四种猪病原菌PCR诊断方法的建立与应用

发布时间：2018-02-28 01:12

本文关键词： 猪传染性胸膜肺炎放线杆菌血清型分型基因分型猪呼吸道疾病综合征复合PCR　出处：《华中农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：猪传染性胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae,APP)是猪传染性胸膜肺炎的主要病原菌。猪感染APP后会导致急性纤维素性肺炎、肺出血性坏死乃至死亡,或者演变成慢性感染,虽无明显的临床症状但生长缓慢乃至成为僵猪。发病后幸存下来携带病原菌的猪,可能会成为再次爆发此病的传染源。近年来,猪传染胸膜肺炎放线杆菌继发感染以及与其它病原菌混合感染的情况越来越严重,造成临床上症状愈加复杂而难以区分。迄今为止,根据荚膜多糖,可将APP分为15种血清型。根据培养过程中是否需要加NAD,可将APP分为生物I型和生物II型,我国流行的主要是生物I型。由于APP血清型众多,现有的商品化疫苗没有涵盖所有血清型,加上制备型特异性血清过程复杂且耗时长,导致对该病的诊断和预防带来了很大的困难。因此,APP的鉴定及分型,以及进一步根据病原采取对应的疫苗防疫和药物治疗措施,对该病的综合防控具有重要的意义。本研究利用APP生物I型标准菌株制备了13种标准阳性血清,经琼脂扩散试验(GDT)测定后,标准阳性血清5型的效价是1:8;标准阳性血清2型、4型、9型和15型的效价是1:16;标准阳性血清1型、3型、6型、7型、8型、10型、11型和12型的效价是1:32。近年来,复合PCR方法因快速、简便的特点而发展迅速得到广泛的应用。根据APP的四种外毒素apx I、apx II、apx III和apx IV设计了5对特异性引物,建立了复合PCR分子分型方法研究,通过一步复合PCR方法就可以区分出APP血清型1~13中的7种(1型、3型、4型、5型、8型、10型和12型)。猪呼吸道疾病是导致保育猪和生长猪以及育肥猪死亡的主要原因。猪嗜血杆菌、猪链球菌、猪多杀巴氏杆菌和猪传染性胸膜肺炎放线杆菌是引起呼吸道综合征(PRDC)的主要病原菌。本研究针对副猪嗜血杆菌的16S r RNA基因、猪链球菌的gdh基因、猪多杀巴氏杆菌的kmt基因和猪传染性胸膜肺炎放线杆菌的apx IV基因设计出四对特异性引物,分别扩增出了长度为821bp、689 bp、457 bp、319 bp的特异性片段。在建立了APP单一PCR方法基础上,进一步对影响PCR的几个主要因素(r Taq酶、Mg2+浓度、d NTP浓度和引物浓度)进行优化,建立了复合PCR检测方法。特异性试验结果表明,副猪嗜血杆菌、猪链球菌、猪多杀巴氏杆菌和猪传染性胸膜肺炎放线杆菌复合PCR只能扩增出四种特异性的目的条带,其它结果为阴性;敏感性试验中,最低检测量分别是副猪嗜血杆菌为112.5pg/μL,猪链球菌为11.7pg/μL,猪多杀巴氏杆菌为8.7ng/μL,猪传染性胸膜肺炎放线杆菌为267pg/μL;重复性试验中,通过9次重复试验的结果表明此方法重复性好。利用该方法对湖北省部分地区采集的579份病料进行了初步检测,结果有59株HPS(10.2%),74株SS(12.8%),35株Pm(6%),24株APP(4.1%),与单一PCR方法检测结果相比,平均阳性符合率为95%。表明该方法实际可行,也为PRDC病原菌的检测和疫病防控提供了技术支撑。
[Abstract]:Actinobacillus pleuropneumoniae is the main pathogen of swine infectious pleural pneumonia. Infection with APP can lead to acute cellulosic pneumonia, pulmonary hemorrhage necrosis or death, or to chronic infection. Although there are no obvious clinical symptoms, but the growth is slow to become stiff pigs. The pigs survived to carry the pathogen after the disease, may become a source of infection for another outbreak of the disease in recent years, Actinobacillus pleuropneumoniae secondary infection and co-infection with other pathogens are becoming more and more serious, resulting in more complex and difficult clinical symptoms. APP can be divided into 15 serotypes. APP can be divided into biological type I and biological type II according to whether it is necessary to add NAD in the course of culture. The existing commercial vaccines do not cover all serotypes, and the process of preparing specific serotypes is complicated and time-consuming, which makes it difficult to diagnose and prevent the disease. Therefore, the identification and typing of app. It is of great significance for the comprehensive prevention and control of the disease to take corresponding vaccine prevention and drug treatment measures according to the pathogen. In this study, 13 standard positive sera were prepared by using APP biotype I standard strain. The titer of standard positive serotype 5 was 1: 8, the titer of standard positive serotype 2, serotype 2, serotype 4, and type 15 was 1: 16, and the titer of standard positive serum type 1, serotype 1, serotype 1, serotype 1, serotype 1, serotype 1, serotype 1, serotype 1, serotype 1, serotype 1, type 1, type 1, type 3, type 1, type 7, type 1, type 10, type 10, type 11, and type 12, type 12, were 1: 32 in recent years. The compound PCR method has been widely used because of its rapid and convenient characteristics. Five pairs of specific primers were designed according to apx Iapx IIAPX III and apx IV of four foreign toxins of APP, and the molecular typing method of compound PCR was established. By using one-step composite PCR method, we can distinguish seven types of APP serotype 1, serotype 1, serotype 1, serotype 1, serotype 1, serotype 1, type 1, type 3, type 4, type 5, type 8, type 8, type 10, and type 12. Respiratory diseases in pigs are the main causes of mortality in the care of pigs, growing pigs and fattening pigs, Haemophilus suis, and Haemophilus suis. Streptococcus suis, Pasteurella multocida and Actinobacillus pleuropneumoniae are the main pathogens causing respiratory syndrome. In this study, the 16s r RNA gene of Haemophilus parahaemophilus and the gdh gene of Streptococcus suis were studied. Four pairs of specific primers were designed from the kmt gene of Pasteurella multocida and the apx IV gene of Actinobacillus pleuropneumoniae, respectively, and the specific fragment was amplified from 821 BP, 689 BP, 457 BP, 319bp, respectively. Based on the APP single PCR method, a single PCR method was developed. Furthermore, several main factors affecting PCR, such as NTP concentration and primer concentration, were optimized, and a complex PCR detection method was established. The specific test results showed that Haemophilus parais and Streptococcus suis were detected. Pasteurella multocida and Actinobacillus pleuropneumoniae combined with PCR could only amplify four specific target bands, but the other results were negative. The lowest detections were 112.5 PG / 渭 L for Haemophilus parais, 11.7 PG / 渭 L for Streptococcus suis, 8.7 ng / 渭 L for Pasteurella multocida and 267pg / 渭 L for Actinobacillus pleuropneumoniae. The results of 9 repeated experiments showed that the method was reproducible. The method was used to detect 579 samples collected from some parts of Hubei Province. The results showed that there were 59 strains of HPSN 10.2 and 74 strains of PMS 12.80.35 strains of APP 4.1B, compared with the results of single PCR method. The average positive coincidence rate was 95. The results showed that the method was feasible and provided technical support for the detection of pathogenic bacteria of PRDC and the prevention and control of epidemic diseases.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61

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