[1]孙学飞,倪艳秀,茅爱华,等.猪链球菌、胸膜肺炎放线杆菌和副猪嗜血杆菌多重PCR检测方法的建立和应用[J].江苏农业学报,2016,(05):1094-1099.[doi:10.3969/j.issn.1000-4440.2016.05.022]
 SUN Xue-fei,NI Yan-xiu,MAO Ai-hua,et al.Development and application of a multiplex PCR for detection of Streptococcus suis, Actinobacillus pleuropneumoniae and Haemophilus parasuis[J].,2016,(05):1094-1099.[doi:10.3969/j.issn.1000-4440.2016.05.022]
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猪链球菌、胸膜肺炎放线杆菌和副猪嗜血杆菌多重PCR检测方法的建立和应用()
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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2016年05期
页码:
1094-1099
栏目:
畜牧兽医·水产养殖
出版日期:
2016-11-22

文章信息/Info

Title:
Development and application of a multiplex PCR for detection of Streptococcus suis, Actinobacillus pleuropneumoniae and Haemophilus parasuis
作者:
孙学飞12倪艳秀2茅爱华2 何孔旺12
1.安徽农业大学动物科技学院,安徽合肥230000;2.江苏省农业科学院兽医研究所,江苏南京210014
Author(s):
SUN Xue-fei12NI Yan-xiu2MAO Ai-hua2HE Kong-wang12
1. College of Animal Science and Technology, Anhui Agricultural University, Hefei 230000,China;2. Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing 210014,China
关键词:
猪链球菌胸膜肺炎放线杆菌副猪嗜血杆菌多重PCR
Keywords:
Streptococcus suisActinobacillus pleuropneumoniaeHaemophilus parasuismultiplex PCR
分类号:
S858.282.61
DOI:
10.3969/j.issn.1000-4440.2016.05.022
文献标志码:
A
摘要:
根据已发表的猪呼吸道疾病主要致病菌猪链球菌(SS)、胸膜肺炎放线杆菌(APP)和副猪嗜血杆菌(HPS)基因序列合成3对特异性引物,通过单一PCR和三重PCR体系及条件优化,建立这3种致病菌的三重PCR检测方法,并对200份临床表观健康猪鼻拭子样品用三重PCR方法进行检测。结果显示:建立的三重PCR方法能对同一样品中的SS、APP、HPS 3种病原菌的DNA模板进行扩增,无交叉反应;对猪体内常见的猪多杀性巴氏杆菌、猪大肠杆菌、猪放线杆菌、猪葡萄球菌、猪丹毒杆菌、猪沙门氏菌进行扩增,并无任何扩增产物;对SS、APP、HPS细菌纯培养物的检测敏感性分别为1×10 4CFU/ml、1×103 CFU/ml、1×10 3 CFU/ml;对人工模拟SS、APP、HPS感染组织样品的敏感性均为1×104CFU/ml。三重PCR方法对200份临床表观健康猪鼻拭子样品的检测结果显示:SS阳性160份,阳性率80.0%;APP阳性3份,阳性1.5%;HPS阳性45份,阳性22.5%。分别用细菌分离法和三重PCR法对30份临床病料进行检测,两者检测结果的符合率达到93%。建立的三重PCR可在4.5 h左右得到检测结果。
Abstract:
To achieve the purpose of simultaneously detecting three kinds of pathogenic bacteria in porcine respiratory diseases, Streptococcus suis (SS), Actinobacillus pleuropneumoniae (APP) and Haemophilus parasuis (HPS), three pairs of specific primers were synthesized from published SS, APP and HPS gene sequences, and the multiplex PCR method for SS, APP, and HPS was established. The multiplex PCR method could simultaneously amplify SS, APP and HPS in the same sample, without cross reaction. Pasteurella Multocida, Escherichia coli, A. suis, Staphylococcus aureus, Salmonella and swine erysipelas were detected by the multiplex PCR respectively and no specific products were amplified. The minimum detection concentrations of SS, APP and HPS were 1×104 CFU/ml, 1×103 CFU/ml and 1×103 CFU/ml, respectively. The sensitivity of the multiplex PCR method to artificially infected samples of SS, APP and HPS was 104 CFU/ml. Among 200 nasal swabs of apparently healthy pigs from Jiangsu province, the positive rates for SS, APP and HPS were 80.0% (160/200), 1.5% (3/200) and 22.5% (45/200), respectively. The total coincidence rate between the bacterial isolation and the multiplex PCR for 30 clinical diseased samples was 93% (28/30). This multiplex PCR is a sensitive, specific and rapid method for the detection and identification of major bacteria in porcine respiratory disease complex, taking only 4.5 h to get the results.

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备注/Memo

备注/Memo:
收稿日期:2016-01-29 基金项目:公益性行业(农业)科研专项经费项目(201303034-8) 作者简介:孙学飞(1990-),男,江苏南京人,硕士,研究方向为动物传染病。(E-mail)sxf514524582@126.com 通讯作者:何孔旺,(E-mail)kwh2003@263.net
更新日期/Last Update: 2016-11-22