基于绵羊肺炎支原体tuf基因的荧光定量PCR方法的建立与应用

发布时间：2018-05-26 19:31

本文选题：绵羊肺炎支原体 + tuf　；参考：《西南民族大学》2015年硕士论文

【摘要】：绵羊肺炎支原体(Mycoplasma ovipneumoniae,Mo)是造成绵羊和山羊慢性非进行性间质性肺炎的一个主要呼吸道病原。目前Mo在世界范围内进行广泛的流行,造成了全球范围内养羊业的经济损失。由于Mo基因序列存在极大的异质性,给PCR检测方法的建立造成较大的困扰。已有普通PCR和荧光定量PCR检测Mo的报道,但仍然存在敏感度不高和通用性不强等缺陷。延伸因子Tu(Elongation factor Tu,EF-Tu)由tuf基因编码,广泛存在于真核生物、原核生物及古细菌中,在蛋白质合成过程中对多肽链的延伸至关重要。tuf基因序列保守,已广泛用于真菌、细菌和支原体的生物分类鉴定和系统进化分析,但目前还未见关于绵羊肺炎支原体tuf基因结构特征和遗传多样性的报道。本研究旨在通过对Mo tuf基因的分子特征分析研究,设计引物,建立通用性更好、敏感度更高的荧光定量PCR检测方法,并用该方法进行Mo感染的病原流行病学调查。1绵羊肺炎支原体tuf基因的分子特性研究根据GenBank中Mo tuf基因编码区(CDS区)序列自行设计引物,扩增Mo参考株Y98和11株Mo临床分离株CDS区,测序和序列分析发现:12株Mo的tuf基因CDS区长1209 bp,编码402个氨基酸,其核苷酸序列及氨基酸序列同源性均为93.3%~100%,G+C含量为39.45%~40.12%;CDS两端保守,中间区域存在较大的变异,其中21~960bp区域碱基突变率仅为8.93%,突变率高达26.9%的位点重点分布于961~1189bp区域内,但是在138~309bp和1047~1196bp区域内保守。有117个单核苷酸突变位点,有41个无义突变,76个有义突变,导致其编码的53个氨基酸发生突变,突变氨基酸主要集中在325~354位点。其中2株分离株含两个TGA密码子,而其他分离株只含一个TGA密码子。TGA是原核生物的终止密码子,而在支原体中却被翻译成色氨酸。基于tuf基因的遗传进化分析发现mo与猪肺炎支原体亲源关系最近,其次为结膜炎支原体,与基于全基因组的遗传进化分析结果完全吻合,但与基于16srrna建立的遗传关系不同,说明motuf基因作为分子靶标进行遗传进化分析优于16srrna。对12株mo的分析发现y98单独聚为一支,而11株分离株全部聚为一支,却又分为两个亚支。有趣的是,来自z地的3株分离株聚在一个亚支,来自j地的4株分离株聚在一个亚支,来自l地的分离株则不规则的分布在这两个亚支中。从本实验现有的样本数量观察发现,来自z地和来自j地羊场的菌株似乎有地域性的倾向,若要证实这个现象还需进一步的扩大样本的数量。2基于绵羊肺炎支原体tuf基因的荧光定量pcr方法的建立由于支原体基因组的g+c含量一般都较低且不同菌株之间存在很大的异质性而限制了分子检测方法的建立。目前mo的分子检测方法有基于16srrna、hsp70建立的pcr方法但是灵敏度不高,易造成假阴性结果的出现。前期研究发现,mo的tuf基因在138~309bp和1047~1196bp区域内与其他感染羊的支原体差异较大,但种内保守,有作为设计检测mo的分子靶点的潜力。因此用primer5软件设计多对特异性引物并筛选到一对最佳引物,扩增150bp大小片段,扩增位点为1047~1196bp区域,以获得最低的ct值分别对反应条件及体系进行优化。结果显示,以mo的核酸为模板通过基于tuf基因的荧光定量pcr扩增后的产物在熔解温度为81.5℃±0.3℃,呈现特异性单峰,无非特异扩增峰及二聚体。该方法在5×109~5×104拷贝/反应线性范围良好,相关系数为0.996,扩增效率为94.1%,标准方程为y=-3.473x+43.411。检测下限为5copies/ul,相当于169ccu/ml。该方法对33株mo分离株能全部检出,并对常见感染羊的支原体及呼吸道易混感的细菌均不检出,由此可见该方法具有良好的敏感性及很高的灵敏度。用50份羊鼻腔棉拭子和134份羊肺组织用本研究建立的荧光定量pcr与基于p113基因的荧光定量pcr进行检出率的比较,发现肺组织检出率一致,但是鼻腔棉拭子多检出2个并经过测序确定为mo;比基于16srrna及hsp70为靶标的普通pcr的鼻腔棉拭子、肺组织检出率分别高12%~42%、9%~42%。该方法不仅可以定性,还能对mo进行精确定量,为mo感染的流行病学调查提供了新方法。3基于tuf基因建立荧光定量PCR方法的应用3.1人工感染样本在肺组织不同病变部位检出率的比较本试验选择6只3月龄Mo抗原抗体双阴性山羊,气管注射5×l09 CCU/只的参考株Y98,山羊在接种后一周左右出现阵发性咳嗽、流泪、流鼻涕等典型症状,病程长,病羊进行性消瘦,于感染后30d扑杀病羊,剖检可见渗出性纤维素胸膜肺炎、胸腔有少量清亮的黄色积液等典型病变,逐只采集每个病羊肺脏病变部位、病健交界部位及无病变健康部位的肺组织,用本研究建立的荧光定量PCR方法进行检测,结果显示,在同一份肺组织中,病健交界处的检出率最高(6/6),健康处次之(4/6),完全病变的部位检出率最低或根本检测不到(1/6)。表明病健交界处是PCR检测Mo的最佳部位。3.2四川、新疆、青海三个地区Mo的病原流行病学调查用本研究建立的检测Mo的荧光定量PCR方法对我国西部四川(n=126)、新疆(n=163)、青海(n=113)三个省份在2013年10月~2014年10月采集的439份表观健康的成年山羊及绵羊的肺组织样本进行检测,结果表明这三个地区的病原阳性率分别为56%、63%、75%,表明这三个地区羊群中Mo的感染严重。
[Abstract]:Mycoplasma ovipneumoniae (Mo) is a major respiratory tract pathogen causing chronic and non progressive interstitial pneumonia in sheep and goats. Currently, Mo is widely prevalent in the world, resulting in the economic loss of sheep industry worldwide. Because of the great heterogeneity in the Mo based sequence, the PCR detection method is given. The establishment of a common PCR and fluorescence quantitative PCR for Mo has been reported, but there are still shortcomings of low sensitivity and poor generality. The extension factor Tu (Elongation factor Tu, EF-Tu) is encoded by the tuf gene and is widely used in eukaryotes, prokaryotes and palaeobacteria in the process of protein synthesis. The extended critical.Tuf gene sequence is conservative and has been widely used in the biological taxonomy and phylogenetic analysis of fungi, bacteria and mycoplasma, but there is no report on the structural characteristics and genetic diversity of the tuf gene