【摘要】：近年來全球豬腹瀉病頻發(fā),發(fā)病規(guī)模大,導(dǎo)致經(jīng)濟(jì)損失慘重。豬流行性腹瀉病毒(Porcine epidemic diarrhea virus, PEDV)、豬傳染性胃腸炎病毒(Transmissible gastroenteritis virus, TGEV )、豬輪狀病毒 A 型(Porcine group A rotavi ruses,GAR )、豬輪狀病毒 C 型(Porcine group C rotaviruses, GCR)、豬圓環(huán)病毒 2 型(Porcine circovirus2,PCV2)被認(rèn)為是最主要的豬病毒性腹瀉病原。這5種病毒感染率高、危害大,且存在混合感染現(xiàn)象,現(xiàn)有檢測(cè)手段存在一定缺陷且難以對(duì)混合感染情況進(jìn)行有效檢測(cè)。本研究分別建立了針對(duì)該5種腹瀉病毒的普通多重PCR和EvaGreen多重實(shí)時(shí)熒光定量PCR方法并應(yīng)用于臨床樣品檢測(cè)。普通PCR在檢測(cè)中有著其獨(dú)特優(yōu)勢(shì)而被廣泛使用。本實(shí)驗(yàn)首先通過GenBank下載5種病毒基因序列,經(jīng)軟件序列比對(duì)后,在保守區(qū)分別設(shè)計(jì)5對(duì)普通PCR引物。通過對(duì)反應(yīng)體系和反應(yīng)條件進(jìn)行優(yōu)化,單重和多重普通PCR均可產(chǎn)生大小分別為 126bp(GCR)、242bp(GAR)、319bp(TGEV)、394bp(PEDV)、508bp(PCV2)目的產(chǎn)物片段。而陰性對(duì)照和非靶標(biāo)陰性對(duì)照沒有相應(yīng)的產(chǎn)物產(chǎn)生,表明建立的單重和多重普通PCR方法具有良好的特異性。單重PCR檢測(cè)GCR、GAR、TGEV、PEDV 和 PCV2 的最低限分別為 5 copies、50 copies、5 copies、5 copies、5 copies。多重普通PCR檢測(cè)混合病毒模板情況的靈敏度都可以達(dá)到50 copies,和單重PCR相同或低一個(gè)數(shù)量級(jí),但相比于先前研究報(bào)道的多重反轉(zhuǎn)錄PCR靈敏度(104-103 copies)提高了約2個(gè)數(shù)量級(jí)。將建立的普通多重PCR方法用于172份臨床樣品檢測(cè)。GCR、GAR、TGEV、PEDV 和 PCV2 陽性率分別為 5.81%、2.32%、8.72%、20.92%和 46.51%,總混合感染率為22.09%,其中主要是PCV2與其它病毒存在混合感染現(xiàn)象,PEDV和PCV2混合感染率較高,占16.86%。挑選64個(gè)樣品進(jìn)行多重PCR和單重PCR對(duì)比,結(jié)果顯示兩者檢出率相近,多重PCR檢出率略低。實(shí)時(shí)熒光定量PCR包括多重實(shí)時(shí)熒光定量PCR是通過實(shí)時(shí)監(jiān)測(cè)熒光信號(hào)的一種核酸片段定性定量分析方法,近來被廣泛用于病原微生物檢測(cè)多重實(shí)時(shí)熒光定量PCR技術(shù)在豬病毒性腹瀉上的應(yīng)用很少。本研究根據(jù)5種腹瀉病毒擴(kuò)增產(chǎn)物Tm值,在保守區(qū)設(shè)計(jì)5對(duì)定量PCR引物,經(jīng)反應(yīng)體系和參數(shù)優(yōu)化,建立了基于EvaGreen熒光染料和熔解曲線分析檢測(cè)這5種病毒的單重和多重?zé)晒舛縋CR方法。結(jié)果顯示設(shè)計(jì)的5對(duì)病毒引物能夠形成特異的熔解峰,TGEV、GAR、GCR、PEDV 和 PCV2 對(duì)應(yīng)產(chǎn)物 Tm 值為 75.07 ± 0.15℃、77.77± 0.06℃、79.63±0.25℃、83.60 ±0.17℃和88.63 ±0.06℃,和多重?zé)晒舛縋CR各病毒溶解峰Tm值類似,而非目的基因?qū)φ蘸完幮詫?duì)照未形成影響實(shí)驗(yàn)分析的非特異峰,表明建立的單重和多重?zé)晒舛縋CR方法特異性良好。批內(nèi)批間重復(fù)性實(shí)驗(yàn)結(jié)果顯示Tm值變異系數(shù)小于0.5%,Ct值變異系數(shù)小于3.5%,表明重復(fù)性良好。TGEV、GAR、GCR、PEDV和PCV2單重?zé)晒舛縋CR的檢測(cè)靈敏度分別是5、5、5、5和50copies。多重實(shí)時(shí)熒光定量PCR對(duì)單個(gè)病毒TGEV、GAR、GCR、PEDV和PCV2的檢測(cè)靈敏度分別為5、5、50、5和50 copies,對(duì)5個(gè)病毒混合模板的檢測(cè)靈敏度都達(dá)到50 copies。將建立的EvaGreen多重實(shí)時(shí)熒光定量PCR方法應(yīng)用于172份臨床樣品檢測(cè)。TGEV、GAR、GCR、PEDV 和 PCV2 陽性率分別為 5.23%、3.48%、7.56%、25.58%和44.76%。存在兩種和三種病毒的混合感染,總混合陽性率為22.66%,其中PEDV和PCV2混合感染率較高,達(dá)到18.02%。檢測(cè)結(jié)果和普通PCR相近。對(duì)其中90份臨床樣品分別進(jìn)行各病毒單重實(shí)時(shí)熒光定量PCR檢測(cè),兩者檢出率比較接近。表明本研究建立的多重實(shí)時(shí)熒光定量PCR方法可較好地用于臨床樣品5種腹瀉病毒檢測(cè)。
[Abstract]:In recent years, the global swine diarrhoea has been frequently occurring, with large scale and heavy economic loss. Swine epidemic diarrhea virus (Porcine epidemic diarrhea virus, PEDV), swine infectious gastroenteritis virus (Transmissible gastroenteritis virus, TGEV), porcine rotavirus A type (Porcine group), porcine rotavirus type Roup C rotaviruses, GCR), the porcine circovirus 2 (Porcine circovirus2, PCV2) is considered to be the most important pathogen of swine viral diarrhea. These 5 viruses have high infection rate, great harm and mixed infection. The existing detection methods have some defects and are difficult to detect the mixed infection effectively. This study established the needle respectively. The common multiplex PCR and EvaGreen multiple real-time fluorescent quantitative PCR methods for the 5 kinds of diarrhea viruses were applied to the detection of clinical samples. The common PCR has its unique advantages and is widely used. This experiment first downloads 5 virus gene sequences by GenBank. After the software sequence ratio, 5 pairs of ordinary PCR are designed in the conservative region. By optimizing the reaction system and reaction conditions, single weight and multiple common PCR can produce fragments of 126bp (GCR), 242bp (GAR), 319bp (TGEV), 394bp (PEDV), 508bp (PCV2), while negative and non