一种基于HMBS FRET-qPCR的哺乳动物DNA内源性内部控制系统的建立及应用

发布时间：2018-05-13 16:17

本文选题：哺乳动物DNA + 内源性内部控制系统　；参考：《扬州大学》2015年硕士论文

【摘要】：聚合酶链式反应(polymerase chain reaction, PCR)作为一种特异灵敏的分子诊断工具,被广泛应用于生命科学研究的各个领域。然而从样品采集、运输、保存、核酸提取到PCR检测的整个过程中都可能存在一些因素,影响被检核酸的质量和水平,造成检测结果的假阳性或假阴性。为提高PCR检测结果的可信度,非常有必要建立一种内源性内部控制(endogenous internal control, EIC)系统,实现从样品采集到PCR检测的全程质量监控。在本论文的研究一,我们设计了一套针对哺乳动物羟甲基胆色烷合成酶(hydroxymethylbilane synthase, HMB S)基因的荧光共振能量转移(fluorescence resonance energy transfer, FRET).定量PCR (quantitative PCR, qPCR)的EIC系统。在研究二及研究三,应用建立的EIC作为质量控制系统,保证分子检测结果的准确度和可信性,从而开展对哥斯达黎加和尼加拉瓜的七种虫媒疾病的分子流行病学调查。本研究建立的EIC系统可扩增人和12种哺乳动物(犬、猫、牛、小鼠、猪、驴、马、猴子、绵羊、山羊、狮子、猎豹)、小鼠的11种组织或脏器(心、肝、脑、脾、肺、脂肪、肾、肌肉、毛发、骨、皮肤)的DNA。此方法也可方便地用于蚊子的吸血体积及对应血液的宿主来源的判定。蚊子样本PCR扩增产物的测序结果发现,本研究采集的蚊子吸取了6种哺乳动物的血液。本系统的灵敏度高(0.05 ng组织或0.5 nl血液/反应体系)特异性好,并可区别不同的哺乳动物物种,因此可成为哺乳动物核酸PCR检测的理想EIC系统,同时也是研究吸血节肢动物的有用工具。虫媒疾病为热带地区犬发病和死亡的重要原因,但是关于这类疾病在中美洲地区的流行信息却十分有限。本研究应用了FRET-qPCR检测犬的全血核酸样本,调查七种虫媒疾病在尼加拉瓜和哥斯达黎加犬中的流行情况。在尼加拉瓜,犬全血核酸样本(n=39)携带有5种病原体：猫立克次体(阳性率5%)、巴贝斯虫(26%)、犬肝簇虫(51%)、扁平无浆体(13%)、犬埃立克次体(56%),而贝纳柯克斯体及犬心丝虫的检测结果为阴性。其中共有30只犬(80%)检测结果为阳性,12只(31%)携带一种病原体,11只(28%)携带两种病原体,3只(8%)携带三种病原体,4只(10%)携带四种病原体。在哥斯达黎加,犬全血核酸样本(n=40)中没有检测到猫立克次体及贝纳柯克斯体的核酸,但另外5种虫媒疾病病原体的检测结果分别为：犬心丝虫(阳性率22.5%)、犬肝簇虫(37.5%)、巴贝斯虫(25%)、扁平无浆体(7.5%)、犬埃立克次体(50%)。9只犬(22.5%)的全血核酸中没有检测到虫媒疾病病原体,14只(35%)感染有一种病原体,11只(27.5%)感染有两种病原体,4只(10%)感染有三种病原体,1只(2.5%)感染有五种病原体。本研究成功地建立了一套基于HMBS基因FRET-qPCR的EIC系统,该系统灵敏度高、特异性强,可实现PCR检测过程的全程监控,并适用于所有哺乳动物DNA样本。进行犬虫媒疾病分子流行病学调查时,本研究首次报道了在中美洲地区吉氏巴贝斯虫的存在。调查结果也表明犬虫媒疾病在尼加拉瓜、哥斯达黎加的犬中的流行十分普遍,且有些疾病可以感染人类。
[Abstract]:Polymerase chain reaction (PCR), as a specific and sensitive molecular diagnostic tool, is widely used in all fields of life science research. However, the whole process of sample collection, transportation, preservation, nucleic acid extraction and PCR detection may be stored in some factors, affecting the quality and level of the detected nucleic acid. In order to improve the reliability of the results of PCR detection, it is very necessary to establish an endogenous internal control (endogenous internal control, EIC) system to realize the whole process quality monitoring from sample collection to PCR detection. In this paper, we designed a set of mammal hydroxymethyl bile. The fluorescence resonance energy transfer (fluorescence resonance energy transfer, FRET) of the hydroxymethylbilane synthase (HMB S) gene. In study two and study three, the application established as a quality control system to ensure the accuracy and credibility of molecular detection results, A molecular epidemiological survey of seven insect borne diseases in Costa Rica and Nicaragua was conducted. The EIC system established in this study could amplify human and 12 mammals (dogs, cats, cattle, mice, pigs, donkeys, horses, monkeys, sheep, goats, lions, cheetahs), and 11 tissues or organs of mice (heart, liver, brain, spleen, lung, fat, kidney, muscle, hair, bone, The DNA. method of skin can also be conveniently used for the bloodsucking volume of mosquitoes and the determination of the source of the host of the blood. The sequencing of the PCR amplification products of the mosquito sample found that the mosquitoes collected in this study have suck up the blood of 6 mammals. The sensitivity of the system is high (0.05 ng or 0.5 NL blood / reaction system), and the specificity is good and can be used. Different mammal species can be used as an ideal EIC system for the detection of mammalian nucleic acid PCR, and it is also a useful tool for studying blood sucking arthropods. Insect vector disease is an important cause of the disease and death of dogs in the tropics, but the epidemic information about this kind of disease in the Central American region is very limited. FRET-qPCR tested the whole blood nucleic acid samples of the dog and investigated the prevalence of seven insect borne diseases in Nicaragua and Costa Rica. In Nicaragua, the dog whole blood nucleic acid samples (n=39) carried 5 pathogens: Rickettsia cat (positive rate 5%), Babes (26%), canine liver tutua (51%), flat no pulp (13%), and dog Erik subbody (56%). There were 30 dogs (80%) tested positive, 12 (31%) carrying one pathogen, 11 (28%) carrying two pathogens, 3 (8%) carrying three pathogens and 4 (10%) carrying four kinds of Mycoplasma. In Costa Rica, n=40 (n=40) was not detected in Costa Rica, and the dog whole blood nucleic acid sample (n=40) was not detected. The nucleic acid of Rickettsia cat and the Bernard Kirk body, but the other 5 kinds of insect vector disease pathogens were detected respectively: dog heart filariasis (positive rate 22.5%), canine liver tutua (37.5%), Babes worm (25%), flat no plasma (7.5%), dog Erik subbody (50%).9 only dog (22.5%) of the whole blood nucleic acid did not detect the pathogen of insect vector disease, 14 (35%) Infection has a pathogen, 11 (27.5%) infected two pathogens, 4 (10%) infected with three pathogens, 1 (2.5%) infected with five pathogens. This study successfully established a set of EIC based on the HMBS gene FRET-qPCR system, the system has high sensitivity and specificity, and can realize the whole process monitoring of the PCR detection process, and it is suitable for all nursing. DNA samples of mammary animals. For the first time in the molecular epidemiology investigation of canine vector diseases, this study was the first to report the presence of the jir BABEI in Central America. The results also showed that the prevalence of canine vector diseases in Nicaragua and Costa Rica was very common, and some diseases could infect humans.

【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：Q503;S855

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