本文選題：口蹄疫病毒 + 水皰性口炎病毒　； 參考：《四川農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：口蹄疫、豬水皰病和水皰性口炎分別是由口蹄疫病毒(Foot-and-mouth disease virus,FMDV)、豬水皰病病毒(Swine vesicular disease virus,SVDV)和水皰性口炎病毒(Vesicular stomatitis virus,VSV)引起的急性動(dòng)物傳染病,都可以感染豬,且由于發(fā)病癥狀相似,從發(fā)病癥狀上鑒別難度較大,因此構(gòu)建一種能同時(shí)鑒別診斷三種疾病和區(qū)分口蹄疫三種血清型的高通量聯(lián)合檢測(cè)方法具有重要意義。本研究以FMDV O型、A型、Asia 1型、VSV和SVDV為研究對(duì)象,建立了寡核苷酸基因芯片檢測(cè)體系。研究?jī)?nèi)容如下：1.共檢基因芯片的靶基因引物和探針的構(gòu)建：參考GenBank中已公布的基因組序列,根據(jù)基因芯片引物設(shè)計(jì)要求,選擇三種病毒的保守區(qū)域設(shè)計(jì)引物和探針,對(duì)引物和探針進(jìn)行了驗(yàn)證,構(gòu)建了基于口蹄疫病毒的A、O、Asia 1型VP1序列、VSV的L基因和SVDV VP1序列的pMD19-T克隆質(zhì)粒并測(cè)序鑒定。結(jié)果顯示,克隆的靶基因與NCBI公布的序列一致性均在95%以上。2.共檢基因芯片的構(gòu)建和優(yōu)化設(shè)計(jì)了基因芯片矩陣和位置指控、雜交質(zhì)控及雜交質(zhì)控探針序列,并對(duì)三種病毒的探針進(jìn)行了篩選。對(duì)芯片點(diǎn)樣后的水化溫度與封閉條件、PCR退火溫度和循環(huán)數(shù)、熒光基團(tuán)標(biāo)記引物的使用量、雜交溫度等進(jìn)行了優(yōu)化。結(jié)果表明：使用醛基基片,在18℃-37℃水化過夜,使用含0.25% BH4Na,25%乙醇的封閉液進(jìn)行封閉所制的芯片效果較好；PCR擴(kuò)增時(shí)選擇58℃退火溫度,一般使用2μL熒光基團(tuán)標(biāo)記引物,擴(kuò)增30個(gè)循環(huán)便可得到足夠的產(chǎn)物；在臨床檢測(cè)時(shí)為提高靈敏度可使用4-8μL熒光基團(tuán)標(biāo)記引物,擴(kuò)增40-50個(gè)循環(huán),雜交溫度達(dá)到42℃便可獲得有意義的結(jié)果,為保證特異性,在52℃進(jìn)行雜交效果更好。3.共檢基因芯片的效果評(píng)價(jià)對(duì)所構(gòu)建的基因芯片檢測(cè)方法進(jìn)行了靈敏性試驗(yàn)、特異性試驗(yàn)、重復(fù)性試驗(yàn)和穩(wěn)定性試驗(yàn)。結(jié)果表明,本研究所設(shè)計(jì)的FMDV-O引物PCR檢測(cè)限為0.1180ng/mL, FMDV-A為0.0184ng/mL, FMDV-Asia I為0.129ng/mL, VSV為0.0267ng/mL, SVDV為0.0247ng/mL,芯片檢測(cè)方法靈敏度至少比PCR檢測(cè)高一個(gè)數(shù)量級(jí)；特異性良好,所設(shè)計(jì)引物沒有出現(xiàn)非特異性擴(kuò)增,能準(zhǔn)確區(qū)分檢測(cè)出FMDV、VSV、SVDV,并能區(qū)分出口蹄疫病毒的三種血清型；應(yīng)用了兩家公司所生產(chǎn)的不同批次醛基基片進(jìn)行重復(fù)性驗(yàn)證,均能夠較好地實(shí)現(xiàn)重復(fù)實(shí)驗(yàn)；將點(diǎn)制好的芯片抽真空后于4℃保存,至少能保存四個(gè)月。
[Abstract]:Foot-and-mouth disease (FMD), porcine blisters disease (BMD) and vesicular stomatitis (BMD) are acute animal infections caused by foot-and-mouth disease virus (Foot-and-mouth disease virus), Swine vesicular disease virus (SVDVV), and vesicular stomatitis virus (Vesicular stomatitis virus VSVV), all of which can infect pigs and have similar symptoms. It is difficult to identify the symptoms of the disease, so it is of great significance to establish a high throughput combined detection method which can simultaneously differentiate and diagnose the three diseases and distinguish the three serotypes of foot-and-mouth disease (FMD). In this study, the detection system of oligonucleotide microarray was established using VSV and SVDV of FMDV O and Asia 1. The research is as follows: 1. Construction of target gene primers and probes for co-detection of gene chips: according to the genomic sequence published in GenBank and according to the design requirements of gene chip primers, the primers and probes of three kinds of viruses were selected to design, and the primers and probes were verified. The L gene of VSV and pMD19-T of VP1 sequence of SVDV based on foot-and-mouth disease virus (FMDV) were constructed and sequenced. The results showed that the identity of the cloned target gene with the NCBI published sequence was more than 95%. The construction and optimization of the gene chip were used to construct and optimize the gene chip matrix, position control, hybridization quality control and hybridization quality control probe sequences, and the probes of three viruses were screened. The hydration temperature, PCR annealing temperature and cycle number, the amount of fluorescent group labeled primers and hybridization temperature were optimized. The results showed that the chip made by using aldehyde substrate, hydrating overnight at 18 鈩，

本文編號(hào)：2004508