【摘要】：目的： 建立基于Taqman探針實(shí)時(shí)熒光定量PCR快速檢測(cè)布魯氏菌的方法，并對(duì)其在臨床上的應(yīng)用進(jìn)行初步的探索；并為將實(shí)時(shí)熒光定量PCR方法應(yīng)用于布魯菌病患者治療后的追蹤隨訪提供實(shí)驗(yàn)室依據(jù)。 方法： 選用布魯氏菌高度保守的16S rDNA序列設(shè)計(jì)引物，并選用屬特異性位點(diǎn)設(shè)計(jì)了FAM、BHQ1標(biāo)記的TaqMan熒光探針，根據(jù)實(shí)時(shí)熒光定量PCR的原理和技術(shù)，建立了布魯氏菌實(shí)時(shí)熒光定量PCR檢測(cè)方法，并構(gòu)建了布魯氏菌熒光定量PCR絕對(duì)定量的標(biāo)準(zhǔn)品，通過(guò)對(duì)該反應(yīng)體系進(jìn)行優(yōu)化，制作標(biāo)準(zhǔn)曲線。應(yīng)用上述建立的基于TaqMan布魯氏菌實(shí)時(shí)熒光定量PCR方法對(duì)吉林大學(xué)第一醫(yī)院感染科2012年7月~2013年8月間明確診斷為布魯氏菌病的50例住院患者的100份外周血樣本和同期隨機(jī)選擇的30位健康志愿者的60份外周血樣本（對(duì)照組）進(jìn)行檢測(cè)，并與標(biāo)準(zhǔn)試管凝集試驗(yàn)和細(xì)菌血培養(yǎng)法做對(duì)比分析；對(duì)8例經(jīng)實(shí)時(shí)熒光定量PCR檢測(cè)為陽(yáng)性的布魯氏菌病患者進(jìn)行跟蹤隨訪，在其確診時(shí)，治療1個(gè)月和治療療程結(jié)束三個(gè)時(shí)間點(diǎn)分別采集患者外周血標(biāo)本行布魯氏菌實(shí)時(shí)熒光定量PCR檢測(cè)，繼而監(jiān)測(cè)其布魯氏菌DNA載量的變化。 結(jié)果： 本實(shí)驗(yàn)成功的以16S rDNA設(shè)計(jì)了布魯氏菌引物及TaqMan探針，并構(gòu)建了布魯氏菌的陽(yáng)性標(biāo)準(zhǔn)品，制作標(biāo)準(zhǔn)曲線，其相關(guān)系數(shù)為R2=0.996。布魯氏菌實(shí)時(shí)熒光定量PCR檢測(cè)反應(yīng)的靈敏度為102copies/μL，對(duì)參考菌株及陰性對(duì)照物均為特異性擴(kuò)增，且組內(nèi)及組間重復(fù)性好。對(duì)50例經(jīng)血培養(yǎng)和/或凝集試驗(yàn)確診為布魯氏菌病患者進(jìn)行檢測(cè)，發(fā)現(xiàn)所建立的布魯氏菌實(shí)時(shí)熒光定量PCR檢測(cè)結(jié)果為陽(yáng)性的患者48例（96%），布魯氏菌實(shí)時(shí)熒光定量PCR的敏感性、特異性、陽(yáng)性預(yù)測(cè)值、陰性預(yù)測(cè)值分別為96%，100%，100%，93.8%。實(shí)時(shí)熒光定量PCR和試管凝集試驗(yàn)組合檢測(cè)的陽(yáng)性一致率高達(dá)92%，一致性檢測(cè)的可信度較高，其Kappa系數(shù)為0.468（P=0.00，有統(tǒng)計(jì)學(xué)意義）。對(duì)8例實(shí)時(shí)熒光定量陽(yáng)性患者進(jìn)行隨訪，發(fā)現(xiàn)布魯氏菌DNA載量在治療1個(gè)月后明顯下降，治療療程結(jié)束后仍有3例患者仍可檢測(cè)到布魯氏菌DNA載量，盡管臨床癥狀消失。 結(jié)論： 本研究所建立的基于TaqMan探針布魯氏菌實(shí)時(shí)熒光定量PCR方法具有敏感度高、特異性好等特點(diǎn)，而且快速、準(zhǔn)確，在對(duì)臨床標(biāo)本檢測(cè)中，其敏感性、特異性、陽(yáng)性預(yù)測(cè)值（PPV）、陰性預(yù)測(cè)值（NPV）均高于試管凝集試驗(yàn)和血培養(yǎng)方法，可以用于臨床上布魯氏菌病的診斷和流行病學(xué)的監(jiān)測(cè)，此外，由于RT-PCR/SAT組合的陽(yáng)性一致率及可信度較高，因此，，該方法適用于檢測(cè)布魯氏菌病早期中經(jīng)血培養(yǎng)或凝集試驗(yàn)陰性的患者，以及凝集試驗(yàn)的確證試驗(yàn)。本次所建立的實(shí)時(shí)熒光定量PCR方法可以用于布魯氏菌病患者治療后的跟蹤隨訪，同時(shí)可通過(guò)監(jiān)測(cè)患者體內(nèi)布魯氏菌DNA載量的變化，為布魯氏菌病臨床分期、抗布病藥物的療效觀察、布魯氏菌病復(fù)發(fā)的監(jiān)測(cè)等提供實(shí)驗(yàn)室方面的依據(jù)。
[Abstract]:Objective: to establish a method for rapid detection of brucella based on real-time fluorescence quantitative PCR (PCR) with Taqman probe and to explore its clinical application. It also provides laboratory basis for the follow-up of brucellosis patients with real-time fluorescence quantitative PCR. Methods: primers were designed with 16s rDNA sequence of Brucella, and FAM,BHQ1 labeled TaqMan fluorescence probe was designed at specific site. According to the principle and technique of real-time fluorescence quantitative PCR, the primer was used to design 16s rDNA sequence of Brucella. A real-time fluorescence quantitative PCR method for the detection of brucella was established, and the absolute quantitative standard of brucella fluorescence quantitative PCR was constructed. The standard curve was made by optimizing the reaction system. Using the real-time fluorescent quantitative PCR method based on TaqMan, 100 peripheral blood samples from 50 inpatients diagnosed with brucellosis from July 2012 to August 2013 were collected from Department of nosocomial infection, Jilin