本文選題：綿羊肺炎支原體 + tuf��； 參考：《西南民族大學(xué)》2015年碩士論文

【摘要】：綿羊肺炎支原體(Mycoplasma ovipneumoniae,Mo)是造成綿羊和山羊慢性非進(jìn)行性間質(zhì)性肺炎的一個(gè)主要呼吸道病原。目前Mo在世界范圍內(nèi)進(jìn)行廣泛的流行,造成了全球范圍內(nèi)養(yǎng)羊業(yè)的經(jīng)濟(jì)損失。由于Mo基因序列存在極大的異質(zhì)性,給PCR檢測(cè)方法的建立造成較大的困擾。已有普通PCR和熒光定量PCR檢測(cè)Mo的報(bào)道,但仍然存在敏感度不高和通用性不強(qiáng)等缺陷。延伸因子Tu(Elongation factor Tu,EF-Tu)由tuf基因編碼,廣泛存在于真核生物、原核生物及古細(xì)菌中,在蛋白質(zhì)合成過程中對(duì)多肽鏈的延伸至關(guān)重要。tuf基因序列保守,已廣泛用于真菌、細(xì)菌和支原體的生物分類鑒定和系統(tǒng)進(jìn)化分析,但目前還未見關(guān)于綿羊肺炎支原體tuf基因結(jié)構(gòu)特征和遺傳多樣性的報(bào)道。本研究旨在通過對(duì)Mo tuf基因的分子特征分析研究,設(shè)計(jì)引物,建立通用性更好、敏感度更高的熒光定量PCR檢測(cè)方法,并用該方法進(jìn)行Mo感染的病原流行病學(xué)調(diào)查。1綿羊肺炎支原體tuf基因的分子特性研究根據(jù)GenBank中Mo tuf基因編碼區(qū)(CDS區(qū))序列自行設(shè)計(jì)引物,擴(kuò)增Mo參考株Y98和11株Mo臨床分離株CDS區(qū),測(cè)序和序列分析發(fā)現(xiàn):12株Mo的tuf基因CDS區(qū)長(zhǎng)1209 bp,編碼402個(gè)氨基酸,其核苷酸序列及氨基酸序列同源性均為93.3%~100%,G+C含量為39.45%~40.12%;CDS兩端保守,中間區(qū)域存在較大的變異,其中21~960bp區(qū)域堿基突變率僅為8.93%,突變率高達(dá)26.9%的位點(diǎn)重點(diǎn)分布于961~1189bp區(qū)域內(nèi),但是在138~309bp和1047~1196bp區(qū)域內(nèi)保守。有117個(gè)單核苷酸突變位點(diǎn),有41個(gè)無義突變,76個(gè)有義突變,導(dǎo)致其編碼的53個(gè)氨基酸發(fā)生突變,突變氨基酸主要集中在325~354位點(diǎn)。其中2株分離株含兩個(gè)TGA密碼子,而其他分離株只含一個(gè)TGA密碼子。TGA是原核生物的終止密碼子,而在支原體中卻被翻譯成色氨酸�；趖uf基因的遺傳進(jìn)化分析發(fā)現(xiàn)mo與豬肺炎支原體親源關(guān)系最近,其次為結(jié)膜炎支原體,與基于全基因組的遺傳進(jìn)化分析結(jié)果完全吻合,但與基于16srrna建立的遺傳關(guān)系不同,說明motuf基因作為分子靶標(biāo)進(jìn)行遺傳進(jìn)化分析優(yōu)于16srrna。對(duì)12株mo的分析發(fā)現(xiàn)y98單獨(dú)聚為一支,而11株分離株全部聚為一支,卻又分為兩個(gè)亞支。有趣的是,來自z地的3株分離株聚在一個(gè)亞支,來自j地的4株分離株聚在一個(gè)亞支,來自l地的分離株則不規(guī)則的分布在這兩個(gè)亞支中。從本實(shí)驗(yàn)現(xiàn)有的樣本數(shù)量觀察發(fā)現(xiàn),來自z地和來自j地羊場(chǎng)的菌株似乎有地域性的傾向,若要證實(shí)這個(gè)現(xiàn)象還需進(jìn)一步的擴(kuò)大樣本的數(shù)量。2基于綿羊肺炎支原體tuf基因的熒光定量pcr方法的建立由于支原體基因組的g+c含量一般都較低且不同菌株之間存在很大的異質(zhì)性而限制了分子檢測(cè)方法的建立。