本文選題：環(huán)介導(dǎo)等溫?cái)U(kuò)增 + 乙型肝炎病毒　； 參考：《承德醫(yī)學(xué)院》2017年碩士論文

【摘要】：乙型肝炎病毒(Hepatitis B virus,HBV)和丙型肝炎病毒(Hepatitis C virus,HCV)感染是全球公共衛(wèi)生問題,尤其是在實(shí)驗(yàn)設(shè)備匱乏的發(fā)展中國家和地區(qū)。HBV和HCV核酸檢測是診斷和療效檢測的最佳方法,國內(nèi)大、中型醫(yī)院普遍使用熒光定量PCR(Fluorescence quantitative PCR,FQ-PCR),然而,FQ-PCR操作流程煩瑣,試劑、儀器均較昂貴,對操作人員的技術(shù)要求高,不適用于在醫(yī)療設(shè)備匱乏的場所進(jìn)行操作。本研究建立了依賴鈣黃綠素和羥基萘酚藍(lán)(hydroxynaphthol blue,HNB)的可視化環(huán)介導(dǎo)等溫?cái)U(kuò)增(loop-mediated isothermal amplification,LAMP)技術(shù),以利于在實(shí)驗(yàn)設(shè)備匱乏的環(huán)境下進(jìn)行HBV和HCV檢測。第一部分應(yīng)用環(huán)介導(dǎo)等溫?cái)U(kuò)增技術(shù)可視化檢測乙型肝炎病毒目的:建立可視化檢測HBV的LAMP方法。方法:首先,根據(jù)NCBI網(wǎng)站上公布的HBV S區(qū)段保守序列設(shè)計(jì)LAMP引物。第二,收集臨床血清樣本,分別按照試劑盒法和煮沸法提取DNA,用FQ-PCR對病毒進(jìn)行定量。第三,對反應(yīng)條件進(jìn)行優(yōu)化,通過對非特異模板擴(kuò)增實(shí)驗(yàn)和內(nèi)切酶消化實(shí)驗(yàn)以驗(yàn)證擴(kuò)增特異性;通過檢測試劑盒提取后倍比稀釋的HBV模板以評價(jià)LAMP的靈敏性;通過LAMP和PCR擴(kuò)增煮沸血清后結(jié)果的比較,以評價(jià)LAMP的抗干擾性。同時(shí),根據(jù)電泳結(jié)果比較依賴SYBR Green I的和依賴HNB的可視化檢測效果。最后,用LAMP和PCR擴(kuò)增臨床樣本,用SPSS 17.0統(tǒng)計(jì)學(xué)軟件分別與FQ-PCR(金標(biāo)準(zhǔn))進(jìn)行比較。結(jié)果:經(jīng)過一系列預(yù)實(shí)驗(yàn),建立了最適LAMP反應(yīng)條件,LAMP特異性高,沒有產(chǎn)生非特異性擴(kuò)增。無論使用哪種核酸提取方法,LAMP靈敏性均為10拷貝/管,由此可得出LAMP的抗干擾性較好。依賴HNB可視化檢測的效果和SYBR Green I、電泳分析相當(dāng),卻不似染料SYBR Green I需要開蓋加入而造成氣溶膠污染。另外,lamp對于煮沸血清的檢測結(jié)果和fq-pcr沒有統(tǒng)計(jì)學(xué)差異(p0.05),且一致性較好(kappa=0.762)。然而,pcr對于煮沸血清的檢測結(jié)果和fq-pcr有統(tǒng)計(jì)學(xué)差異(p0.05),且一致性較差(kappa=0.186)。結(jié)論:本研究針對hbvs區(qū)段,設(shè)計(jì)了針對我國主要的四種hbv基因型lamp通用引物,通過設(shè)計(jì)環(huán)引物,提高了擴(kuò)增效率。另外,使用煮沸血清為模板,簡化了操作流程,從而建立了依賴hnb可視化檢測hbv的lamp技術(shù)。第二部分應(yīng)用反轉(zhuǎn)錄環(huán)介導(dǎo)等溫?cái)U(kuò)增技術(shù)可視化檢測丙型肝炎病毒目的:建立可視化檢測hcv的rt-lamp方法。方法:首先,收集并用fq-pcr(金標(biāo)準(zhǔn))檢測75例臨床樣本。根據(jù)9種hcv基因亞型的5’非編碼區(qū)(5’utr)設(shè)計(jì)通用rt-lamp引物。然后,通過比較傳統(tǒng)rt-lamp系統(tǒng)和加入taqdna聚合酶的系統(tǒng)以優(yōu)化體系,通過非特異模板擴(kuò)增實(shí)驗(yàn)以評價(jià)其特異性,通過rt-lamp和rt-pcr同時(shí)檢測倍比稀釋的hcv模板以評價(jià)其靈敏性。用電泳分析擴(kuò)增情況,同時(shí)用鈣黃綠素和hnb可視化檢測產(chǎn)物。最后,用rt-lamp和rt-pcr檢測臨床樣本,用spss17.0統(tǒng)計(jì)學(xué)軟件分別與fq-pcr進(jìn)行比較。結(jié)果:結(jié)果表明向傳統(tǒng)rt-lamp體系中加入taqdna聚合酶可提高20min的擴(kuò)增效率。rt-lamp的特異性好,沒有非特異性擴(kuò)增現(xiàn)象。經(jīng)檢測稀釋模板,rt-lamp的靈敏性為10iu/管,比rt-pcr高10倍。另外,鈣黃綠素和hnb的可視效果與電泳分析相一致。經(jīng)檢測75例臨床樣本,rt-lamp顯示了和fq-pcr較好的一致性(p0.05,kappa=0.762)。結(jié)論:本研究針對hcv5’utr區(qū)段,設(shè)計(jì)了檢測hcv9種基因亞型的rt-lamp通用引物,建立了依賴鈣黃綠素或hnb可視化檢測hcv的rt-lamp技術(shù)。第三部分應(yīng)用反轉(zhuǎn)錄等溫?cái)U(kuò)增技術(shù)對丙型肝炎病毒1b和2a基因型檢測目的:建立對HCV 1b和2a基因型分型的RT-LAMP方法。方法:首先,收集74例陽性臨床采樣標(biāo)本并用FQ-PCR方法分型和定量。第二,分別根據(jù)HCV 1b和2a序列各自的5’UTR設(shè)計(jì)相應(yīng)的RT-LAMP引物。第三,利用與非特異模板擴(kuò)增實(shí)驗(yàn)和酶切實(shí)驗(yàn)以評價(jià)各引物的特異性。第四,擴(kuò)增倍比稀釋的模板以評價(jià)各引物的靈敏性,并用依賴鈣黃綠素的可視化方法判定結(jié)果。最后,對所有臨床樣本分別用HCV 1b和2a分型引物進(jìn)行兩組平行反應(yīng),應(yīng)用SPSS 17.0統(tǒng)計(jì)學(xué)軟件比較RT-LAMP和FQ-PCR的一致性。結(jié)果:結(jié)果表明RT-LAMP引物特異性好,于非特異模板無交叉反應(yīng)且HCV1b和2a型酶切產(chǎn)物大小與預(yù)期相一致。RT-LAMP法最低檢測HCV 1b和2a的靈敏度為100 IU/mL,且依賴鈣黃綠素的可視化方法和電泳分析相一致。經(jīng)對臨床樣本進(jìn)行平行實(shí)驗(yàn),RT-LAMP檢測HCV 1b型的陽性率為97.37%,檢測HCV 2a型的陽性率為94.44%。SPSS 17.0統(tǒng)計(jì)學(xué)軟件表明RT-LAMP和FQ-PCR沒有統(tǒng)計(jì)學(xué)差異(P0.05)。結(jié)論:本研究通過比較多組HCV基因序列,設(shè)計(jì)了兩套分型引物,建立了依賴鈣黃綠素可視化分型檢測HCV的RT-LAMP技術(shù)。
