本文選題：口蹄疫病毒 + 水皰性口炎病毒��； 參考：《四川農業(yè)大學》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,因此構建一種能同時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合檢測方法具有重要意義。本研究以FMDV O型、A型、Asia 1型、VSV和SVDV為研究對象,建立了寡核苷酸基因芯片檢測體系。研究內容如下：1.共檢基因芯片的靶基因引物和探針的構建：參考GenBank中已公布的基因組序列,根據基因芯片引物設計要求,選擇三種病毒的保守區(qū)域設計引物和探針,對引物和探針進行了驗證,構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克隆質粒并測序鑒定。結果顯示,克隆的靶基因與NCBI公布的序列一致性均在95%以上。2.共檢基因芯片的構建和優(yōu)化設計了基因芯片矩陣和位置指控、雜交質控及雜交質控探針序列,并對三種病毒的探針進行了篩選。對芯片點樣后的水化溫度與封閉條件、PCR退火溫度和循環(huán)數、熒光基團標記引物的使用量、雜交溫度等進行了優(yōu)化。結果表明：使用醛基基片,在18℃-37℃水化過夜,使用含0.25% BH4Na,25%乙醇的封閉液進行封閉所制的芯片效果較好；PCR擴增時選擇58℃退火溫度,一般使用2μL熒光基團標記引物,擴增30個循環(huán)便可得到足夠的產物；在臨床檢測時為提高靈敏度可使用4-8μL熒光基團標記引物,擴增40-50個循環(huán),雜交溫度達到42℃便可獲得有意義的結果,為保證特異性,在52℃進行雜交效果更好。3.共檢基因芯片的效果評價對所構建的基因芯片檢測方法進行了靈敏性試驗、特異性試驗、重復性試驗和穩(wěn)定性試驗。結果表明,本研究所設計的FMDV-O引物PCR檢測限為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,芯片檢測方法靈敏度至少比PCR檢測高一個數量級；特異性良好,所設計引物沒有出現非特異性擴增,能準確區(qū)分檢測出FMDV、VSV、SVDV,并能區(qū)分出口蹄疫病毒的三種血清型；應用了兩家公司所生產的不同批次醛基基片進行重復性驗證,均能夠較好地實現重復實驗；將點制好的芯片抽真空后于4℃保存,至少能保存四個月。
[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：2004508