基于HAND系统的腹泻病原体多重PCR检测方法的建立和应用研究

发布时间：2018-05-19 21:38

本文选题：腹泻病原体 + HAND系统　；参考：《南方医科大学》2014年硕士论文

【摘要】：研究背景和目的急性感染性腹泻(简称腹泻)一直是危害人类健康特别是婴幼儿健康的常见病和多发病,是全球范围内影响儿童和成人的重要公共卫生问题。引起腹泻的病原体主要是病毒和细菌,但具体种类繁多,且常常伴有多种腹泻病原体复合感染的情况,在实际检测中往往需要从这一系列的怀疑对象中确定腹泻病原体,为腹泻病的快速诊断带来了极大的困扰。因此,建立一种高通量、高效的腹泻病原体快速检测技术对于诊断、控制和及时治疗腹泻病具有重要意义。然而,国内外对腹泻病原体实验室检测还很大程度依赖传统分离培养和生化鉴定,但是分离培养和生化鉴定敏感性低、费时费力,需要一周左右才能完成,难以达到暴发流行时快速检测的要求,而且有的病原体(如诺如病毒)目前还没有合适的细胞体系和动物模型进行培养。一些免疫学方法(如酶联免疫吸附试验)也被广泛的应用到腹泻病原体的检测,但其敏感性低,假阴性率高,容易造成漏检,而且对于细菌来说需要预先将样本中的目标菌进行浓缩和纯化,短时间内也很难得到结果,难以达到快速检测的要求。随着分子生物学检测方法的发展,基于腹泻病原体的分子生物学诊断技术如PCR、RT-PCR、Real-Time PCR、LAMP等在腹泻病原体的检测以及腹泻病诊断方面发挥着重大作用,克服了以上的缺点,特异性和敏感性较高,已成为目前腹泻病原体快速诊断的常规手段,但这些方法一次只能检测一种病原体,达不到高通量的检测要求,对于复合感染的病例也容易漏检,而且Real-Time PCR仪器费用昂贵,检测成本较高,操作复杂,不利于在基层实验室推广。基因芯片技术虽然具有高通量的特点,但也存在技术成本昂贵、复杂、检测灵敏度低且重复性差等难以解决的问题。多重PCR是一种相对省时、省力方法,可以在一个反应体系中同时检测多个病原体,具有高通量、低成本的特点,但由于多重PCR是多种不同引物混合,容易形成引物间的相互干扰和引物二聚体,使得反应体系扩增效率不均衡,稳定性差,而基于HAND系统多重PCR可以有效的解决这些问题。相同标签辅助-无引物二聚体(Homo-Tag Assisted Non-Dimer,HAND)系统,亦称同源加尾系统,是1997年Browine为了消除PCR中引物二聚体的产生而设计的实验方法。该系统采用两种引物,即加尾引物(3’端为特异性结合序列,5’端为添加的通用尾巴序列)和尾巴引物(序列与加尾引物5’端添加的通用尾巴相同)。其基本原理首先是低浓度的加尾引物与模板特异性结合,扩增形成两端均含有尾巴引物相同序列的初始PCR产物；此时若加尾引物之间形成引物二聚体,由于两端存在互补的序列,引物二聚体的单链会各自形成一个稳定的发夹结构,而该结构难以成为下一循环的扩增模板,从而大大的减少了引物二聚体的形成；其次是待低浓度的加尾引物消耗完,高浓度的尾巴引物以初始PCR产物为模板进行特异性结合,直至扩增完成。所以将HAND系统与多重PCR结合的优点是可以有效地抑制引物二聚体的产生,提高多重PCR体系引物的容纳数量,减小各基因扩增效率的差异,使扩增均衡高效和稳定。因此,本研究第一部分拟构建针对常见腹泻病毒、肠道致病菌、致病性弧菌三组腹泻病原体的基于HAND系统多重PCR检测方法,旨在为腹泻病的诊断寻求广谱高效的快速检测方法。弧菌是一类菌体短小,直或弯曲状的革兰氏阴性细菌,兼性厌氧,广泛分布于自然界,以淡水及海水中最多。弧菌有很多种,其中已经证明对人类具有致病性的弧菌主要有霍乱弧菌(vibrio cholera)、副溶血弧菌(vibrioparahaemolyticus)、创伤弧菌(vibrio vulnificus)、拟态弧菌(vibrio mimicus)和溶藻弧菌(vibrio alginolyticus)等。致病性弧菌广泛存在于温带和热带地区的海水、海底沉积物、浮游生物和水产品中,而因各种致病性弧菌污染水产品导致的问题也越来越多。人类可以通过食用被弧菌污染的水产品而造成急性胃肠炎和败血症等疾病,在影响人类的健康同时还可能因弧菌污染影响水产品养殖业的发展。此外,水产品中的分离的致病性弧菌大多数是非流行株,但它们却是一些弧菌毒素基因的保存库,而这些毒素基因的水平转移可能造成非流行株与流行株的转变。因此,开展致病性弧菌的分子流行病学调查,可为防控致病性弧菌感染引起的疾病提供有效信息。基于此,本研究第二部分拟利用构建的基于HAND系统致病性弧菌多重PCR方法对2012.8—2013.7期间珠海、中山两地供澳水产品中的致病性弧菌进行描述性研究,并了解弧菌的相关毒素基因的分布。方法 1.基于HAND系统腹泻病原体多重PCR检测方法的建立首先选择A组轮状病毒的VP6基因、诺如病毒的RDRP基因、星状病毒的NSP基因、札如病毒的caspid基因作为4种常见腹泻病毒的靶基因,选择志贺氏菌的virA基因、沙门氏菌的invA基因、小肠结肠炎耶尔森菌的ail基因、金黄色葡萄球菌的nuc基因、大肠杆菌0157：H7的rfbE基因作为5种肠道致病菌的靶基因,选择创伤弧菌的vvhA基因、霍乱弧菌的ompW基因、副溶血弧菌的toxR基因、拟态弧菌的VMH基因、溶藻弧菌的gyrB基因作为5种致病性弧菌的靶基因,根据靶基因的保守序列设计特异性引物并在5’端加上尾巴序列,通过优化加尾引物和尾巴引物的浓度、Mg2+浓度、DMSO浓度、循环参数构建三组基于HAND系统腹泻病原体多重PCR反应体系,再分析其稳定性、特异性和检测下限值,并运用于模拟的临床粪便样本检测,盲法试验评价方法的实用性。 