应用非序列依赖单引物PCR（SISPA-PCR）技术筛查腹泻相关的病毒

发布时间：2018-05-10 05:37

本文选题：去游离核酸的随机PCR + 新病毒　；参考：《中国疾病预防控制中心》2010年硕士论文

【摘要】：背景：随着生物学技术的快速发展,新病毒及一些熟知病毒的新型别被不断发现。近年来发现的许多新发病毒均对人类健康有着极大的威胁,造成了非常严重的社会公共卫生问题,如艾滋病毒、丙型肝炎病毒、SARS、禽流感病毒等。据估计,在急性上呼吸道感染中约70%为病毒感染引起,其中有部分可能为未知病毒感染引起；尽管腹泻病为人类常见和多发的疾病,但仍旧有20-40%的腹泻病例的病原不清楚。明确病原,对传染病的预防控制意义重大。目前,用于新病毒发现的技术也不断涌现,由形态学、抗原抗体检测到核酸序列的测定,由随机PCR方法,到高通量测序技术的运用等,都大大缩短了新病毒发现的进程。目的：1.建立应用非序列依赖单引物PCR(SISPA-PCR)技术筛查腹泻相关病毒的技术平台：2、发现与腹泻相关的新病毒。方法：1、使用常规的RT-PCR方法筛查常见腹泻相关的已知病毒；2、应用改进的随机PCR技术(DNase-SISPA-PCR)发现和鉴定病毒：该技术是通过过滤的方法去除标本中的细菌、细胞等,通过超速离心富集病毒颗粒,使用DNA酶和RNA酶消化游离的核酸,去除游离核酸的干扰,然后使用含有已知片断的随机引物引发PCR扩增,并且对500bp以上的PCR产物进行回收,转化克隆,测定序列,对序列进行生物信息学分析,确定是否为新病毒；3、使用染色体步移技术(Genome Walking)获得新病毒的未知序列；使用RACE方法获得新病毒5’和3’末端序列；4、应用生物信息学分析新病毒序列特征和功能。结果：1.在动物(90天以下的幼猪)粪便标本中发现了4株新病毒,包括一株博卡样病毒(PBoV1),两株新星状病毒(PAstV1和PAstV2)和一株小RNA病毒(PPicV)。经过生物信息学分析,发现这几株病毒具有相应病毒的一些序列特征,在健康幼猪粪便标本中,PAstVl检出率为1.51%和PAstV2的检出率为1.26%,博卡样病毒的检出率为12.59%,小RNA病毒的检出率为3.02%。2.获得了PBoVl、PAstVl的全基因序列,PAstV2和PPicV的部分序列。结论：1.建立了应用非序列依赖单引物PCR (SISPA-PCR)技术筛查腹泻相关病毒的技术平台。研究结果表明DNase-SISPA-PCR是一种发现新病毒的很有效的方法。Genome Walking技术和DNase-SISPA-PCR都能很好的扩增未知序列,将两者结合起来运用,能够很快的获得一株病毒的全基因序列。2.应用上述技术平台在猪的粪便中发现了四株全新的病毒PBoVl、PAstVl、PAstV2和PPicV. 3.生物信息学分析发现PBoVl在进化关系上与人博卡病毒较近,具备一些细小病毒的特征；PAstVl在进化关系上与人星状病毒较近,具备星状病毒的一些特征。
[Abstract]:Background: with the rapid development of biological technology, new viruses and new types of viruses have been discovered. In recent years, many new viruses have been found to be a great threat to human health, resulting in very serious social and public health problems, such as HIV, hepatitis C virus SARSs, avian influenza virus and so on. It is estimated that about 70% of acute upper respiratory tract infections are caused by viral infections, some of which may be caused by unknown viral infections; although diarrhoeal diseases are common and common in humans, the pathogens of 20 to 40% of diarrhoeal cases are still unknown. It is of great significance for the prevention and control of infectious diseases to identify the pathogen. At present, new virus detection techniques have been emerging. The detection of nucleic acid sequences by morphology, antigen-antibody detection, random PCR method, and the application of high-throughput sequencing technology have greatly shortened the process of new virus discovery. Purpose 1. A non-sequence-dependent single-primer PCR SISPA-PCR technique was established to screen diarrhea related virus (CDV). A new diarrhea related virus was found. Methods: 1, routine RT-PCR method was used to screen known viruses associated with common diarrhea, and improved random PCR technique was used to detect and identify viruses. This technique was used to filter out bacteria, cells, etc. Virus particles were enriched by ultracentrifugation, the free nucleic acid was digested by DNA enzyme and RNA enzyme, the interference of free nucleic acid was removed, then the PCR amplification was initiated by random primers containing known fragments, and the PCR products above 500bp were recovered. Transformation cloning, sequencing, bioinformatics analysis of the sequence to determine whether it is a new virus, using chromosomal walking technique to obtain the unknown sequence of the new virus. The 5 'and 3' terminal sequences of the new virus were obtained by RACE method. The sequence characteristics and functions of the new virus were analyzed by bioinformatics. The result is 1: 1. Four new viruses were found in faecal specimens of young pigs under 90 days of animal growth, including a Boca-like virus (PBoV1), two nova viruses (PASTV1 and PAstV2) and a small strain of RNA virus (PPicV1). By bioinformatics analysis, it was found that these viruses had some sequence characteristics of the corresponding viruses. The positive rates of PASTVl and PAstV2 were 1.51%, 1.26%, 12.59% and 3.02% in fecal specimens of healthy young pigs, respectively, and the detection rates of small RNA viruses were 3.02% and 1.26%, respectively. The complete gene sequence of PBoVlPastVl and partial sequence of PPicV and PAstV2 were obtained. Conclusion 1. A technique platform for the screening of diarrhoea-associated virus by using non-sequence-dependent single primer PCR SISPA-PCR was established. The results show that DNase-SISPA-PCR is a very effective method to find new virus. Genome Walking and DNase-SISPA-PCR can amplify the unknown sequence very well. By combining the two methods, the whole gene sequence of a virus can be obtained quickly. Using the above technology platform, four new strains of virus PBoVlSPAstV2 and PPicV2 were found in pig feces. 3. Bioinformatics analysis showed that PBoVl was close to human Boca virus in evolutionary relationship, and had some characteristics of parvovirus. In evolutionary relationship, PAstVl was close to human stellate virus and had some characteristics of stellate virus.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R373

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