猪腹泻病原多重PCR检测方法的建立及主要病毒进化分析
发布时间：2019-05-13 17:55
【摘要】：猪萨佩罗病毒(Porcine sapelovirus,PSV)、猪嵴病毒(Porcine kobuvirus,PKV)、猪星状病毒(Porcine astrovirus,PAstV)、猪德尔塔冠状病毒病(Porcine Deltacoronavirus,PDCoV)、猪环曲病毒(Porcine torovirus,PToV)、猪捷申病毒(Porcine teschvoirus,PTV)、猪札幌病毒(Porcine sapovirus,PSaV)是一类均可引起猪消化道疾病的病毒,主要导致猪出现一系列的腹泻症状,而且常表现为两种以上病原的混合感染。多重PCR方法具有敏感性和特异性高且可以同时检测多种病原等优点,比较适合用于混合感染的快速诊断。因此,本研究针对七种病毒的保守序列分别设计一对特异性引物,建立了可以同时检测这七种病毒感染的多重PCR方法,并将此方法用于临床样品的检测,证实建立的方法灵敏度好,特异性强,能用于临床鉴别诊断。对猪腹泻粪便中七种病毒的临床检验分析发现PSV、PKV、PAstV的阳性率较高,为了了解这三种病毒的进化特点,挑选3株PSV和PKV,2株PAstV进行全基因组序列测定及分析。1.PSV、PKV、PAstV、PDCoV、PToV、PTV和PSaV多重RT-PCR检测方法的建立及初步应用合成了七对特异性引物分别用于扩增PSV、PKV、PAstV、PDCoV、PToV、PTV和PSaV基因序列,目的片段的大小分别为624bp、280bp、249bp、504bp、421bp、176bp、340bp。通过优化多重RT-PCR的反应条件,建立了可以同时检测这七种病毒的多重RT-PCR方法。该多重RT-PCR的特异性、重复性均良好,灵敏性可达到pg级水平。使用该方法检测492份来自上海地区的猪腹泻粪便样品,其中PKV的感染率最高达到41.9%(206/492),PAstV和PSV的感染率次之,分别为31.1%(153/492)、15.7%(77/492)。对检测结果统计发现,492份临床样品中有28.7%(141/492)的样品中存在2种及以上的病毒感染,主要是PKV和PAstV;13.0%(64/492)的样品中存在3种及以上的病毒感染,主要是PKV、PAstV和PSV;3.2%(16/492)的样品中存在4种及以上的病毒感染,主要是PKV、PAstV、PSV与PDCoV;2份(0.6%)样品中存在PKV、PAstV、PSV、PSaV与PDCoV混合感染,;1份样品中存在PKV、PAstV、PSV、PSaV、PDCoV与PTV混合感染。2.PSV、PKV、PAstV全基因组序列的扩增及进化分析鉴于PSV、PKV、PAstV有较高的感染率,为深入了解这3种病毒的基因组分子特性,实验从阳性样品中挑选3株PSV和PKV、2株PAstV,进行全基因组序列的扩增,并进行遗传进化分析。测序结果显示:我们的3株PSV全基因组序列与国内报道的毒株序列亲缘性较近,而与美国、德国、韩国代表株的遗传距离较远,并对VP1基因进行序列比对分析,表明这3个毒株的VP1基因与德国分离株Po5116同源性最低,而与中国分离株YC2011相似性最高。3株PKV之间的全基因组同源性不高,仅达88.1%-89.1%,说明上海不同地区流行的PKV毒株之间存在着一定的变异,但VP1基因进化树表明,它们与日本株PKV的亲缘性最近,由此推测,近年来我国流行的PKV和日本株PKV之间有着一定的相似性,很有可能是从日本株变异而来。另外,2株PAstV的全基因组同源性仅73.8%,归属于不同的亚群,与不同国家的PAstV相似性为40.5%~77.1%,同源性普遍较低;对PAstV的ORF2基因进行序列比对分析,表明我国流行的PAstV与美国株PAstV之间有一定的相似性,很有可能是由美国株变异而来。
[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.
【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.651
[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.
【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.651
【参考文献】
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