【作者】 许静；

【导师】 夏超明；

【作者基本信息】 苏州大学 ， 病原生物学， 2008， 硕士

【摘要】 本研究拟在课题组在已成功建立PCR法检测日本血吸虫特异性DNA的研究基础上,进一步探索核酸检测技术在日本血吸虫病早期诊断及疗效考核中的应用。首先验证了PCR法对日本血吸虫感染家兔模型的不同组织样本(包括日本血吸虫成虫、虫卵、感染家兔的肝组织、粪便和外周血清)中日本血吸虫特异性DNA的检测结果,且进一步确定了PCR法检测的敏感性和特异性,结果显示其敏感性为80fg/μL的DNA水平,所检测的DNA片段是日本血吸虫特异的,与曼氏血吸虫及华支睾吸虫均无交叉反应。对感染家兔的血清DNA的检测结果证明了PCR法对日本血吸虫感染的早期诊断和疗效考核价值,即可从感染后第1w的血清中扩增出特异条带,于治疗后第12w即转为阴性,与以往研究结果基本一致。在已建立的PCR法的基础上,本研究进一步建立了一种新型快速、灵敏的核酸检测法,即环媒介导的恒温扩增法(简称LAMP法),并与PCR法比较检测的敏感性和特异性,结果表明,LAMP法的敏感性显著高于PCR法,约为PCR法的104倍,可扩增出极微量水平的DNA(0.08fg/μl),亦未见与曼氏血吸虫、华支睾吸虫的交叉反应。在日本血吸虫感染家兔模型的早期诊断和疗效考核的实验中,LAMP法同样可从感染后第1w的血清中检测出阳性结果,对疗效考核的评价表明LAMP法在治疗后第13w时即转为阴性,较PCR法晚一周转阴,显示出其具有更高的敏感性。为深入探讨日本血吸虫感染宿主血清中核酸物质的来源,本实验建立了单、双性感染小鼠模型进行研究。结果初步证实在日本血吸虫感染早期,血清中的核酸物质可能主要来源于移行过程中死亡的童虫以及虫体发育过程中更新脱落的表皮组织,而在感染的急性期和慢性期,小鼠血清中的核酸物质可能主要来源于虫卵的分解产物。从而为应用核酸检测法早期诊断日本血吸虫感染,尤其是为疗效考核提供了理论依据。在动物模型实验研究的基础上,应用两种核酸检测法检测了30例日本血吸虫病慢性期病人血清。结果显示,PCR法可从30例病人血清中检测出18例阳性结果,阳性率为60%,而LAMP法可检测出29例阳性结果,阳性率为96.7%。研究结果表明LAMP法是一种特异而又灵敏的快速核酸检测技术,且其操作简便,结果判断直观,具有应用于血吸虫病的早期诊断和疗效考核的潜在价值,值得进一步深入研究。更多还原

【Abstract】 In order to observe the stability of PCR assay for early diagnosis and therapy evaluation in S. japonicum infection, PCR assay was repeatedly used to detect different samples from rabbit infected with S. japonicum, including adult worms, liver homogenate, eggs, feces and sera. The results showed that a specific DNA fragment of S. japonicum was amplified by PCR when the different samples DNA was used as templates, and had no cross-reactivity between S. mansoni and C. sinensis. The minimum amounts of DNA detectable using PCR assay were 800fg/μl. As to the early diagnosis and therapy evaluation, PCR assay could detect the specific DNA at the 1st week post-infection, and then the DNA in serum became negative at the 12th week post-treatment. These data were consistent with the former findings.On the basis of PCR assay, a new nucleic acid assay was developed and evaluated in this research, which was called Loop-mediated isothermal amplification (LAMP). It had provided higher sensitivity and specificity than PCR assay. The minimum amounts of DNA detectable using LAMP assay was 0.08fg/μl, which was 104 times more sensitive than PCR assay. Similarly, no cross-reactivity was observed between S. mansoni and C. sinensis. And the results of LAMP detection, in the sera of rabbit infected with S. japonicum from 1w to 30w, were compared with PCR assay. LAMP could also amplify the specific DNA fragment in sera at the 1st week post-infection for the early diagnosis, and the DNA detection became negative at the 13th week post-treatment for the therapy evaluation. One week later than PCR assay, due to its higher sensitivity. However, LAMP assay was easy to perform, reacted rapidly and was inexpensive, it may therefore be applied in the routine diagnosis in human.In order to investigate the source of nucleic acid in the sera of host infected with S. japonicum, single or dual sex cercariae of S. Japonicum infected mice models were set up for further research. And the results showed that in the early infectious period, the nucleic acids may mainly come from schistosomula and the epidermis tissue of worm, however, in the acute and chronic stages, they may essentially come from tissues with broken or dead eggs. It concluded that nucleic acid assays were useful methods for early detection, and had potential value for therapy evaluation.At the base of the encouraging results from animal model, both methods were used to detect 30 cases sera of patients, and the results showed that PCR assay could detect 18 positive cases(positive rate was 60.0%), while LAMP could detect 29 positive cases(positive rate was 96.7%). All these data suggested that LAMP assay was more sensitive than PCR assay, meanwhile it was easy to perform, and the results were intuitionistic, therefore it is a potential tool for early diagnosis and therapy evaluation in S. japonicum infection.更多还原