結(jié)核分枝桿菌環(huán)介導(dǎo)等溫?cái)U(kuò)增實(shí)時(shí)熒光與反向斑點(diǎn)雜交檢測(cè)方法的研究
發(fā)布時(shí)間：2018-07-22 15:23
【摘要】：結(jié)核病是一種嚴(yán)重威脅人類健康的傳染病，是我國(guó)重點(diǎn)防控的疾病之一，也是全世界重點(diǎn)關(guān)注的公共衛(wèi)生和社會(huì)問(wèn)題。據(jù)世界衛(wèi)生組織（WHO）統(tǒng)計(jì)，全球目前約有860萬(wàn)人罹患結(jié)核病，130萬(wàn)人死于結(jié)核病。結(jié)核病的發(fā)病率以平均每年增加0.4%的速度增長(zhǎng)。因此，尋找一種快速準(zhǔn)確的結(jié)核分枝桿菌檢測(cè)方法已刻不容緩。近年來(lái)興起的環(huán)介導(dǎo)等溫?cái)U(kuò)增技術(shù)具有快速、敏感性高、特異性強(qiáng)等優(yōu)點(diǎn)，而反向斑點(diǎn)雜交技術(shù)結(jié)合了基因擴(kuò)增與分子雜交兩項(xiàng)技術(shù)，可準(zhǔn)確、直觀的觀察結(jié)果，操作簡(jiǎn)便。本研究建立了結(jié)核分枝桿菌環(huán)介導(dǎo)等溫?cái)U(kuò)增技術(shù)和反向斑點(diǎn)雜交技術(shù)，可以滿足現(xiàn)場(chǎng)快速檢測(cè)的需求，為結(jié)核病的防控提供了技術(shù)支持。 本研究分為四部分： 第一部分：結(jié)核分枝桿菌插入序列IS6110基因的克隆與序列分析 選擇結(jié)核分枝桿菌特異性較好的插入序列IS6110片段作為靶序列，根據(jù)GenBank上公布的的結(jié)核分枝桿菌IS6110序列(GenBank登錄號(hào)為:X17348.1)設(shè)計(jì)一對(duì)特異性引物。將PCR擴(kuò)增后的目的片段與pGEM-T載體相連接，連接產(chǎn)物轉(zhuǎn)化至大腸桿菌感受態(tài)細(xì)胞，通過(guò)藍(lán)白斑篩選得到克隆質(zhì)粒pGEM-T-IS6110。克隆質(zhì)粒經(jīng)PCR及測(cè)序鑒定，結(jié)果證實(shí)獲得了結(jié)核分枝桿菌IS6110基因。 第二部分：結(jié)核分枝桿菌環(huán)介導(dǎo)等溫?cái)U(kuò)增實(shí)時(shí)熒光檢測(cè)方法的建立 針對(duì)結(jié)核分枝桿菌特有的插入序列IS6110，自行設(shè)計(jì)并合成結(jié)核分枝桿菌的環(huán)介導(dǎo)等溫?cái)U(kuò)增引物組(包括一對(duì)外引物以及一對(duì)內(nèi)引物)，，將引物組中的各引物按照一定的摩爾濃度比例配制成引物組溶液，并利用環(huán)介導(dǎo)等溫?cái)U(kuò)增實(shí)時(shí)熒光檢測(cè)系統(tǒng)建立結(jié)核分枝桿菌的環(huán)介導(dǎo)等溫?cái)U(kuò)增檢測(cè)方法，同時(shí)對(duì)內(nèi)外引物摩爾濃度比例、反應(yīng)溫度進(jìn)行優(yōu)化得到最佳反應(yīng)條件，結(jié)果通過(guò)環(huán)介導(dǎo)等溫?cái)U(kuò)增實(shí)時(shí)熒光曲線進(jìn)行分析。通過(guò)分析環(huán)介導(dǎo)等溫?cái)U(kuò)增實(shí)時(shí)熒光曲線可知，整個(gè)反應(yīng)用時(shí)1h，大多數(shù)反應(yīng)在30min內(nèi)開(kāi)始擴(kuò)增。同時(shí)，對(duì)10倍梯度稀釋的結(jié)核分枝桿菌DNA模板進(jìn)行檢測(cè)，環(huán)介導(dǎo)等溫?cái)U(kuò)增實(shí)時(shí)熒光檢測(cè)方法檢測(cè)限可達(dá)2.4fg/反應(yīng)，比PCR高100倍，對(duì)其他常見(jiàn)病原菌的DNA無(wú)檢出，靈敏度和特異性均較好，具有廣闊的臨床應(yīng)用前景。 第三部分：結(jié)核分枝桿菌反向斑點(diǎn)雜交檢測(cè)方法的建立 針對(duì)結(jié)核分枝桿菌特有的插入序列IS6110，自行設(shè)計(jì)并合成生物素標(biāo)記引物及特異性寡核苷酸探針。以結(jié)核分枝桿菌DNA為模板，通過(guò)PCR擴(kuò)增，制備生物素標(biāo)記的目的片段，產(chǎn)物變性與固定在帶正電荷的尼龍膜上的特異性寡核苷酸探針在65℃進(jìn)行雜交，每次雜交反應(yīng)設(shè)有陽(yáng)性對(duì)照及陰性對(duì)照。