目前mo的分子檢測(cè)方法有基于16srrna、hsp70建立的pcr方法但是靈敏度不高,易造成假陰性結(jié)果的出現(xiàn)。前期研究發(fā)現(xiàn),mo的tuf基因在138~309bp和1047~1196bp區(qū)域內(nèi)與其他感染羊的支原體差異較大,但種內(nèi)保守,有作為設(shè)計(jì)檢測(cè)mo的分子靶點(diǎn)的潛力。因此用primer5軟件設(shè)計(jì)多對(duì)特異性引物并篩選到一對(duì)最佳引物,擴(kuò)增150bp大小片段,擴(kuò)增位點(diǎn)為1047~1196bp區(qū)域,以獲得最低的ct值分別對(duì)反應(yīng)條件及體系進(jìn)行優(yōu)化。結(jié)果顯示,以mo的核酸為模板通過基于tuf基因的熒光定量pcr擴(kuò)增后的產(chǎn)物在熔解溫度為81.5℃±0.3℃,呈現(xiàn)特異性單峰,無非特異擴(kuò)增峰及二聚體。該方法在5×109~5×104拷貝/反應(yīng)線性范圍良好,相關(guān)系數(shù)為0.996,擴(kuò)增效率為94.1%,標(biāo)準(zhǔn)方程為y=-3.473x+43.411。檢測(cè)下限為5copies/ul,相當(dāng)于169ccu/ml。該方法對(duì)33株mo分離株能全部檢出,并對(duì)常見感染羊的支原體及呼吸道易混感的細(xì)菌均不檢出,由此可見該方法具有良好的敏感性及很高的靈敏度。用50份羊鼻腔棉拭子和134份羊肺組織用本研究建立的熒光定量pcr與基于p113基因的熒光定量pcr進(jìn)行檢出率的比較,發(fā)現(xiàn)肺組織檢出率一致,但是鼻腔棉拭子多檢出2個(gè)并經(jīng)過測(cè)序確定為mo;比基于16srrna及hsp70為靶標(biāo)的普通pcr的鼻腔棉拭子、肺組織檢出率分別高12%~42%、9%~42%。該方法不僅可以定性,還能對(duì)mo進(jìn)行精確定量,為mo感染的流行病學(xué)調(diào)查提供了新方法。3基于tuf基因建立熒光定量PCR方法的應(yīng)用3.1人工感染樣本在肺組織不同病變部位檢出率的比較本試驗(yàn)選擇6只3月齡Mo抗原抗體雙陰性山羊,氣管注射5×l09 CCU/只的參考株Y98,山羊在接種后一周左右出現(xiàn)陣發(fā)性咳嗽、流淚、流鼻涕等典型癥狀,病程長(zhǎng),病羊進(jìn)行性消瘦,于感染后30d撲殺病羊,剖檢可見滲出性纖維素胸膜肺炎、胸腔有少量清亮的黃色積液等典型病變,逐只采集每個(gè)病羊肺臟病變部位、病健交界部位及無病變健康部位的肺組織,用本研究建立的熒光定量PCR方法進(jìn)行檢測(cè),結(jié)果顯示,在同一份肺組織中,病健交界處的檢出率最高(6/6),健康處次之(4/6),完全病變的部位檢出率最低或根本檢測(cè)不到(1/6)。表明病健交界處是PCR檢測(cè)Mo的最佳部位。3.2四川、新疆、青海三個(gè)地區(qū)Mo的病原流行病學(xué)調(diào)查用本研究建立的檢測(cè)Mo的熒光定量PCR方法對(duì)我國(guó)西部四川(n=126)、新疆(n=163)、青海(n=113)三個(gè)省份在2013年10月~2014年10月采集的439份表觀健康的成年山羊及綿羊的肺組織樣本進(jìn)行檢測(cè),結(jié)果表明這三個(gè)地區(qū)的病原陽性率分別為56%、63%、75%,表明這三個(gè)地區(qū)羊群中Mo的感染嚴(yán)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.
【學(xué)位授予單位】：西南民族大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.62

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