猪呼吸道病毒性疫病多重PCR方法的建立与应用研究

发布时间：2018-01-23 03:52

本文关键词： 猪呼吸道病毒性疫病多重PCR 混合感染临床检测　出处：《贵州大学》2015年硕士论文　论文类型：学位论文

【摘要】：猪呼吸道疾病是影响养猪业发展的主要疾病之一,是目前我国规模化猪场的常发病、多发病,已经引起人们的普遍重视,给我国养猪业带来了巨大的危害和损失。猪伪狂犬病病毒、猪圆环病毒2型、猪蓝耳病病毒、猪瘟病毒、猪细小病毒和猪流感病毒是最为多见的猪病毒性呼吸道疾病病原,且在临床上常呈混合或继发感染。猪群中,多病原混合感染已是疾病发生的普遍特点,两种或以上病原体共同感染导致猪群发病率和死亡率增高,使得疾病诊断和防治难度加大。为实现临床多病原混合感染的快速确诊,本研究建立了针对上述6种病毒的多重PCR鉴别诊断方法,为相关疫病的快速诊断和防控提供了有效的技术手段。主要研究内容如下:1、PRV、PCV2、PRRSV、CSFV、PPV、SIV的单项PCR/RT-PCR方法的建立参照NCBI中PRV g B(AF257079)、PCV2 ORF2(NC_005148)、PRRSV ORF6(NC_001961)、CSFV C(AY805221)、PPV NS1(NC_001718)、SIV M(GU086140)等基因序列,应用相关软件各设计了1对特异性引物,分别用于扩增PRV g B基因、PCV2 ORF2基因、PRRSV ORF6基因、CSFV C基因、PPV NS1基因和SIV M基因的部分序列,扩增的目的片段大小分别为192bp、255bp、364 bp、530 bp、759 bp和981bp,通过对目的基因的测序鉴定以及反应的条件优化,建立了PRV、PCV2、PRRSV、CSFV、PPV、SIV的单项PCR/RT-PCR方法,为摸索建立多重PCR方法的条件打下基础。2、三重PCR及三重RT-PCR鉴别诊断方法的建立与初步应用在建立的PRV、PCV2、PRRSV、CSFV、PPV和SIV的单项PCR/RT-PCR方法的基础上,以阳性病毒核酸为模板进行多重PCR扩增及反应体系和反应条件的优化,分别得到与设计相符合的3条特异性条带,建立了能同时检测PRV、PCV2和PPV三种DNA病毒的三重PCR方法和能同时检测PRRSV、CSFV和SIV三种RNA病毒的三重RT-PCR方法。试验结果表明该方法具有较好的敏感性和特异性,且均在56℃时检测效果最好。该方法对临床病料的检测结果与单项PCR结果一致。3、猪呼吸道病毒性疫病多重PCR鉴别诊断方法的建立与初步应用在建立的三重PCR方法及三重RT-PCR方法的基础上,通过对多重PCR反应条件的优化,以及临床应用检测,成功建立了能同时检测PRV、PCV2、CSFV和SIV四种病毒的多重PCR方法,能同时检测PRV、PCV2、PRRSV、CSFV和SIV五种病毒的多重PCR方法,以及同时检测PRV、PCV2、PRRSV、CSFV、PPV和SIV六种病毒的多重PCR方法,敏感性试验表明,建立的多重PCR最低检测限均达到pg级水平,特异性结果表明均只能检测出目的条带,对其他病毒扩增结果呈阴性。该方法对48份临床病料的检测结果表明其能快速、准确地对病料进行检测。
[Abstract]:Pig respiratory disease is one of the main diseases affecting the development of pig industry. It is a frequent and frequent disease in large-scale pig farms in China, which has caused widespread attention. Pig pseudorabies virus, porcine circovirus type 2, swine blue ear disease virus, swine fever virus. Porcine parvovirus and swine influenza virus are the most common pathogens of swine viral respiratory diseases, and often present mixed or secondary infection in clinic. The co-infection of two or more pathogens leads to higher morbidity and mortality in pigs, which makes it more difficult to diagnose and prevent diseases. In this study, a multiplex PCR differential diagnosis method for the above 6 viruses was established, which provided an effective technical means for the rapid diagnosis and prevention and control of related epidemic diseases. The main research contents are as follows: 1. The establishment of a single PCR/RT-PCR method for PCV2 / PRRSVN CSFV / PPVSIV referred to PRV g Bn AF257079 in NCBI. PCV2 ORF2 / NCZ 005148 / PRRSV ORF6 / NCSTP 001961CSFV / AY805221). A pair of specific primers were designed by using the software of PPV NS1, NC-1 1818, SIV MIV, GU086140) and other gene sequences. They were used to amplify PRV g B gene and PCV2 ORF2 gene and PRRSv ORF6 gene and CSFVC gene respectively. The size of the target fragment of PPV NS1 gene and SIV M gene was 255bp, 364bp and 530bp, respectively. 759bp and 981bp. by sequencing the target gene and optimizing the reaction conditions, the PRVV PCV2 / PRRSVG CSFVV PPV was established. The single PCR/RT-PCR method of SIV lays a foundation for establishing the condition of multiplex PCR method. Establishment and preliminary application of differential diagnosis of triple PCR and triple RT-PCR in the established PRV / PCV2 / PRRSVV / CSFV. On the basis of the single PCR/RT-PCR method of PPV and SIV, the positive viral nucleic acid was used as template for multiplex PCR amplification, and the reaction system and reaction conditions were optimized. Three specific bands were obtained which were consistent with the design, and a triple PCR method and a simultaneous detection method for PRRSV were established, which could simultaneously detect DNA viruses of PRV-1 PCV2 and PPV. Triplex RT-PCR method for CSFV and SIV RNA virus. The results show that the method has good sensitivity and specificity. The results of this method were consistent with that of single PCR. 3. The establishment and preliminary application of multiple PCR differential diagnosis method for swine respiratory virus blight were based on the established triple PCR method and triple RT-PCR method. By optimizing the reaction conditions of multiplex PCR and detecting the clinical application, a multiplex PCR method was successfully established to detect four kinds of viruses (PRV / PCV _ 2 and SIV) at the same time. A multiplex PCR method for the simultaneous detection of PRV / PCV2 / PRRSVV / CSFV and PRV / PCV2 / PRRSVV / CSFV was developed. The sensitivity test of multiplex PCR method for PPV and SIV showed that the minimum detection limit of multiplex PCR reached PG level, and the specific results showed that only the target bands could be detected. The results of 48 clinical samples showed that the method could be used to detect the samples quickly and accurately.
【学位授予单位】：贵州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.28

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