[Abstract]:Hepatitis B virus (Hepatitis B virus, HBV) and hepatitis C virus (Hepatitis C virus, HCV) infection are the global public health problems, especially in the developing countries and regions with lack of experimental equipment,.HBV and HCV nucleic acid detection is the best method for diagnosis and efficacy detection. Large and medium-sized hospitals are widely used in medium size hospitals with fluorescent quantitative PCR (Fluorescenc). E quantitative PCR, FQ-PCR), however, the FQ-PCR operation process is tedious, the reagent, the instrument are all expensive, the technical requirement of the operator is high, it is not suitable for the operation in the place where the medical equipment is scarce. This study established the visual loop mediated isothermal amplification (loop-mediat) depending on the calc and the hydroxyl naphthol blue (hydroxynaphthol blue, HNB). Ed isothermal amplification, LAMP) technology to facilitate the detection of HBV and HCV in the environment of lack of experimental equipment. The first part uses ring mediated isothermal amplification to visualize hepatitis B virus purpose: to establish a LAMP method for visual detection of HBV. Method: first, to design L according to the HBV S section published on NCBI site. AMP primers. Second, the clinical serum samples were collected, the DNA was extracted according to the kit method and the boiling method respectively. The virus was quantified by FQ-PCR. Third, the reaction conditions were optimized. The non specific template amplification experiment and the digestion experiment of endonuclease were used to verify the specificity. The sensitivity of valence LAMP; comparing the results of boiling serum after LAMP and PCR amplification to evaluate the anti-interference of LAMP. At the same time, according to the results of electrophoresis, the results of SYBR Green I and HNB depend on the visual detection results. Finally, the clinical samples were amplified by LAMP and PCR and compared with FQ-PCR (gold standard) by SPSS 17 system software. Fruit: after a series of pre experiments, the optimum LAMP reaction conditions were established, and the specificity of LAMP was high and no non specific amplification was produced. No matter which nucleic acid extraction method was used, the sensitivity of LAMP was 10 copies / tubes. Thus, the anti-interference of LAMP was better. The effect of HNB visual detection and SYBR Green I, electrophoretic analysis, were not similar to dyed. SYBR Green I needed open cover to cause aerosol pollution. In addition, there was no statistical difference between the results of detection of boiling serum by lamp and FQ-PCR (P0.05), and the consistency was better (kappa=0.762). However, the results of PCR for boiling serum were statistically different from FQ-PCR (P0.05), and the consistency was poor (kappa=0.186). Conclusion: This study According to the HBVs section, we designed four main HBV genotypes lamp universal primers for our country. By designing ring primers, the amplification efficiency was improved. In addition, using boiling serum as a template, the operation process was simplified, and the lamp technology that depended on the visual detection of HBV by HNB was established. The second part applied inversion ring mediated isothermal amplification technology to be visible. Detection of hepatitis C virus Objective: to establish a RT-LAMP method for visual detection of HCV. Method: first, collect and use FQ-PCR (gold standard) to detect 