梅毒螺旋体核酸检测及基于微滴式数字PCR技术的梅毒螺旋体核酸绝对定量方法学研究

发布时间：2018-05-15 09:29

本文选题：巢式PCR + 微滴式数字PCR　；参考：《安徽医科大学》2017年硕士论文

【摘要】：第一部分各期梅毒外周血中梅毒螺旋体核酸检出率的研究目的利用巢式PCR技术探究各期梅毒患者外周血中梅毒螺旋体DNA的检出率方法临床收集192例梅毒患者外周血样本,其中包括37例一期梅毒,92例二期梅毒,63例潜伏梅毒,利用TP0105和TP0574基因片段特异的内外引物,行巢式PCR技术对样本进行检测。结果一期梅毒患者外周血中梅毒螺旋体的检出率为45.9%(TP0105)和40.5%(TP0574),两基因的总检出率为48.6%;二期梅毒患者外周血中梅毒螺旋体的检出率为52.2%(TP0105)和52.2%(TP0574),两基因的总检出率为62.0%;潜伏梅毒患者外周血中梅毒螺旋体的检出率为6.3%(TP0105)和4.8%(TP0574),两基因的总检出率为7.9%。两基因的检出率在统计学上没有差异(P0.05),Kappa值为0.714。结论梅毒螺旋体基因可以在螺旋体感染的各个阶段的梅毒患者外周血中检测到,其中在一期梅毒和二期梅毒中的检出率最高。巢式PCR技术在早期梅毒的诊断中起到了重要的作用,可以在临床上用于暗视野镜检和血清学检测的补充诊断标准。第二部分基于微滴式数字PCR技术的梅毒螺旋体核酸绝对定量方法的建立及应用微滴式数字PCR技术是近年来迅速发展起来的一项基于单分子PCR方法来进行计数的核酸分子绝对定量技术,目前基于该技术的梅毒螺旋体绝对定量方法还未有文献报道。本研究建立了微滴式数字PCR梅毒螺旋体核酸绝对定量检测平台,从特异性、灵敏度、可信度、重复性几个方面对平台进行评估,又将建立好的dd PCR检测平台应用于TP感染兔模型外周血的核酸检测,确定该技术的可行性。本研究设计了针对基因分别为TP0105和TP0574的特异性探针,利用ddPCR技术检测了健康人、非梅毒病人、正常兔、鼠血浆中的目的基因片段,确定了该技术的空白下限值(LOB)为3.2copies(TP0105)和1.4copies(TP0574),为后续对于临床样本的检测确定了cut-off值。用dd PCR技术对各稀释梯度的质粒进行核酸定量检测,确定该检测技术的高灵敏度,最低检测下限(LOD)为单拷贝。将每个梯度质粒经dd PCR检测出的实际拷贝数与计算出的理论拷贝数进行相关分析,发现二者呈高度相关性,说明dd PCR技术的检测结果是可信的。为了评估dd PCR的重复性,我们对各稀释梯度兔睾丸悬液的检测均进行了三次重复,对三次拷贝数结果进行分析研究发现每个稀释梯度的RSD值均在可接受的范围内,表明dd PCR具有很好的可重复性。在dd PCR与q PCR的对比研究中我们发现dd PCR具有比q PCR更低的检测下限,灵敏度更高,且dd PCR具有比q PCR更好的重复性,特别是在目的基因处于低拷贝的情况下。本研究利用dd PCR技术动态监测了兔模型对TP的感染情况,发现感染兔外周血中核酸水平的出现早于血清学水平,证实了dd PCR技术的可行性,为后续对于临床样本的核酸绝对定量研究奠定了基础,对指导用于极早期梅毒、胎传梅毒、神经梅毒等早期诊断具有重要意义。
[Abstract]:Part I study on detection rate of Treponema pallidum nucleic acid in peripheral blood of patients with syphilis objective to investigate the detection rate of Treponema pallidum DNA in peripheral blood of patients with syphilis by nested PCR. Among them, there were 37 cases of primary syphilis, 92 cases of secondary syphilis and 63 cases of latent syphilis. The samples were detected by nested PCR using TP0105 and TP0574 gene fragment specific internal and external primers. Results the detection rates of Treponema pallidum in peripheral blood of primary syphilis patients were 45.9% TP0105) and 40.5% respectively, the total detection rate of the two genes was 48.60.The detection rate of Treponema pallidum in peripheral blood of patients with secondary syphilis was 52.2% TP0105) and 52.2% TP057444.The total detection rate of the two genes was 62.0%. The detection rate of Treponema pallidum in peripheral blood of patients with syphilis was 6.3% TP0105) and 4.8% TP0574. The total detection rate of the two genes was 7.9%. There was no statistical difference in the detection rate between the two genes. The Kappa value of P0. 05 was 0. 714. Conclusion Treponema pallidum gene can be detected in peripheral blood of patients with Treponema pallidum infection in all stages, among which the detection rate is the highest in primary syphilis and secondary syphilis. Nested PCR technique plays an important role in the diagnosis of early syphilis and can be used as a supplementary diagnostic standard for dark field microscopy and serological examination. Part two Establishment and Application of absolute quantitative method of Treponema pallidum Nucleic Acid based on Microdrop Digital PCR technique; Microdrop Digital PCR technique is a newly developed kernel which is based on single molecule PCR method to count the nucleic acid of Treponema pallidum. Acid molecule absolute quantitative technique, At present, the absolute quantitative method of Treponema pallidum based on this technique has not been reported. In this study, an absolute quantitative detection platform for Treponema pallidum by microdrop digital PCR was established. The platform was evaluated in terms of specificity, sensitivity, reliability and repeatability. The dd PCR detection platform was established to detect nucleic acid in peripheral blood of rabbit model infected with TP, and the feasibility of this technique was confirmed. In this study, specific probes targeting TP0105 and TP0574 genes were designed to detect the target gene fragments in the plasma of healthy subjects, non-syphilis patients, normal rabbits and mice by using ddPCR technique. The blank lower limit (LOB) of this technique was determined to be 3.2copiesl TP0105), and the cut-off value was determined for subsequent clinical sample detection. The dd PCR technique was used to detect the nucleic acid of the diluted gradient plasmids. The high sensitivity of the detection technique and the lowest detection limit were determined as a single copy. The correlation analysis between the actual copy number of each gradient plasmid detected by dd PCR and the calculated theoretical copy number shows that there is a high correlation between them, which indicates that the detection results of dd PCR technique are reliable. In order to evaluate the repeatability of dd PCR, the test of testicular suspension of each diluted gradient rabbit was repeated three times, and the results of three copies number were analyzed. The results showed that the RSD value of each dilution gradient was within the acceptable range. The results show that dd PCR has good repeatability. In the comparative study of dd PCR and Q PCR, we found that dd PCR has lower detection limit and higher sensitivity than Q PCR, and dd PCR has better repeatability than Q PCR, especially when the target gene is in low copy. In this study, dd PCR technique was used to dynamically monitor TP infection in rabbit model. It was found that nucleic acid level in peripheral blood of infected rabbits was earlier than serological level, which confirmed the feasibility of dd PCR technique. It is of great significance for the early diagnosis of very early syphilis, fetal syphilis, neurosyphilis and so on.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R759.1

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