of SS. Then, the annealing temperature and primer concentration reaction conditions were optimized. The results of specificity assay showed that the method could only amplify 687 bp specific sequence from SS samples, and there was no cross reaction with Actinobacillus pleuropneumoniae (APP), Haemophilus parasuis (H. parasuis), swine Escherichia coli (swine E. coli), Salmonella suis (S. suis) and (S. hyicus). Moreover, the method also could detected the main serotypes 1, 2, 7 and 9 type of SS, respectively. The results of sensitivity assay showed that the lowest SS concentration detected by the nano PCR method was 10 cfu/mL, and its sensitivity was 100 times higher than ordinary PCR of SS. The SS nucleic acid was detected three times, and the results were consistent. Finally, 44 suspected SS infection samples which collected from clinical cases were detected by nano PCR and routine PCR, respectively. The clinical sample testing showed that the positive rates were 72.7% (32/44), 54.5% (24/44), respectively. In conclusion, the nano PCR detection method of SS was established in this study can accurately and efficiently detect SS, which provides technical support for the clinical diagnosis and epidemiological investigation of S. suis disease.

Key words: Streptococcus suis, Glutamate dehydrogenase gene, Nano PCR, Clinical diagnosis

中图分类号:

S855.1

张昆丽, 林本夫, 席振军, 杨冬霞, 卞志标, 宋帅, 蒋智勇, 蔡汝健, 李春玲. 猪链球菌纳米PCR检测方法的建立和应用[J]. 华北农学报, 2021, 36(4): 212-217. doi: 10.7668/hbnxb.20192014.

ZHANG Kunli, LIN Benfu, XI Zhenjun, YANG Dongxia, BIAN Zhibiao, SONG Shuai, JIANG Zhiyong, CAI Rujian, LI Chunling. Establishment of a Nano PCR Assay for Detection of the Streptococcus suis[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2021, 36(4): 212-217. doi: 10.7668/hbnxb.20192014.

http://www.hbnxb.net/CN/Y2021/V36/I4/212

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