target negative controls have no corresponding products, indicating that single and multiple common PCR methods are established. Good specificity. The minimum limits for single weight PCR detection of GCR, GAR, TGEV, PEDV and PCV2 are 5 copies, 50 copies, 5 copies, 5 copies, and 5 copies. multiple common PCR tests can reach 50, the same or one order of magnitude as single or low, but compared to the multiple reverse transcripts reported previously. The PCR sensitivity (104-103 copies) increased by about 2 orders of magnitude. The common multiple PCR method will be established for 172 clinical samples to detect.GCR, GAR, TGEV, PEDV and PCV2, respectively, 5.81%, 2.32%, 8.72%, 20.92% and 46.51%, and the total mixed infection rate is 22.09%, mainly the presence of mixed infection between PCV2 and other viruses, PEDV and PCV2. The rate of mixed infection was high, and 64 samples were selected for 16.86%. to compare multiple PCR and single PCR. The results showed that the detection rate was similar, and the detection rate of multiple PCR was slightly lower. Real time fluorescence quantitative PCR including multiple real-time quantitative PCR was a qualitative and quantitative analysis method of nucleic acid segments through real-time monitoring of fluorescence signals, and recently it was widely used for disease. The application of multiple real-time fluorescence quantitative PCR in the detection of viral diarrhea in swine was very few. This study was based on the Tm value of 5 kinds of diarrhea virus, and designed 5 pairs of quantitative PCR primers in the conservative region. Through the optimization of the reaction system and parameters, a single and multiple detection of these 5 viruses was established based on the EvaGreen fluorescent dye and the fusion curve analysis. The results of heavy fluorescence quantitative PCR showed that the designed 5 pairs of primers could form a specific fusion peak. The Tm values of the products of TGEV, GAR, GCR, PEDV and PCV2 were 75.07 + 0.15, 77.77 + 0.06, 79.63 + 0.25, 83.60, 0.17 and 88.63 + 75.07, similar to those of the multiple fluorescence quantitative PCR viruses, but not the target genes. The control and negative control did not form the non specific peak of the experimental analysis, indicating that the established single weight and multiple fluorescence quantitative PCR method had good specificity. The results of interbatch reproducibility test showed that the Tm value variation coefficient was less than 0.5% and the Ct value variation coefficient was less than 3.5%, indicating that the reproducibility of good.TGEV, GAR, GCR, PEDV and PCV2 single heavy fluorescence quantitative PCR were detected. The sensitivity of 5,5,5,5 and 50copies. multiplex real-time fluorescent quantitative PCR for single virus TGEV, GAR, GCR, PEDV and PCV2 are 5,5,50,5 and 50 copies respectively. The sensitivity of the 5 virus mixed templates is 50 copies. and the EvaGreen multiple realtime fluorescence quantitative method is applied to 172 clinical samples. The positive rates of.TGEV, GAR, GCR, PEDV and PCV2 were 5.23%, 3.48%, 7.56%, 25.58% and 44.76%. were mixed with two and three viruses, and the total mixed positive rate was 22.66%. The mixed infection rate of PEDV and PCV2 was higher, and the result of 18.02%. detection was similar to that of ordinary PCR. The virus single weight of 90 clinical samples was carried out in real time respectively. The detection rate of fluorescence quantitative PCR was close. It showed that the multiple real-time fluorescence quantitative PCR method established in this study could be better used for the detection of 5 kinds of diarrhoea virus in clinical samples.
【學(xué)位授予單位】：浙江理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S852.651

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本文編號(hào)：2150848