通過(guò)對(duì)探針濃度、鏈酶親和素標(biāo)記堿性磷酸酶的稀釋倍數(shù)、雜交時(shí)間、酶促反應(yīng)時(shí)間四方面進(jìn)行優(yōu)化得到反向斑點(diǎn)雜交的最優(yōu)反應(yīng)條件，結(jié)果通過(guò)對(duì)膜顯色后是否出現(xiàn)藍(lán)紫色斑點(diǎn)來(lái)判定陽(yáng)性或者陰性。本研究建立的反向斑點(diǎn)雜交的特異性好，與其他常見(jiàn)病原菌無(wú)交叉反應(yīng)，對(duì)10倍梯度稀釋的結(jié)核分枝桿菌DNA模板進(jìn)行檢測(cè)可達(dá)到24fg/反應(yīng)的檢測(cè)限。與PCR相比，該方法的檢測(cè)限提高了10倍，可實(shí)現(xiàn)結(jié)核分枝桿菌與其他常見(jiàn)病原菌的鑒別診斷。 第四部分：環(huán)介導(dǎo)等溫?cái)U(kuò)增實(shí)時(shí)熒光檢測(cè)方法與反向斑點(diǎn)雜交方法在臨床標(biāo)本中的應(yīng)用 以臨床上確診為結(jié)核病患者的痰標(biāo)本的DNA為模板，分別進(jìn)行環(huán)節(jié)導(dǎo)等溫?cái)U(kuò)增實(shí)時(shí)熒光檢測(cè)和反向斑點(diǎn)雜交檢測(cè)。所有痰標(biāo)本均出現(xiàn)熒光擴(kuò)增曲線并出現(xiàn)藍(lán)紫色斑點(diǎn)，該結(jié)果表明本研究建立的環(huán)介導(dǎo)等溫?cái)U(kuò)增實(shí)時(shí)熒光檢測(cè)結(jié)合反向斑點(diǎn)雜交方法可成功的檢測(cè)出結(jié)核分枝桿菌，該方法靈敏、特異，不需要精密儀器，適合在臨床實(shí)驗(yàn)室推廣使用。
[Abstract]:Tuberculosis is one of the infectious diseases that seriously threaten human health. It is one of the key prevention and control diseases in our country. It is also a public health and social problem that is the key concern of the world. According to the WHO (WHO), about 8 million 600 thousand people worldwide are now suffering from tuberculosis and 1 million 300 thousand people die from tuberculosis. The incidence of tuberculosis is increased by an average of 0.4% per year. Therefore, it is urgent to find a rapid and accurate detection method for Mycobacterium tuberculosis. In recent years, the ring mediated isothermal amplification technology has the advantages of fast, high sensitivity and strong specificity, and the reverse dot blot hybridization technique combines two techniques of gene amplification and molecular hybridization, which can be accurately and intuitively observed. It is easy to operate. This study has established a ring mediated isothermal amplification technique of Mycobacterium tuberculosis and reverse dot blot hybridization technology, which can meet the needs of rapid detection in the field and provide technical support for the prevention and control of tuberculosis.