of Mycoplasma sheeppneumoniae. The aim of this study is to design primers by analyzing the molecular characteristics of the Mo tuf gene. A better general purpose, more sensitive fluorescence quantitative PCR detection method, and using this method to investigate the pathogenic epidemiology of Mo infection, the molecular characterization of the tuf gene of Mycoplasma sheeppneumoniae.1 was designed according to the sequence of Mo tuf gene coding region (CDS region) sequence in GenBank, and the Mo reference strain Y98 and 11 Mo clinical isolates were amplified and tested. Sequence and sequence analysis found that 12 Mo tuf gene CDS region length 1209 BP, encoding 402 amino acids, its nucleotide sequence and amino acid sequence homology are 93.3%~100%, G+C content is 39.45%~40.12%; CDS at both ends of the conservative, the middle region of the existence of large variation, 21~960bp region base mutation rate is only 8.93%, the mutation rate up to 26.9% of the site The focus is distributed in the 961~1189bp region, but conserved in the 138~309bp and 1047~1196bp regions. There are 117 single nucleotide mutation sites, 41 non sense mutations and 76 sense mutations, resulting in the mutation of the 53 amino acids in its encoding and the main concentration of the mutant amino acids at the 325~354 locus. 2 isolates contain two TGA codons and other fractions. Only one TGA codon.TGA is the terminating codon of the prokaryotes, but it is translated into tryptophan in Mycoplasma. The genetic evolution analysis based on the tuf gene found that the affinity between Mo and Mycoplasma pneumoniae is closest, followed by Mycoplasma conjunctivitis, which is in complete agreement with the whole genome based genetic evolution analysis, but it is based on 16S The genetic relationship established by rRNA is different, indicating that the genetic evolution analysis of the motuf gene as a molecular target is superior to 16srrna. for the analysis of 12 strains of Mo, and it is found that y98 is separated into one branch alone, while the 11 isolates are all together to one branch, but they are divided into two subbranches. It is interesting that the 3 isolates from Z land are clustered in one subbranch and from 4 isolates from J. The isolated strains from L were distributed irregularly in the two subbranches of a Subbranch. From the number of existing samples from this experiment, it was found that the strains from Z and j sheep farms seemed to have a regional tendency to further expand the number of samples based on the tuf gene of Mycoplasma pneumoniae. The establishment of the molecular detection method is limited by the low g+c content of the Mycoplasma genome and the existence of large heterogeneity among the different strains of the Mycoplasma genome. At present, the molecular detection methods of Mo are based on the PCR method based on the HSP70, but the sensitivity is not high, and the false negative results are easily caused by Mo. The study found that the tuf gene of Mo is different from other Mycoplasma in the 138~309bp and 1047~1196bp regions, but it is conservative and has the potential to be designed to detect the molecular targets of Mo. Therefore, a number of pairs of specific primers are designed with primer5 software and a pair of optimal primers are screened and 150bp fragments are amplified and the amplification site is 1047~119 The reaction conditions and systems were