本文選題：去游離核酸的隨機(jī)PCR + 新病毒　； 參考：《中國(guó)疾病預(yù)防控制中心》2010年碩士論文

【摘要】：背景：隨著生物學(xué)技術(shù)的快速發(fā)展,新病毒及一些熟知病毒的新型別被不斷發(fā)現(xiàn)。近年來發(fā)現(xiàn)的許多新發(fā)病毒均對(duì)人類健康有著極大的威脅,造成了非常嚴(yán)重的社會(huì)公共衛(wèi)生問題,如艾滋病毒、丙型肝炎病毒、SARS、禽流感病毒等。據(jù)估計(jì),在急性上呼吸道感染中約70%為病毒感染引起,其中有部分可能為未知病毒感染引起；盡管腹瀉病為人類常見和多發(fā)的疾病,但仍舊有20-40%的腹瀉病例的病原不清楚。明確病原,對(duì)傳染病的預(yù)防控制意義重大。目前,用于新病毒發(fā)現(xiàn)的技術(shù)也不斷涌現(xiàn),由形態(tài)學(xué)、抗原抗體檢測(cè)到核酸序列的測(cè)定,由隨機(jī)PCR方法,到高通量測(cè)序技術(shù)的運(yùn)用等,都大大縮短了新病毒發(fā)現(xiàn)的進(jìn)程。 目的：1.建立應(yīng)用非序列依賴單引物PCR(SISPA-PCR)技術(shù)篩查腹瀉相關(guān)病毒的技術(shù)平臺(tái)：2、發(fā)現(xiàn)與腹瀉相關(guān)的新病毒。 方法：1、使用常規(guī)的RT-PCR方法篩查常見腹瀉相關(guān)的已知病毒；2、應(yīng)用改進(jìn)的隨機(jī)PCR技術(shù)(DNase-SISPA-PCR)發(fā)現(xiàn)和鑒定病毒：該技術(shù)是通過過濾的方法去除標(biāo)本中的細(xì)菌、細(xì)胞等,通過超速離心富集病毒顆粒,使用DNA酶和RNA酶消化游離的核酸,去除游離核酸的干擾,然后使用含有已知片斷的隨機(jī)引物引發(fā)PCR擴(kuò)增,并且對(duì)500bp以上的PCR產(chǎn)物進(jìn)行回收,轉(zhuǎn)化克隆,測(cè)定序列,對(duì)序列進(jìn)行生物信息學(xué)分析,確定是否為新病毒；3、使用染色體步移技術(shù)(Genome Walking)獲得新病毒的未知序列；使用RACE方法獲得新病毒5’和3’末端序列；4、應(yīng)用生物信息學(xué)分析新病毒序列特征和功能。 結(jié)果：1.在動(dòng)物(90天以下的幼豬)糞便標(biāo)本中發(fā)現(xiàn)了4株新病毒,包括一株博卡樣病毒(PBoV1),兩株新星狀病毒(PAstV1和PAstV2)和一株小RNA病毒(PPicV)。經(jīng)過生物信息學(xué)分析,發(fā)現(xiàn)這幾株病毒具有相應(yīng)病毒的一些序列特征,在健康幼豬糞便標(biāo)本中,PAstVl檢出率為1.51%和PAstV2的檢出率為1.26%,博卡樣病毒的檢出率為12.59%,小RNA病毒的檢出率為3.02%。2.獲得了PBoVl、PAstVl的全基因序列,PAstV2和PPicV的部分序列。 結(jié)論：1.建立了應(yīng)用非序列依賴單引物PCR (SISPA-PCR)技術(shù)篩查腹瀉相關(guān)病毒的技術(shù)平臺(tái)。研究結(jié)果表明DNase-SISPA-PCR是一種發(fā)現(xiàn)新病毒的很有效的方法。Genome Walking技術(shù)和DNase-SISPA-PCR都能很好的擴(kuò)增未知序列,將兩者結(jié)合起來運(yùn)用,能夠很快的獲得一株病毒的全基因序列。2.應(yīng)用上述技術(shù)平臺(tái)在豬的糞便中發(fā)現(xiàn)了四株全新的病毒PBoVl、PAstVl、PAstV2和PPicV. 3.生物信息學(xué)分析發(fā)現(xiàn)PBoVl在進(jìn)化關(guān)系上與人博卡病毒較近,具備一些細(xì)小病毒的特征；PAstVl在進(jìn)化關(guān)系上與人星狀病毒較近,具備星狀病毒的一些特征。
[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.
【學(xué)位授予單位】：中國(guó)疾病預(yù)防控制中心
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R373

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