This study is divided into four parts:
Part one: cloning and sequence analysis of Mycobacterium tuberculosis insertion sequence IS6110 gene
The specific insertion sequence IS6110 fragment of Mycobacterium tuberculosis was selected as the target sequence. A pair of specific primers were designed according to the IS6110 sequence of Mycobacterium tuberculosis published on the GenBank (GenBank login number: X17348.1). The target fragment of the PCR amplification was connected with the pGEM-T vector, and the connection product was transformed to the Escherichia coli feeling state fine. The cloned plasmid pGEM-T-IS6110. was screened by blue spot and identified by PCR and sequencing. The results showed that the IS6110 gene of Mycobacterium tuberculosis was obtained.
The second part: the establishment of loop mediated isothermal amplification real-time fluorescence detection for Mycobacterium tuberculosis.
In view of the specific insertion sequence IS6110 of Mycobacterium tuberculosis, a ring mediated isothermal amplification primer group (including one external primer and one pair of primers) was designed and synthesized by ourselves. The primers in the primer group were prepared according to a certain molar concentration to a primer group solution, and the ring mediated isothermal amplification was used for real-time fluorescence detection. The system established the method of isothermal amplification detection of Mycobacterium tuberculosis by loop mediated isothermal amplification. At the same time, the optimum reaction conditions were obtained by optimizing the molar concentration ratio of the internal and external primers and the reaction temperature. The results were analyzed by the loop mediated isothermal amplification of real time fluorescence curve. 1H, most of the reactions began to expand in 30min. At the same time, the 10 times gradient dilution of Mycobacterium tuberculosis DNA template was detected. The detection limit of the ring mediated isothermal amplification real-time fluorescence detection method was 100 times higher than PCR, and the sensitivity and specificity were better for other common pathogenic bacteria, and had a broad clinical application. Prospects.
The third part: establishment of reverse dot blot hybridization method for detection of Mycobacterium tuberculosis.
Biotin marker primers and specific oligonucleotide probes were designed and synthesized for the specific insertion sequence of Mycobacterium tuberculosis (IS6110). The target fragment of biotin was prepared by PCR amplification with DNA as a template. The specific oligonucleotide probes of product denaturation and immobilization on the nylon membrane with positive charge were 65 The optimum reaction conditions of the reverse dot blot hybridization were obtained by four aspects of the concentration of the probe, the dilution times of the chain enzyme avidin labeled alkaline phosphatase, the time of hybridization and the reaction time of the enzyme. It was positive or negative. The specificity of the reverse dot blot established in this study was good, and there was no cross reaction with other common pathogens. The detection limit of DNA template for Mycobacterium tuberculosis with 10 times gradient dilution could reach the detection limit of 24fg/ reaction. Compared with PCR, the detection limit of this method was 10 times higher than that of the method, which could realize Mycobacterium tuberculosis and others. Differential diagnosis of common pathogenic bacteria.
The fourth part: the application of loop mediated isothermal amplification real-time fluorescence detection and reverse dot blot hybridization in clinical specimens.
The DNA of sputum specimens of patients diagnosed with tuberculosis was used as a template. The real-time fluorescence detection and reverse dot blot detection were carried out respectively. All sputum specimens showed fluorescence amplification curve and blue purple spots. The results showed that the ring mediated isothermal amplification real-time fluorescence detection combined with reverse spot was established in this study. Dot blot can successfully detect Mycobacterium tuberculosis. The method is sensitive and specific, and does not require precision instruments. It is suitable for clinical laboratories.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R52;R440
本文編號(hào)：2137896
[Abstract]:Tuberculosis is one of the infectious diseases that seriously threaten human health. It is one of the key prevention and control diseases in our country. It is also a public health and social problem that is the key concern of the world. According to the WHO (WHO), about 8 million 600 thousand people worldwide are now suffering from tuberculosis and 1 million 300 thousand people die from tuberculosis. The incidence of tuberculosis is increased by an average of 0.4% per year. Therefore, it is urgent to find a rapid and accurate detection method for Mycobacterium tuberculosis. In recent years, the ring mediated isothermal amplification technology has the advantages of fast, high sensitivity and strong specificity, and the reverse dot blot hybridization technique combines two techniques of gene amplification and molecular hybridization, which can be accurately and intuitively observed. It is easy to operate. This study has established a ring mediated isothermal amplification technique of Mycobacterium tuberculosis and reverse dot blot hybridization technology, which can meet the needs of rapid detection in the field and provide technical support for the prevention and control of tuberculosis.
