布鲁氏菌实时荧光定量PCR快速检测方法的建立和临床应用的初步研究

发布时间：2018-09-10 06:33

【摘要】：目的：建立基于Taqman探针实时荧光定量PCR快速检测布鲁氏菌的方法，并对其在临床上的应用进行初步的探索；并为将实时荧光定量PCR方法应用于布鲁菌病患者治疗后的追踪随访提供实验室依据。方法：选用布鲁氏菌高度保守的16S rDNA序列设计引物，并选用属特异性位点设计了FAM、BHQ1标记的TaqMan荧光探针，根据实时荧光定量PCR的原理和技术，建立了布鲁氏菌实时荧光定量PCR检测方法，并构建了布鲁氏菌荧光定量PCR绝对定量的标准品，通过对该反应体系进行优化，制作标准曲线。应用上述建立的基于TaqMan布鲁氏菌实时荧光定量PCR方法对吉林大学第一医院感染科2012年7月~2013年8月间明确诊断为布鲁氏菌病的50例住院患者的100份外周血样本和同期随机选择的30位健康志愿者的60份外周血样本（对照组）进行检测，并与标准试管凝集试验和细菌血培养法做对比分析；对8例经实时荧光定量PCR检测为阳性的布鲁氏菌病患者进行跟踪随访，在其确诊时，治疗1个月和治疗疗程结束三个时间点分别采集患者外周血标本行布鲁氏菌实时荧光定量PCR检测，继而监测其布鲁氏菌DNA载量的变化。结果：本实验成功的以16S rDNA设计了布鲁氏菌引物及TaqMan探针，并构建了布鲁氏菌的阳性标准品，制作标准曲线，其相关系数为R2=0.996。布鲁氏菌实时荧光定量PCR检测反应的灵敏度为102copies/μL，对参考菌株及阴性对照物均为特异性扩增，且组内及组间重复性好。对50例经血培养和/或凝集试验确诊为布鲁氏菌病患者进行检测，发现所建立的布鲁氏菌实时荧光定量PCR检测结果为阳性的患者48例（96%），布鲁氏菌实时荧光定量PCR的敏感性、特异性、阳性预测值、阴性预测值分别为96%，100%，100%，93.8%。实时荧光定量PCR和试管凝集试验组合检测的阳性一致率高达92%，一致性检测的可信度较高，其Kappa系数为0.468（P=0.00，有统计学意义）。对8例实时荧光定量阳性患者进行随访，发现布鲁氏菌DNA载量在治疗1个月后明显下降，治疗疗程结束后仍有3例患者仍可检测到布鲁氏菌DNA载量，尽管临床症状消失。结论：本研究所建立的基于TaqMan探针布鲁氏菌实时荧光定量PCR方法具有敏感度高、特异性好等特点，而且快速、准确，在对临床标本检测中，其敏感性、特异性、阳性预测值（PPV）、阴性预测值（NPV）均高于试管凝集试验和血培养方法，可以用于临床上布鲁氏菌病的诊断和流行病学的监测，此外，由于RT-PCR/SAT组合的阳性一致率及可信度较高，因此，，该方法适用于检测布鲁氏菌病早期中经血培养或凝集试验阴性的患者，以及凝集试验的确证试验。本次所建立的实时荧光定量PCR方法可以用于布鲁氏菌病患者治疗后的跟踪随访，同时可通过监测患者体内布鲁氏菌DNA载量的变化，为布鲁氏菌病临床分期、抗布病药物的疗效观察、布鲁氏菌病复发的监测等提供实验室方面的依据。
[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.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R440;R516.7

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