optimized in the 6BP region to obtain the lowest CT values. The results showed that the products of the Mo nucleic acid as the template were amplified by the tuf gene based fluorescence quantitative PCR at the melting temperature of 81.5 and 0.3 C, showing a specific single peak, no specific amplification peak and two polymer. The method was in 5 x 109~5 x 104 copy / reaction. The linear range is good, the correlation coefficient is 0.996, the amplification efficiency is 94.1%, the standard equation is y=-3.473x+43.411. detection limit 5copies/ul, the equivalent of 169ccu/ml. method to 33 strains of Mo isolate can be all detected, and the common infected sheep mycoplasma and respiratory tract susceptible bacteria are not detected, thus the method has good sensitivity. Sensitivity and high sensitivity. The detection rate of fluorescence quantitative PCR from 50 sheep's nasal swabs and 134 parts of the lung tissue was compared with that based on p113 based fluorescent quantitative PCR. The detection rate of lung tissue was the same, but 2 of the nasal swabs were detected and Mo was determined through the sequencing, and the target was 16SrRNA and HSP70 as the target. The normal PCR nasal cotton swabs, the detection rate of lung tissue is high 12%~42%, 9%~42%. method not only can be qualitative, but also accurate quantitative Mo, it provides a new method for the epidemiological investigation of Mo infection,.3 based on the tuf gene based fluorescent quantitative PCR method, the detection rate of 3.1 Artificial Infection Samples in different parts of the lung tissue In this experiment, 6 3 month old Mo antigen antibody double negative goats were selected and the trachea was injected with 5 x l09 CCU/ reference strain Y98. The goats appeared paroxysmal cough, tears, runny nose and other typical symptoms about a week after the inoculation, the disease course was long, the disease sheep had sex emaciation, and the 30d culling sheep after infection, and the exudative cellulosic pleuritis and thoracic cavity were found. Thoracic cavity was found to be seen in the thoracic cavity of exudative cellulosic pleural pneumonia and thoracic cavity. With a small number of clear yellow hydrops and other typical lesions, the lung tissues of each sick sheep, the adjacent parts of the disease and the lung tissue without the lesion were detected by the fluorescence quantitative PCR method established in this study. The results showed that in the same lung tissue, the detection rate was the highest (6/6) in the same lung tissue (4/6). The detection rate of the complete lesion was the lowest or the basic detection (1/6). It was indicated that the junction of the disease was the best part of the PCR detection Mo,.3.2 Sichuan, Xinjiang, Qinghai, three regions, the epidemiological investigation of Mo was used to detect Mo by the fluorescence quantitative PCR method for the detection of Mo in Sichuan (n=126), Xinjiang (n=163), Qinghai (n=113) in Western China. The lung tissue samples from 439 healthy adult goats and sheep were detected in October ~2014 in October 2013. The results showed that the positive rates of pathogens in these three regions were 56%, 63% and 75% respectively, indicating that the infection of Mo in the flocks of the three areas was serious.
【学位授予单位】：西南民族大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.62

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