本文選題：豬病毒性腹瀉　切入點：多重RT-PCR　出處：《內蒙古農業(yè)大學》2015年碩士論文　論文類型：學位論文

【摘要】：豬流行性腹瀉病毒(PEDV)、豬傳染性胃腸炎病毒(TGEV)、豬輪狀病毒(PoRV)是目前造成豬病毒性腹瀉的三種主要病原,且在臨床癥狀、病理變化以及流行病學上極為相似。傳統(tǒng)檢測方法雖能對三者進行鑒別診斷,但因操作復雜、檢測周期長、靈敏度低等缺點而在實踐中并不常用。本試驗針對三種病毒的保守序列分別設計特異性引物,并在明確各病毒單項RT-PCR最優(yōu)反應條件的前提下,逐步優(yōu)化多重RT-PCR反應條件,最終建立了可同時檢測上述三種病毒的多重RT-PCR檢測方法。特異性、靈敏性及一致性試驗結果表明,多重RT-PCR方法特異性強,靈敏度高,與常規(guī)RT-PCR方法的整體符合率為97.5%,適用于三種病毒的鑒別診斷和流行病學調查。應用已建立的多重RT-PCR方法,對2013～2015年間內蒙古9個盟市的413份腹瀉樣品和115份無腹瀉癥狀樣品進行PEDV、TGEV、PoRV三種病原的檢測。結果顯示,受檢的413份腹瀉病料中有260份呈現PEDV陽性,陽性率為62.95%；未發(fā)現TGEV感染病例；PoRV僅于個別豬場與PEDV昆合感染,陽性率為1.94%。在未發(fā)生腹瀉的豬群中同樣存在PEDV感染,陽性率為41.74%,未發(fā)現TGEV感染,僅發(fā)現一例PoRV感染豬只。不同日齡豬群均存在PEDV感染,以哺乳仔豬陽性率最高,達72.03%；其次是母豬。此外,不同類型樣品的病原檢出率有所差異,腸道樣品中PEDV檢出率最高,達87.50%,其次是肛門拭子,糞便中PEDV陽性率最低,為52.50%。檢測結果表明,PEDV是引起內蒙古地區(qū)豬群病毒性腹瀉的主要病原,并主要危害哺乳仔豬。對各盟市PEDV檢測為陽性的病料進行M、S基因的克隆、測序,并與國內外已報道的57條基因序列進行比對和遺傳進化分析。同源性及遺傳進化分析結果表明,CH/NMG/HLBE株、CH/NMG/BYNE株、CH/NMG/CF株和CH/NMG/EEDS株、CH/NMG/BT株、CH/NMG/HHHT株同源性較高,并與近年來流行的“亞美型”毒株親緣關系較近,同位于G1分支上；CH/NMG/WLCB株和CH/NMG/XLGL株與疫苗株Vaccine CV777同源性較高,同位于G3分支上；CH/NMG/TL株則與吉林CH/JLGZ/2011株親緣關系最為密切,同位于G2分支。核苷酸及氨基酸序列比對結果表明,PEDV的M基因較為保守,但其S基因變異明顯,且主要集中于S1區(qū)的N’端。
[Abstract]:Porcine epidemic diarrhea virus (PEDVV), transmissible gastroenteritis virus (TGEV) and porcine rotavirus (PoRV) are the three main pathogens causing porcine viral diarrhea. The pathological changes and epidemiology are very similar. Although the traditional detection method can differentiate the three, but because of the complexity of the operation, the detection period is long. In this experiment, specific primers were designed for the conserved sequences of three viruses, and the conditions of multiple RT-PCR reaction were optimized step by step on the premise of determining the optimal reaction conditions of individual RT-PCR of each virus. Finally, a multiplex RT-PCR detection method was established for simultaneous detection of the above three viruses. The results of specificity, sensitivity and consistency test showed that the multiplex RT-PCR method had strong specificity and high sensitivity. The overall coincidence rate with the conventional RT-PCR method is 97.50.It is suitable for the differential diagnosis and epidemiological investigation of three viruses. 413 diarrhea samples and 115 diarrhea free samples from 9 cities in Inner Mongolia between 2013 and 2015 were tested for three pathogens, PEVV TGEV and PoRV. The results showed that 260 of 413 samples were positive for PEDV. The positive rate of TGEV infection was 62.95, and the positive rate was 1.94.The positive rate was 1.94.The positive rate of PEDV infection was 41.74 in the pigs without diarrhea, and no TGEV infection was found. Only one PoRV infected pig was found. The positive rate of PEDV was 72.03 in suckling piglets, 72.03 in suckling piglets, and 72.03 in sows. In addition, the positive rate of PEDV was the highest in different types of samples and the highest in intestinal samples. The positive rate of PEDV in feces was the lowest (52.50). The results showed that PEDV was the main pathogen causing viral diarrhea in pigs in Inner Mongolia. The PEDV positive samples were cloned and sequenced. The results of homology and genetic evolution analysis showed that CHP / NMG- / BYNE and CH/NMG/EEDS / NMG- / BT were highly homologous to CHR / NMG- / HHT, respectively, and the results of homology and genetic evolution analysis showed that CHR / NMG- / HLBE was highly homologous to CHP / NMG- BYNE and CHP / NMG- / BYNE, respectively, and there was a high homology between CHR / NMGP-HHT and CH-NMGP-HHT. It was closely related to the prevalent "Asian American" virus strains in recent years, and had the highest homology with Vaccine CV777 in G _ 1 branch and CH/NMG/XLGL strain, and was the most closely related to Jilin CH/JLGZ/2011 strain on the same G3 branch. The results of nucleotide and amino acid sequence alignment showed that the M gene of PEDV was conserved, but its S gene mutation was obvious, and it was mainly concentrated at the N' end of S1 region.
【學位授予單位】：內蒙古農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S858.28