2.供澳水产品中致病性弧菌分子流行病学调查 2012.8--2013.7期间,采用随机抽样的方法,每月抽取大约125份珠海、中山两地的供澳水产品样,利用建立的基于HAND系统致病性弧菌多重PCR方法进行初筛,再将初筛阳性样品在TCBS培养上分离培养和生化鉴定进行验证,所得结果进行致病性弧菌的季节性、地域性和水产品种类分析；对分离到的阳性菌株利用PCR方法检测ctxA、zot、ace、tcpA、tdh、trh?tlh、viuB8个弧菌毒素基因。结果 1.基于HAND系统腹泻病原体多重PCR检测方法的建立 (1)成功构建基于HAND系统腹泻病毒多重RT-PCR检测方法。特异性分析显示四种腹泻病毒间无交叉反应,敏感性分析显示轮状病毒、诺如病毒、星状病毒和札如病毒的检测下限分别达到48、9.6、1.92和48pg；盲法试验评价结果显示符合率100%； (2)成功构建基于HAND系统肠道致病菌多重PCR检测方法。特异性分析显示五种肠道致病菌无交叉反应,特异性100%,敏感性分析显示志贺氏菌、大肠杆菌0157：H7、小肠结肠炎耶尔森菌三种致病菌的检测下限值均为100cfu/ml,金黄色葡萄球菌的检测下限值为1000cfu/ml,沙门氏菌的检测下限值为10cfu/ml；盲法试验评价结果显示符合率100%； (3)成功构建基于HAND系统致病性弧菌多重PCR检测方法。特异性分析显示五种致病性弧菌无交叉反应,特异性100%,敏感性分析显示创伤弧菌、霍乱弧菌、副溶血弧菌、溶藻弧菌四种致病性弧菌的检测下限值均为100cfu/ml,拟态弧菌的检测下限值为10cfu/ml；盲法试验评价结果显示符合率100%。 2.供澳水产品中致病性弧菌分子流行病学特征 (1)基本情况：共收集1510份供澳水产品样本,检出898份阳性,弧菌阳性率为59.5%；复合感染样本563份,复合感染率37.3%,其中副溶血弧菌和溶藻弧菌复合感染样本293份,占52%;具体霍乱弧菌、副溶血弧菌、创伤弧菌、拟态弧菌和溶藻弧菌的阳性数分别为：329份(21.8%)、535份(35.4%)、56份(3.7%)、108份(7.2%)、611份(40.5%),其中霍乱弧菌中仅有2株O1群小川型,5株O1群稻叶型,其余为非O1/非0139群； (2)三间分布：在一年中的5-11月份致病性弧菌总阳性率较高,而1月份和12月份的致病性弧菌总阳性率较低,一年中每月弧菌的阳性率差异有统计学意义(χ2=198.26,P=0.000),供澳水产品中致病性弧菌主要在夏秋季流行；珠海地区总弧菌阳性率69.5%高于中山的51.9%,两地总弧菌阳性率差异有统计学意义(χ2=47.42,P=0.000),珠海供澳水产品中弧菌的污染现象比中山地区严重；贝类的总弧菌阳性率70.6%高于鱼类的54.4%,两类水产品总弧菌阳性率差异有统计学意义(χ2=36.65,P=0.000),贝类中致病性弧菌的污染现象比鱼类严重； (3)毒素基因分布：329株霍乱弧菌中,ctxA均为阴性,1株ace+、zot+、tcpA+14株ace+、zot+、tcpA-,6株ace-、zot+、tcpA-,5株ace-、zot-、tcpA+；535株副溶血弧菌中,tlh均阳性,7株tdh阳性,检出率1.3%；9株trh阳性,检出率1.6%；56株创伤弧菌中,12株viuB阳性,检出率21.4%；108株拟态弧菌中,ctxA、ace、tcpA均阴性,2株zot阳性；611株溶藻弧菌中,146株tlh阳性,检出率23.9%,tdh、trh均为阴性。结论 1.所建立的三组基于HAND系统腹泻病原体多重PCR检测方法,具有快速、稳定、特异、灵敏和低成本的特点,非常适用于基层医学实验室； 2.供澳水产品中致病性弧菌污染的现象比较严重,且普遍存在两种或三种致病性弧菌的多重污染,要加大致病性弧菌的监测力度,以防致病性弧菌的感染和流行； 3.环境中分离的致病性弧菌大多数是非流行株的,却是已知毒素基因的重要保存库。
[Abstract]:Background and purpose of research
Acute infectious diarrhea (abbreviated diarrhoea) is a common and frequently occurring disease that endangers human health, especially infant health. It is an important public health problem that affects children and adults worldwide. The pathogens causing diarrhoea are mainly viruses and bacteria, but the specific species are complex and often accompanied by multiple diarrhea pathogens compound infection. In actual testing, it is often necessary to identify diarrhoea pathogens from this series of sceptics and cause great trouble for the rapid diagnosis of diarrhoea. Therefore, the establishment of