本文選題：巢式PCR + 微滴式數(shù)字PCR�。� 參考：《安徽醫(yī)科大學(xué)》2017年碩士論文

【摘要】：第一部分各期梅毒外周血中梅毒螺旋體核酸檢出率的研究目的利用巢式PCR技術(shù)探究各期梅毒患者外周血中梅毒螺旋體DNA的檢出率方法臨床收集192例梅毒患者外周血樣本,其中包括37例一期梅毒,92例二期梅毒,63例潛伏梅毒,利用TP0105和TP0574基因片段特異的內(nèi)外引物,行巢式PCR技術(shù)對(duì)樣本進(jìn)行檢測(cè)。結(jié)果一期梅毒患者外周血中梅毒螺旋體的檢出率為45.9%(TP0105)和40.5%(TP0574),兩基因的總檢出率為48.6%;二期梅毒患者外周血中梅毒螺旋體的檢出率為52.2%(TP0105)和52.2%(TP0574),兩基因的總檢出率為62.0%;潛伏梅毒患者外周血中梅毒螺旋體的檢出率為6.3%(TP0105)和4.8%(TP0574),兩基因的總檢出率為7.9%。兩基因的檢出率在統(tǒng)計(jì)學(xué)上沒(méi)有差異(P0.05),Kappa值為0.714。結(jié)論梅毒螺旋體基因可以在螺旋體感染的各個(gè)階段的梅毒患者外周血中檢測(cè)到,其中在一期梅毒和二期梅毒中的檢出率最高。巢式PCR技術(shù)在早期梅毒的診斷中起到了重要的作用,可以在臨床上用于暗視野鏡檢和血清學(xué)檢測(cè)的補(bǔ)充診斷標(biāo)準(zhǔn)。第二部分基于微滴式數(shù)字PCR技術(shù)的梅毒螺旋體核酸絕對(duì)定量方法的建立及應(yīng)用微滴式數(shù)字PCR技術(shù)是近年來(lái)迅速發(fā)展起來(lái)的一項(xiàng)基于單分子PCR方法來(lái)進(jìn)行計(jì)數(shù)的核酸分子絕對(duì)定量技術(shù),目前基于該技術(shù)的梅毒螺旋體絕對(duì)定量方法還未有文獻(xiàn)報(bào)道。本研究建立了微滴式數(shù)字PCR梅毒螺旋體核酸絕對(duì)定量檢測(cè)平臺(tái),從特異性、靈敏度、可信度、重復(fù)性幾個(gè)方面對(duì)平臺(tái)進(jìn)行評(píng)估,又將建立好的dd PCR檢測(cè)平臺(tái)應(yīng)用于TP感染兔模型外周血的核酸檢測(cè),確定該技術(shù)的可行性。本研究設(shè)計(jì)了針對(duì)基因分別為T(mén)P0105和TP0574的特異性探針,利用ddPCR技術(shù)檢測(cè)了健康人、非梅毒病人、正常兔、鼠血漿中的目的基因片段,確定了該技術(shù)的空白下限值(LOB)為3.2copies(TP0105)和1.4copies(TP0574),為后續(xù)對(duì)于臨床樣本的檢測(cè)確定了cut-off值。用dd PCR技術(shù)對(duì)各稀釋梯度的質(zhì)粒進(jìn)行核酸定量檢測(cè),確定該檢測(cè)技術(shù)的高靈敏度,最低檢測(cè)下限(LOD)為單拷貝。將每個(gè)梯度質(zhì)粒經(jīng)dd PCR檢測(cè)出的實(shí)際拷貝數(shù)與計(jì)算出的理論拷貝數(shù)進(jìn)行相關(guān)分析,發(fā)現(xiàn)二者呈高度相關(guān)性,說(shuō)明dd PCR技術(shù)的檢測(cè)結(jié)果是可信的。為了評(píng)估dd PCR的重復(fù)性,我們對(duì)各稀釋梯度兔睪丸懸液的檢測(cè)均進(jìn)行了三次重復(fù),對(duì)三次拷貝數(shù)結(jié)果進(jìn)行分析研究發(fā)現(xiàn)每個(gè)稀釋梯度的RSD值均在可接受的范圍內(nèi),表明dd PCR具有很好的可重復(fù)性。在dd PCR與q PCR的對(duì)比研究中我們發(fā)現(xiàn)dd PCR具有比q PCR更低的檢測(cè)下限,靈敏度更高,且dd PCR具有比q PCR更好的重復(fù)性,特別是在目的基因處于低拷貝的情況下。本研究利用dd PCR技術(shù)動(dòng)態(tài)監(jiān)測(cè)了兔模型對(duì)TP的感染情況,發(fā)現(xiàn)感染兔外周血中核酸水平的出現(xiàn)早于血清學(xué)水平,證實(shí)了dd PCR技術(shù)的可行性,為后續(xù)對(duì)于臨床樣本的核酸絕對(duì)定量研究奠定了基礎(chǔ),對(duì)指導(dǎo)用于極早期梅毒、胎傳梅毒、神經(jīng)梅毒等早期診斷具有重要意義。
[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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R759.1

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