本文選題：腺病毒 + 分型��； 參考：《河北北方學(xué)院》2017年碩士論文

【摘要】：人腺病毒(Human adenovirus,HAdV)最早發(fā)現(xiàn)于人體扁桃體組織中,是一種無包膜立體對稱的二十面體雙鏈DNA病毒顆粒。腺病毒的致病譜與血清型有關(guān),不同血清型可引起不同疾病,所以我們需要能夠確定其型別的檢測方法。根據(jù)文獻(xiàn)報道,目前腺病毒有68個型別,國際病毒學(xué)分類委員會(The International Committee on Taxonomy of Viruses,ICTV)將1-52型劃分為7個亞屬(A-G),B亞屬主要包括3、7、14、11和55型,可引起呼吸道疾病。近年來,腺病毒引起的呼吸道感染疫情時有暴發(fā),疫情發(fā)展快、感染性強(qiáng),病情嚴(yán)重者可危及生命,其早期檢測和分型也成為醫(yī)務(wù)工作者及研究者們面臨的重要問題。目前的檢測和分型方法中,病毒分離法耗時太長,免疫學(xué)方法并不能區(qū)分具體的血清型別。PCR擴(kuò)增法通過擴(kuò)增特異性基因片段,結(jié)合測序法確定其型別,但測序法靈敏度不足,主要用于實(shí)驗(yàn)室研究和新型腺病毒的確定。環(huán)介導(dǎo)等溫擴(kuò)增雖靈敏度高,特異性好,但其引物設(shè)計要求較高且易污染,不能實(shí)現(xiàn)高通量的要求。因此,迫切需要建立一種快速、簡便的腺病毒分型檢測方法。本文旨在建立一種腺病毒分型化學(xué)發(fā)光基因芯片檢測方法,可對腺病毒3、7、14、11和55型準(zhǔn)確分型,與病毒分離等方法相比,可縮短檢測時間,提高檢測效率,為腺病毒分型檢測提供一種新的方法。本實(shí)驗(yàn)通過查閱文獻(xiàn)確定腺病毒的靶基因,從GenBank中下載特異性基因序列,利用Primer5.0、Clustalx1.8.msw、Alignmen等生物學(xué)軟件設(shè)計引物和探針,對引物和探針進(jìn)行合成和篩選,制備寡核苷酸基因芯片。運(yùn)用多重不對稱PCR法擴(kuò)增特異性基因片段,將擴(kuò)增產(chǎn)物與基因芯片上的特異性探針雜交,經(jīng)過洗滌、化學(xué)發(fā)光顯色后,進(jìn)行結(jié)果分析。在優(yōu)化的多重PCR體系、雜交條件和化學(xué)發(fā)光檢測條件下,對芯片的特異性、靈敏性和重復(fù)性進(jìn)行評價,通過檢測38例臨床樣本,考核基因芯片的實(shí)用性。本研究選取腺病毒的靶基因?yàn)閔exon基因,共篩選出4對引物和5條探針,3型、7型和14型各一對引物、11和55型腺病毒共用1對引物,5種腺病毒各對應(yīng)1條探針。完成了5種腺病毒質(zhì)粒參考品的構(gòu)建,用質(zhì)粒參考品對芯片的特異性、靈敏度和重復(fù)性進(jìn)行考核,以質(zhì)粒為模板確定五重PCR體系進(jìn)行PCR擴(kuò)增,擴(kuò)增產(chǎn)物與基因芯片上的探針雜交后,特異性良好,相互間無交叉信號,該基因芯片的最低檢測限為3×103copies/μl,同一芯片內(nèi)和不同芯片間的重復(fù)性變異系數(shù)CV值均小于15%。38例臨床樣本的分型結(jié)果與測序法結(jié)果基本一致(37/38)。綜上,本文建立了一種腺病毒化學(xué)發(fā)光基因芯片檢測方法,該方法可同時對3、7、11、14和55型腺病毒準(zhǔn)確分型檢測,該芯片在特異性、靈敏度和重復(fù)性方面均達(dá)到預(yù)期的性能要求,為腺病毒的臨床分型診斷和流行病學(xué)調(diào)查提供了新的高通量檢測方法。
[Abstract]:Human adenovirus (HAdV) was first found in human tonsils, which is an icosahedral double-stranded DNA virus particle with no capsule. The pathogenic spectrum of adenovirus is related to serotype. Different serotypes can cause different diseases, so we need to be able to determine the detection method of adenovirus type. According to the literature, there are 68 types of adenovirus. The International Committee of Classification of Virology (the International Committee on Taxonomy of virus ICTV) divides 1-52 into 7 subgenera A-GGUB, which mainly include 3C7A1411 and 55, which can cause respiratory diseases. In recent years, the respiratory tract infection caused by adenovirus sometimes breaks out, the epidemic develops rapidly, the infection is strong, the serious disease can endanger the life, its early detection and typing also become the important problem that the medical workers and researchers are faced with. In the current detection and typing methods, the virus separation method takes too long, the immunological method can not distinguish the specific serum type. PCR amplification method can amplify the specific gene fragment, combined with sequencing method to determine its type, but the sensitivity of sequencing method is not enough. It is mainly used for laboratory research and identification of new adenovirus. Although the sensitivity and specificity of the loop mediated isothermal amplification were high, the primer design requirements were high and easy to pollute, which could not meet the requirement of high throughput. Therefore, it is urgent to establish a rapid and simple method for the detection of adenovirus. The aim of this paper is to establish a chemiluminescence gene chip detection method for adenovirus-typing, which can be used to accurately type adenovirus type 3H7N14H11 and 55. Compared with the method of virus isolation, it can shorten the detection time and improve the detection efficiency. To provide a new method for the detection of adenovirus typing. In this experiment, the target gene of adenovirus was identified by consulting literature, and the specific gene sequence was downloaded from GenBank. Primers and probes were designed by Primer5.0G Clustalx1.8.mswswAlignmen and other biological software. The primers and probes were synthesized and screened, and oligonucleotide gene chips were prepared. The specific gene fragments were amplified by multiple asymmetric PCR. The products were hybridized with the specific probes on the gene chip. After washing and chemiluminescence, the results were analyzed. The specificity, sensitivity and repeatability of the microarray were evaluated under the optimized multiplex PCR system, hybridization conditions and chemiluminescence detection conditions. The practicability of the gene chip was evaluated by detecting 38 clinical samples. In this study, the target gene of adenovirus was selected as hexon gene. A total of 4 pairs of primers and 5 pairs of probes, one pair of primers, and five pairs of adenovirus probes, were selected in this study. Five kinds of adenovirus plasmid reference materials were constructed. The specificity, sensitivity and repeatability of the microarray were evaluated with the plasmid reference material. The plasmid template was used to determine the five PCR system for PCR amplification. The amplification products were hybridized with the probes on the gene chip, the specificity of the products was good, and there was no cross signal between them. The minimum detection limit of this gene chip is 3 脳 103copies/ 渭 l. The CV value of repeatability within the same chip and between different chips is less than 15.38 clinical samples. In summary, a chemiluminescence gene chip method for detection of adenovirus chemiluminescence gene chip was developed. The method can be used to detect adenovirus type 71114 and 55 at the same time. The chip achieves the expected performance requirements in terms of specificity, sensitivity and repeatability. It provides a new high-throughput detection method for clinical typing diagnosis and epidemiological investigation of adenovirus.
【學(xué)位授予單位】：河北北方學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R440;R511

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