a high throughput and efficient detection technique for diarrhea pathogens is of great significance for the diagnosis, control and treatment of diarrhoea. Laboratory tests for diarrhoea pathogens are also largely dependent on traditional isolation and biochemical identification, but the isolation, culture and biochemical identification are low and time-consuming. It takes about a week to complete, and it is difficult to meet the requirements of rapid detection when the outbreak is outburst, and some pathogens (such as norovirus) have no proper cell system at present. Some immunological methods, such as enzyme linked immunosorbent assay (ELISA), are also widely used in the detection of diarrhoea pathogens, but their sensitivity is low, the false negative rate is high, and it is easy to cause leakage, and for bacteria, it is necessary to concentrate and purify the target bacteria in advance, and it is difficult to get the result in a short time. With the development of molecular biological detection methods, molecular biological diagnostic techniques based on diarrhoea, such as PCR, RT-PCR, Real-Time PCR and LAMP, have played a major role in the detection of diarrhoea pathogens and the diagnosis of diarrhoea, which have overcome the above shortcomings, and have become more specific and sensitive. It is a routine method for the rapid diagnosis of diarrhoea pathogens, but these methods can only detect one kind of pathogen at a time, can not meet the requirements of high flux detection, and it is easy to leak detection for the cases of complex infection, and the Real-Time PCR instrument is expensive, the cost is high, the operation is complex, and it is not conducive to the promotion of the basic laboratory. Although the operation is characterized by high throughput, there are also difficult problems such as high cost, complexity, low sensitivity and poor repeatability. Multiple PCR is a relatively time-saving and labor-saving method to detect multiple pathogens at the same time in a reaction system, with high flux and low cost, but multiple PCR is different With the same primers, the interaction between primers and primer two polymer can be easily formed, which makes the efficiency of the reaction system uneven and the stability is poor, and the multiple PCR based on HAND system can effectively solve these problems.