University. During the same period, 60 peripheral blood samples (control group) were randomly selected from 30 healthy volunteers. Compared with the standard test tube agglutination test and bacterial blood culture method, 8 brucellosis patients who were positive by real-time fluorescence quantitative PCR were followed up. The peripheral blood samples of patients were collected for real-time fluorescence quantitative PCR detection of brucella and then the DNA load of brucella was monitored at one month after treatment and at the end of the course of treatment. Results: the primer and TaqMan probe of brucella were designed successfully with 16s rDNA. The positive standard sample of brucella was constructed and the standard curve was made. The correlation coefficient was 0.996. The sensitivity of real-time fluorescence quantitative PCR assay for brucella was 102copies/ 渭 L, which was specific for both reference strains and negative controls, and had good reproducibility within and between groups. Fifty patients with brucellosis confirmed by blood culture and / or agglutination test were tested. 48 patients (96%) were found to be positive by real-time fluorescence quantitative PCR of brucella. The sensitivity of brucella real-time fluorescence quantitative PCR was found. The specificity, positive predictive value and negative predictive value were 96 / 100 and 93.8% respectively. The positive consistency rate of real-time fluorescence quantitative PCR and test tube agglutination test was as high as 92%, and the reliability of consistency test was high. Its Kappa coefficient was 0.468 (P0. 000, with statistical significance). The results showed that the DNA load of brucella significantly decreased after one month of treatment, and 3 patients could still detect the DNA load of brucella after the end of treatment course, although the clinical symptoms disappeared. Conclusion: the real-time fluorescent quantitative PCR method based on TaqMan probe has the characteristics of high sensitivity, good specificity, fast and accurate, and it is sensitive and specific in the detection of clinical specimens. The positive predictive value of (PPV), negative predictive value (NPV) is higher than that of test tube agglutination test and blood culture method. It can be used in clinical diagnosis and epidemiological surveillance of brucellosis. In addition, because of the high positive consistency rate and credibility of RT-PCR/SAT combination, therefore, This method is suitable for the early detection of brucellosis in patients with negative blood culture or agglutination test, as well as the confirmatory test of agglutination test. The established real-time fluorescent quantitative PCR method can be used to follow up the patients with brucellosis after treatment, and it can be used to monitor the changes of DNA load of brucellosis in patients with brucellosis, and it is a clinical stage of brucellosis. The observation of the curative effect of anti-brucellosis drugs and the monitoring of brucellosis recurrence provide laboratory basis.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R440;R516.7

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