75 clinical samples. General RT-LAMP primers are designed according to the 5 'non coding region (5' UTR) of the 9 HCV subtypes. Then, the system is superior to the traditional RT-LAMP system and the system adding taqdna polymerase. The system was used to evaluate its specificity by nonspecific template amplification experiments. The sensitivity was evaluated by simultaneous detection of double dilution HCV templates by RT-LAMP and RT-PCR. The amplification conditions were analyzed by electrophoretic analysis. At the same time, calcium yellow green and HNB were used to visualize the products. Finally, the clinical samples were detected by RT-LAMP and RT-PCR, and the SPSS17.0 statistical software was used respectively. Results compared with FQ-PCR. Results: the results showed that adding taqdna polymerase to the traditional RT-LAMP system could improve the specificity of 20min amplification efficiency.Rt-lamp without non specific amplification. The sensitivity of RT-LAMP was 10 times higher than that of RT-PCR by detecting the dilution template. Besides, the visual effect and electrophoresis analysis of calcein and HNB Consistent. After testing 75 clinical samples, RT-LAMP showed good consistency with FQ-PCR (P0.05, kappa=0.762). Conclusion: This study designed a RT-LAMP universal primer for detection of hcv9 gene subtypes for hcv5 'UTR section, and established a RT-LAMP technique for the visualization of HCV by the visualization of calcein or HNB. The third part was used for reverse transcription isothermal. Detection of hepatitis C virus 1b and 2A genotypes by amplification: a RT-LAMP method for typing HCV 1b and 2A genotypes. Methods: first, 74 samples of positive clinical samples were collected and classified and quantified by FQ-PCR method. Second, the corresponding RT-LAMP primers were designed according to the 5 'UTR HCV 1b and 2A sequences. Third. Specific template amplification experiments and enzyme cutting experiments were used to evaluate the specificity of the primers. Fourth, the amplification of double dilution templates was used to evaluate the sensitivity of the primers, and the results were determined by the visualization of calcein dependence. Finally, two groups of parallel reactions were performed with HCV 1b and 2A types, respectively, and SPSS 17 statistics were applied to all clinical samples. The software compared the consistency between RT-LAMP and FQ-PCR. Results: the results showed that the specificity of RT-LAMP primers was good, the non specific template was no cross reaction and the sensitivity of.RT-LAMP method to detect HCV 1b and 2a was 100 IU/mL with the same.RT-LAMP method, and the visual method and electrophoresis analysis were the same. Parallel experiments on clinical samples showed that the positive rate of RT-LAMP HCV 1b was 97.37%. The positive rate of HCV 2 A was 94.44%.SPSS 17 statistics software showed that there was no statistical difference between RT-LAMP and FQ-PCR (P0.05). Conclusion: This study set up two sets of type primers by comparing multiple groups of HCV gene sequences, and established the dependence on calcium yellow green. The RT-LAMP technology of HCV was detected by stereotyping.

【學(xué)位授予單位】：承德醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R512.6;R440

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