The same label auxiliary Homo-Tag Assisted Non-Dimer (HAND) system, also known as the homologous tailing system, is an experimental method designed by Browine in 1997 to eliminate the production of primer two polymer in PCR. The system uses two primers, namely, the tail primer (3 'end as a specific binding sequence, and the 5 "end as a general tail order added. Column) and tail primers (the same sequence is the same as the general tail added to the 5 'end of the tail primer). The basic principle is that a low concentration of tail primers and a template specific binding are first amplified to form an initial PCR product at both ends containing the same sequence of the tail primers; at this time, a primer two polymer is formed between the tailed primers, because the two ends are complementary to each other. Sequence, the single strand of primer two polymer will each form a stable hairpin structure, and the structure is difficult to be an amplification template for the next cycle, thus greatly reducing the formation of primer two polymer; secondly, the tail primer for low concentration is consumed, and the high concentration tail primers specifically combine with the initial PCR product as a template. The combination of HAND system and multiple PCR can effectively inhibit the production of primer two polymer, increase the number of primers in multiple PCR system, reduce the difference of gene amplification efficiency and make the amplification balanced and efficient and stable. Therefore, the first part of this study is to construct the common diarrhea virus and intestinal pathogenic bacteria. The multiplex PCR detection method based on HAND system for diarrheal pathogens in three groups of pathogenic vibrio is aimed at finding a broad spectrum and efficient method for the diagnosis of diarrheal diseases.
Vibrio is a group of Gram-negative bacteria with short, straight or curved form of gram negative bacteria. It is widely distributed in nature and is widely distributed in the natural world. There are many Vibrio species in fresh water and sea water. Among them, Vibrio has been proved to be pathogenic Vibrio (Vibrio cholera), Vibrio parahaemolyticus (vibrioparahaemolyticus), Vibrio vulnificus (vibr). IO vulnificus), Vibrio mimetic (Vibrio mimicus) and Vibrio alginolyticus (Vibrio alginolyticus). Pathogenic vibrio is widely found in temperate and tropical seawater, seafloor sediments, plankton and aquatic products, and more and more problems are caused by various pathogenic Vibrio contaminated aquatic products. Human can be contaminated by Vibrio. Diseases such as acute gastroenteritis and septicemia caused by dyed aquatic products may affect human health and may also affect the development of aquatic products in aquatic products. In addition, the isolated Vibrio isolates in aquatic products are mostly non epidemic strains, but they are the storage of some Vibrio toxin genes, and the water of these toxin genes. Therefore, the molecular epidemiological investigation of pathogenic Vibrio can provide effective information for preventing and controlling the disease caused by Vibrio Vibrio infection. Based on this, the second part of this study is to use the constructed multiple PCR method of pathogenic Vibrio based on HAND system for the period of 2012.8 to 2013.7. A descriptive study of pathogenic Vibrio was carried out in Zhuhai and Zhongshan, and the distribution of Vibrio related toxin genes was also studied.
Method
1. establishment of multiplex PCR assay for diarrhea pathogens based on HAND system
First, select the VP6 gene of A rotavirus, the RDRP gene of norovirus, the NSP gene of the stellate virus, the caspid gene of the zzavirus as the target gene for 4 common diarrhea viruses, select the virA gene of Shigella, the invA gene of Salmonella, the ail gene of Jerson bacteria of the enterocolitis, the NUC gene of Staphylococcus aureus, and the large intestine The rfbE gene of bacilli 0157:H7 was used as the target gene for 5 kinds of intestinal pathogenic bacteria, which chose the vvhA gene of Vibrio vulnificus, the ompW gene of Vibrio cholerae, the toxR gene of Vibrio parahaemolyticus, the VMH gene of Vibrio mimici, the gyrB gene of Vibrio alginolyticus as the target gene of 5 pathogenic Vibrio, and the specific primers were designed according to the conservative sequence of the target gene and 5 By adding tail sequence, three groups of multiple PCR reaction systems based on HAND system diarrhea pathogen were constructed by optimizing the concentration of tail and tail primers, concentration of Mg2+, DMSO concentration, and cycle parameters, and then the stability, specificity and detection limit were analyzed, and used for simulated clinical fecal sample detection, and the method of blind test evaluation was real. Use sex.
2. molecular epidemiology of pathogenic Vibrio in Australian aquatic products
During the period of 2012.8--2013.7, a random sampling method was used to extract about 125 seafood samples from Zhuhai and Zhongshan each month. Using the established multiple PCR method based on HAND system pathogenic Vibrio, the initial sifting samples were screened in TCBS culture and verified by biochemical identification. The results were pathogeny arc. The seasonal, regional and aquatic products of the bacteria were analyzed, and the isolates were detected by PCR method to detect ctxA, zot, ACE, tcpA, TDH, TRH? TLH, and viuB8 Vibrio toxin genes.
