【摘要】：豬薩佩羅病毒(Porcine sapelovirus,PSV)、豬嵴病毒(Porcine kobuvirus,PKV)、豬星狀病毒(Porcine astrovirus,PAstV)、豬德爾塔冠狀病毒病(Porcine Deltacoronavirus,PDCoV)、豬環(huán)曲病毒(Porcine torovirus,PToV)、豬捷申病毒(Porcine teschvoirus,PTV)、豬札幌病毒(Porcine sapovirus,PSaV)是一類均可引起豬消化道疾病的病毒,主要導(dǎo)致豬出現(xiàn)一系列的腹瀉癥狀,而且常表現(xiàn)為兩種以上病原的混合感染。多重PCR方法具有敏感性和特異性高且可以同時(shí)檢測多種病原等優(yōu)點(diǎn),比較適合用于混合感染的快速診斷。因此,本研究針對七種病毒的保守序列分別設(shè)計(jì)一對特異性引物,建立了可以同時(shí)檢測這七種病毒感染的多重PCR方法,并將此方法用于臨床樣品的檢測,證實(shí)建立的方法靈敏度好,特異性強(qiáng),能用于臨床鑒別診斷。對豬腹瀉糞便中七種病毒的臨床檢驗(yàn)分析發(fā)現(xiàn)PSV、PKV、PAstV的陽性率較高,為了了解這三種病毒的進(jìn)化特點(diǎn),挑選3株P(guān)SV和PKV,2株P(guān)AstV進(jìn)行全基因組序列測定及分析。1.PSV、PKV、PAstV、PDCoV、PToV、PTV和PSaV多重RT-PCR檢測方法的建立及初步應(yīng)用合成了七對特異性引物分別用于擴(kuò)增PSV、PKV、PAstV、PDCoV、PToV、PTV和PSaV基因序列,目的片段的大小分別為624bp、280bp、249bp、504bp、421bp、176bp、340bp。通過優(yōu)化多重RT-PCR的反應(yīng)條件,建立了可以同時(shí)檢測這七種病毒的多重RT-PCR方法。該多重RT-PCR的特異性、重復(fù)性均良好,靈敏性可達(dá)到pg級水平。使用該方法檢測492份來自上海地區(qū)的豬腹瀉糞便樣品,其中PKV的感染率最高達(dá)到41.9%(206/492),PAstV和PSV的感染率次之,分別為31.1%(153/492)、15.7%(77/492)。對檢測結(jié)果統(tǒng)計(jì)發(fā)現(xiàn),492份臨床樣品中有28.7%(141/492)的樣品中存在2種及以上的病毒感染,主要是PKV和PAstV;13.0%(64/492)的樣品中存在3種及以上的病毒感染,主要是PKV、PAstV和PSV;3.2%(16/492)的樣品中存在4種及以上的病毒感染,主要是PKV、PAstV、PSV與PDCoV;2份(0.6%)樣品中存在PKV、PAstV、PSV、PSaV與PDCoV混合感染,;1份樣品中存在PKV、PAstV、PSV、PSaV、PDCoV與PTV混合感染。2.PSV、PKV、PAstV全基因組序列的擴(kuò)增及進(jìn)化分析鑒于PSV、PKV、PAstV有較高的感染率,為深入了解這3種病毒的基因組分子特性,實(shí)驗(yàn)從陽性樣品中挑選3株P(guān)SV和PKV、2株P(guān)AstV,進(jìn)行全基因組序列的擴(kuò)增,并進(jìn)行遺傳進(jìn)化分析。測序結(jié)果顯示:我們的3株P(guān)SV全基因組序列與國內(nèi)報(bào)道的毒株序列親緣性較近,而與美國、德國、韓國代表株的遺傳距離較遠(yuǎn),并對VP1基因進(jìn)行序列比對分析,表明這3個(gè)毒株的VP1基因與德國分離株P(guān)o5116同源性最低,而與中國分離株YC2011相似性最高。3株P(guān)KV之間的全基因組同源性不高,僅達(dá)88.1%-89.1%,說明上海不同地區(qū)流行的PKV毒株之間存在著一定的變異,但VP1基因進(jìn)化樹表明,它們與日本株P(guān)KV的親緣性最近,由此推測,近年來我國流行的PKV和日本株P(guān)KV之間有著一定的相似性,很有可能是從日本株變異而來。另外,2株P(guān)AstV的全基因組同源性僅73.8%,歸屬于不同的亞群,與不同國家的PAstV相似性為40.5%~77.1%,同源性普遍較低;對PAstV的ORF2基因進(jìn)行序列比對分析,表明我國流行的PAstV與美國株P(guān)AstV之間有一定的相似性,很有可能是由美國株變異而來。
[Abstract]:Porcine saprorovirus (PSV), porcine circovirus (PKV), porcine stellate virus (PTV), porcine delta coronavirus (PTV), porcine delta virus (PDCoV), porcine circovirus (PTV), porcine tetranvirus (PTV), and Sapporo virus (PTV), PsaV) is a kind of virus that can cause the digestive tract disease of the pig, which mainly causes a series of diarrhea symptoms in the pig, and often appears as a mixed infection of more than two pathogens. The multiplex PCR method has the advantages of high sensitivity and specificity and can simultaneously detect various pathogens and the like, and is suitable for rapid diagnosis of mixed infection. Therefore, a pair of specific primers are respectively designed for conserved sequences of seven viruses, multiple PCR methods for simultaneously detecting the seven viral infections are established, and the method is used for the detection of clinical samples, and the established method is good in sensitivity and strong in specificity, Can be used for clinical differential diagnosis. The results showed that the positive rate of PSV, PKV and PASTV was