【參考文獻】

相關期刊論文 前10條

1 田小艷;孫華;鄧雨修;蘇潤環(huán);王東東;宋延華;;3種致豬腹瀉病毒的多重RT-PCR檢測[J];動物醫(yī)學進展;2009年09期

2 郭旋;陳靜;劉歡;侯韶毅;龍鳳;李瑩瑩;曾娟;蘭家暖;劉芳;黃偉堅;;廣西仔豬腹瀉病毒病原流行病學調查[J];南方農業(yè)學報;2013年01期

3 鄭逢梅;霍金耀;趙軍;常洪濤;王曉夢;陳陸;王川慶;;2010～2012年華中地區(qū)豬流行性腹瀉病毒分子特征和遺傳進化分析[J];病毒學報;2013年02期

4 樂正中,肖道彰,廖煥芬,龍躍忠;貴州省豬流行性腹瀉病的調查[J];貴州農業(yè)科學;1989年05期

5 李寶英;鐘細茍;;豬流行性腹瀉調查診斷報告[J];江西畜牧獸醫(yī)雜志;1987年01期

6 鄭新添;楊小燕;戴愛玲;李曉華;陳星星;;豬傳染性胃腸炎病毒、豬流行性腹瀉病毒和豬輪狀病毒三重PCR檢測方法的建立[J];龍巖學院學報;2011年05期

7 王子龍;韓慶安;王玉清;仇國明;李同山;孫繼國;;河北省豬病毒性腹瀉流行病學調查[J];黑龍江畜牧獸醫(yī);2014年10期

8 蔣靜;李健;胡永強;邱璐;熊煒;王巧全;;上海等4省市動物冠狀病毒的流行病學調查[J];畜牧與獸醫(yī);2007年12期

9 吳小偉;豬傳染性胃腸炎的疫情調查及防制報告[J];中國動物檢疫;1998年02期

10 錢永清,王兆久,劉洪云,唐騏駿,嚴仲烈;免疫酶技術證實:國內的豬傳染性胃腸炎病毒與比利時的CLV在血清學上相一致[J];獸醫(yī)科技雜志;1981年08期

相關碩士學位論文 前1條

1 黃海龍;豬傳染性胃腸炎和豬流行性腹瀉二聯RT-PCR診斷方法的建立及初步應用[D];吉林農業(yè)大學;2004年

，

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