This study is divided into four parts:
Part one: cloning and sequence analysis of Mycobacterium tuberculosis insertion sequence IS6110 gene
The specific insertion sequence IS6110 fragment of Mycobacterium tuberculosis was selected as the target sequence. A pair of specific primers were designed according to the IS6110 sequence of Mycobacterium tuberculosis published on the GenBank (GenBank login number: X17348.1). The target fragment of the PCR amplification was connected with the pGEM-T vector, and the connection product was transformed to the Escherichia coli feeling state fine. The cloned plasmid pGEM-T-IS6110. was screened by blue spot and identified by PCR and sequencing. The results showed that the IS6110 gene of Mycobacterium tuberculosis was obtained.
The second part: the establishment of loop mediated isothermal amplification real-time fluorescence detection for Mycobacterium tuberculosis.
In view of the specific insertion sequence IS6110 of Mycobacterium tuberculosis, a ring mediated isothermal amplification primer group (including one external primer and one pair of primers) was designed and synthesized by ourselves. The primers in the primer group were prepared according to a certain molar concentration to a primer group solution, and the ring mediated isothermal amplification was used for real-time fluorescence detection. The system established the method of isothermal amplification detection of Mycobacterium tuberculosis by loop mediated isothermal amplification. At the same time, the optimum reaction conditions were obtained by optimizing the molar concentration ratio of the internal and external primers and the reaction temperature. The results were analyzed by the loop mediated isothermal amplification of real time fluorescence curve. 1H, most of the reactions began to expand in 30min. At the same time, the 10 times gradient dilution of Mycobacterium tuberculosis DNA template was detected. The detection limit of the ring mediated isothermal amplification real-time fluorescence detection method was 100 times higher than PCR, and the sensitivity and specificity were better for other common pathogenic bacteria, and had a broad clinical application. Prospects.
The third part: establishment of reverse dot blot hybridization method for detection of Mycobacterium tuberculosis.
Biotin marker primers and specific oligonucleotide probes were designed and synthesized for the specific insertion sequence of Mycobacterium tuberculosis (IS6110). The target fragment of biotin was prepared by PCR amplification with DNA as a template. The specific oligonucleotide probes of product denaturation and immobilization on the nylon membrane with positive charge were 65 The optimum reaction conditions of the reverse dot blot hybridization were obtained by four aspects of the concentration of the probe, the dilution times of the chain enzyme avidin labeled alkaline phosphatase, the time of hybridization and the reaction time of the enzyme. It was positive or negative. The specificity of the reverse dot blot established in this study was good, and there was no cross reaction with other common pathogens. The detection limit of DNA template for Mycobacterium tuberculosis with 10 times gradient dilution could reach the detection limit of 24fg/ reaction. Compared with PCR, the detection limit of this method was 10 times higher than that of the method, which could realize Mycobacterium tuberculosis and others. Differential diagnosis of common pathogenic bacteria.
The fourth part: the application of loop mediated isothermal amplification real-time fluorescence detection and reverse dot blot hybridization in clinical specimens.
The DNA of sputum specimens of patients diagnosed with tuberculosis was used as a template. The real-time fluorescence detection and reverse dot blot detection were carried out respectively. All sputum specimens showed fluorescence amplification curve and blue purple spots. The results showed that the ring mediated isothermal amplification real-time fluorescence detection combined with reverse spot was established in this study. Dot blot can successfully detect Mycobacterium tuberculosis. The method is sensitive and specific, and does not require precision instruments. It is suitable for clinical laboratories.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R52;R440
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