higher in the stool of pig diarrhea. In order to understand the evolution characteristics of these three viruses,3 strains of PSV and PKV were selected and the whole genome sequence was determined and analyzed by 2 strains of PASTV. Seven pairs of specific primers were used to amplify PSV, PKV, PstV, PDCoV, PTOV, PTV and PSV gene sequences, respectively. The sizes of the target fragments were 624 bp,280 bp,249 bp,504 bp,421 bp,176 bp and 340 bp, respectively. By optimizing the reaction conditions of multiplex RT-PCR, a multiplex RT-PCR method for simultaneous detection of these seven viruses was established. The multiplex RT-PCR has good specificity and repeatability, and the sensitivity can reach the level of pg. The infection rate of PASTV and PSV was 31.1% (153/492) and 15.7% (77/492) respectively. Of the 492 clinical samples, 28.7% (141/492) of the samples were infected with the virus, mainly PKV and PstV; 13.0% (64/492) of the samples were infected with three or more viral infections, mainly PKV, PstV and PSV; and 3.2% (16/492) of the samples had four or more viral infections, PKV, PstV, PSV, PSV and PDCoV were present in 2 (0.6%) samples, and PKV, PstV, PSV, PSV, PDCoV and PTV were present in one sample. PASTV has a high infection rate. In order to get a better understanding of the genomic molecular characteristics of these three viruses,3 strains of PSV and PKV and 2 PASTV are selected from the positive sample, and the whole genome sequence is amplified and the genetic evolution analysis is carried out. The results showed that the whole genome sequence of the 3 PSV was closely related to the sequence of the strains reported in China, but it was far from the genetic distance of the representative strains of the United States, Germany and Korea, and the sequence ratio of the VP1 gene was analyzed. The homology of the VP1 gene of the three strains is the lowest with that of the German isolate, and the similarity with the Chinese isolate YC2011 is the highest. The total genomic homology between the three strains is not high, which is only 88.1%-89.1%, indicating that there is a certain variation between the popular PKV strains in different regions of Shanghai. But the VP1 gene of PKV shows that they are closely related to the Japanese strain PKV, and it is presumed that there is a certain similarity between the PKV and the Japanese strain PKV in China in recent years, and it is likely that it is from the Japanese strain. In addition, the total genomic homology of 2 strains of PstV was only 73.8%, belonging to different subpopulations. The similarity of PAstV in different countries was 40.5%-77.1%, and the homology was generally lower; and the ORF2 gene of PASTV was compared with that of PASTV, indicating that there was a certain similarity between PASTV and PASTV in China. It's probably from the American strain.
【學(xué)位授予單位】：上海海洋大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.651

【參考文獻(xiàn)】

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1 李雨o，

本文編號：2476075