Result
1. establishment of multiplex PCR assay for diarrhea pathogens based on HAND system
(1) successful construction of multiple RT-PCR detection methods based on HAND system diarrhea virus. Specific analysis showed no cross reaction between four kinds of diarrhea viruses. Sensitivity analysis showed that rotavirus, norovirus, stellate virus and zzavirus had a lower limit of 48,9.6,1.92 and 48pg, and the result of blind test showed that the coincidence rate was 100%.
(2) successful construction of multiple PCR detection methods for intestinal pathogenic bacteria based on HAND system. Specific analysis showed that five kinds of intestinal pathogenic bacteria had no cross reaction, specificity 100%. Sensitivity analysis showed that Shigella, Escherichia coli 0157:H7, and three pathogenic bacteria of Jerson bacteria of enterocolitis were both 100cfu/ml and Staphylococcus aureus. The lower limit is 1000cfu/ml, the lower limit of Salmonella detection is 10cfu/ml, and the result of blind test shows that the coincidence rate is 100%.
(3) successful construction of multiple PCR detection methods based on HAND pathogenic Vibrio. Specific analysis showed that five pathogenic Vibrio without cross reaction, specificity 100%, sensitivity analysis showed that the detection limit of Vibrio vulnificus, Vibrio cholerae, Vibrio parahaemolyticus and Vibrio alginolyticus were 100cfu/ml, and the detection limit of Vibrio mimicus The value is 10cfu/ml; the blind test results show that the coincidence rate is 100%..
2. molecular epidemiology of pathogenic Vibrio in Australian aquatic products
(1) the basic situation: a total of 1510 samples of Australian aquatic products were collected, 898 positive were detected, the positive rate of Vibrio was 59.5%, 563 samples of compound infection and 37.3% compound infection rate, including 293 samples of Vibrio parahaemolyticus and Vibrio alginolyticus, 52%, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio Vibrio and Vibrio alginolyticus The numbers were 329 (21.8%), 535 (35.4%), 56 (3.7%), 108 (7.2%) and 611 (40.5%), of which only 2 O1 group of Vibrio cholerae, 5 O1 group rice leaf type, and the other non O1/ non 0139 group;
(2) three distribution: the total positive rate of Vibrio virulence was higher in the month of 5-11, and the total positive rate of pathogenic Vibrio in January and December was lower, and the positive rate of Vibrio was statistically significant in one year (x 2=198.26, P=0.000). The pathogenic Vibrio in Australian aquatic products was mainly in summer and autumn, and the total Vibrio Yang in Zhuhai area The rate of sex was 69.5% higher than that of 51.9% in Zhongshan. The positive rate of Vibrio was statistically significant (x 2=47.42, P=0.000). The pollution of Vibrio in Zhuhai aquatic products was more serious than that in Zhongshan; the positive rate of the total Vibrio in shellfish was 70.6% higher than that of fish, and the positive rate of total arc bacteria in the two types of aquatic products was statistically significant (x 2=36.65, P=0.000). The contamination of pathogenic Vibrio in shellfish is more serious than that in fish.
(3) distribution of toxin gene: among 329 strains of Vibrio cholerae, ctxA was negative, 1 strains of ace+, zot+, tcpA+14 strain ace+, zot+, tcpA-, 6 ace-, zot+, tcpA-, 5 strains of ace-, 535 strains of Vibrio parahaemolyticus, 7 positive, detection rate 1.3%, 9 strains positive, 1.6%, 12, 12, 21.4%; 21.4%; 21.4%; 12 Among the 8 Vibrio mimicus, ctxA, ACE, tcpA were all negative, 2 zot positive, 611 strains of Vibrio alginolyticus, 146 TLH positive, the detection rate was 23.9%, TDH and TRH were negative.
conclusion
1. methods of multiple PCR detection based on HAND system for diarrhea pathogens were established, which have the characteristics of fast, stable, specific, sensitive and low cost, and are very suitable for the basic medical laboratory.
2. the contamination of pathogenic Vibrio in Australian aquatic products is serious, and the multiple contamination of two or three pathogenic vibrio is widespread, and the monitoring of Vibrio vulnificus should be added in order to prevent the infection and epidemic of Vibrio.
3. most pathogenic Vibrio isolated from the environment are non epidemic strains, but are important repositories for known toxin genes.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R512.5

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