本文關(guān)鍵詞： 豬呼吸道 病毒性疫病 多重PCR 混合感染 臨床檢測(cè)　出處：《貴州大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：豬呼吸道疾病是影響?zhàn)B豬業(yè)發(fā)展的主要疾病之一,是目前我國(guó)規(guī)�；i場(chǎng)的常發(fā)病、多發(fā)病,已經(jīng)引起人們的普遍重視,給我國(guó)養(yǎng)豬業(yè)帶來(lái)了巨大的危害和損失。豬偽狂犬病病毒、豬圓環(huán)病毒2型、豬藍(lán)耳病病毒、豬瘟病毒、豬細(xì)小病毒和豬流感病毒是最為多見(jiàn)的豬病毒性呼吸道疾病病原,且在臨床上常呈混合或繼發(fā)感染。豬群中,多病原混合感染已是疾病發(fā)生的普遍特點(diǎn),兩種或以上病原體共同感染導(dǎo)致豬群發(fā)病率和死亡率增高,使得疾病診斷和防治難度加大。為實(shí)現(xiàn)臨床多病原混合感染的快速確診,本研究建立了針對(duì)上述6種病毒的多重PCR鑒別診斷方法,為相關(guān)疫病的快速診斷和防控提供了有效的技術(shù)手段。主要研究?jī)?nèi)容如下:1、PRV、PCV2、PRRSV、CSFV、PPV、SIV的單項(xiàng)PCR/RT-PCR方法的建立參照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,應(yīng)用相關(guān)軟件各設(shè)計(jì)了1對(duì)特異性引物,分別用于擴(kuò)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,擴(kuò)增的目的片段大小分別為192bp、255bp、364 bp、530 bp、759 bp和981bp,通過(guò)對(duì)目的基因的測(cè)序鑒定以及反應(yīng)的條件優(yōu)化,建立了PRV、PCV2、PRRSV、CSFV、PPV、SIV的單項(xiàng)PCR/RT-PCR方法,為摸索建立多重PCR方法的條件打下基礎(chǔ)。2、三重PCR及三重RT-PCR鑒別診斷方法的建立與初步應(yīng)用在建立的PRV、PCV2、PRRSV、CSFV、PPV和SIV的單項(xiàng)PCR/RT-PCR方法的基礎(chǔ)上,以陽(yáng)性病毒核酸為模板進(jìn)行多重PCR擴(kuò)增及反應(yīng)體系和反應(yīng)條件的優(yōu)化,分別得到與設(shè)計(jì)相符合的3條特異性條帶,建立了能同時(shí)檢測(cè)PRV、PCV2和PPV三種DNA病毒的三重PCR方法和能同時(shí)檢測(cè)PRRSV、CSFV和SIV三種RNA病毒的三重RT-PCR方法。試驗(yàn)結(jié)果表明該方法具有較好的敏感性和特異性,且均在56℃時(shí)檢測(cè)效果最好。該方法對(duì)臨床病料的檢測(cè)結(jié)果與單項(xiàng)PCR結(jié)果一致。3、豬呼吸道病毒性疫病多重PCR鑒別診斷方法的建立與初步應(yīng)用在建立的三重PCR方法及三重RT-PCR方法的基礎(chǔ)上,通過(guò)對(duì)多重PCR反應(yīng)條件的優(yōu)化,以及臨床應(yīng)用檢測(cè),成功建立了能同時(shí)檢測(cè)PRV、PCV2、CSFV和SIV四種病毒的多重PCR方法,能同時(shí)檢測(cè)PRV、PCV2、PRRSV、CSFV和SIV五種病毒的多重PCR方法,以及同時(shí)檢測(cè)PRV、PCV2、PRRSV、CSFV、PPV和SIV六種病毒的多重PCR方法,敏感性試驗(yàn)表明,建立的多重PCR最低檢測(cè)限均達(dá)到pg級(jí)水平,特異性結(jié)果表明均只能檢測(cè)出目的條帶,對(duì)其他病毒擴(kuò)增結(jié)果呈陰性。該方法對(duì)48份臨床病料的檢測(cè)結(jié)果表明其能快速、準(zhǔn)確地對(duì)病料進(jìn)行檢測(cè)。
[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.
【學(xué)位授予單位】：貴州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S858.28

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