【作者】 许静；

【导师】 夏超明；

【作者基本信息】 苏州大学 ， 病原生物学， 2011， 博士

【摘要】 研究表明核酸检测法用于日本血吸虫感染家兔血清特异性DNA的检测具有早期诊断和疗效考核价值。本研究成功建立了具有高度敏感性和特异性的LAMP法,并在前期中、高感染度动物模型的研究基础上,进一步探讨LAMP检测法对日本血吸虫低感染度家兔模型早期诊断和疗效考核的价值,并对LAMP体系进行了优化,同时评价了本课题组所建立的3种核酸检测法应用于日本血吸虫病患者的诊断及疗效考核效果。全文共分为五个部分:第一部分LAMP法反应体系的建立及对日本血吸虫感染家兔血清DNA的检测在已建立的两种PCR法核酸检测技术的基础上,本研究进一步建立了一种新型快速、灵敏的核酸检测法,即环介导的恒温扩增法(简称LAMP法),并与两种PCR法比较检测的敏感性和特异性,结果表明,LAMP法的敏感性为nested-PCR法的10倍,为普通PCR法的100倍,可扩增出含有1.02个拷贝的质粒DNA,亦未见与曼氏血吸虫、华支睾吸虫和旋毛虫的交叉反应。在日本血吸虫中高度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3d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17w时即转为阴性,与nested-PCR法转阴时间一致,较PCR法晚1w转阴,显示出其具有更高的敏感性。第二部分LAMP检测法反应体系及反应条件的优化对LAMP反应体系的优化结果表明,LAMP反应体系最佳内引物浓度为0.96μM,最佳Mg2+浓度为4mM,最佳dNTPs浓度为1.0mM。模板抽提法采用性价比较好的酚氯仿萃取法。向LAMP扩增产物中加入SYBR GreenⅠ染料,观察其颜色改变来判断扩增结果较在反应体系中加入荧光染料,通过观察荧光反应来判断扩增结果更为准确。第三部分LAMP检测法对低感染度日本血吸虫病早期诊断及疗效考核效果的评价在前期中度感染动物模型实验研究取得良好效果的基础上,建立了四组更加符合现场病人实际状况的低感染度动物模型,进一步评价核酸检测法特别是LAMP法的实际应用价值。结果显示三种核酸检测法均可从四种不同低感染度家兔模型感染后3d及1w~7w的外周血清中扩增到日本血吸虫特异性DNA片段,尤其是可从感染30条血吸虫尾蚴的家兔外周血清检测出特异性DNA片段,其EPG仅为14。对四种不同低感染度日本血吸虫病家兔模型疗效考核的结果表明,LAMP法对感染200条和100条日本血吸虫尾蚴的家兔均于治疗后第14w(感染后21w)血清中的血吸虫DNA转为阴性,与nested-PCR的阴转时间一致,较普通PCR法晚2w转阴。对感染50条和30条日本血吸虫尾蚴的家兔血清的检测结果则显示,LAMP法于治疗后第10w(感染后17w)宿主血清中的血吸虫DNA转为阴性,与nested-PCR的阴转时间一致,较普通PCR法晚1w转阴。结果表明LAMP法对家兔血清DNA检测的敏感性与nested-PCR法相当,较普通PCR法高,且疗效考核的效果与感染度相关,即感染度越重,血清中血吸虫DNA转阴所需时间越长。且LAMP法较普通PCR法和nested-PCR法结果更加直观,无需进行电泳,肉眼即可判断阳性结果,具有潜在的现场应用前景。第四部分LAMP检测法在日本血吸虫病患者诊断及疗效考核中的应用对110例粪检阳性的慢性病人血清DNA的检测结果表明,LAMP法的阳性率为95.5%(105/110),nested-PCR法的阳性率为89.1%(98/110),普通PCR法的阳性率为87.3%(96/110)。对45例非疫区正常人的检测结果显示,普通PCR法、nested-PCR法和LAMP法的特异性均为100%。三种核酸检测法即普通PCR法、nested-PCR法和LAMP法的阴性预测值(NPV)分别为87.5%、88.9%和95%,阳性预测值(PPV)分别为93.2%,91.6%和91.3%。对47例治疗后三个月、六个月和九个月病人血清的检测结果显示,三种核酸检测法的阴转率均随治疗后时间的延长而增加。LAMP法的阴转率分别为23.4%,61.7%和83.0%,普通PCR法的阴转率分别为31.2%,68.1%和89.4%,nested-PCR法的阴转率分别为27.7%,76.6%和85.1%。而免疫学诊断方法ELISA的阴转率分别为17.0%,19.1%和25.5%,IHA的阴转率分别为23.4%,42.5%和31.9%,三种核酸检测法和两种免疫学诊断法对治疗后九个月病人血清的阴转率存在显著差异,P<0.05。第五部分LAMP检测法对不同感染度日本血吸虫病患者的诊断及疗效考核价值对110例慢性病人血清和47例治疗后病人血清按照EPG水平进行分类,进一步评价LAMP检测法对日本血吸虫病患者的诊断及疗效考核价值。检测结果显示,LAMP法对高感染度病人血清的阳性检出率为100%(4/4),对中感染度的阳性检出率为92.3%(12/13),对低感染度的阳性检出率为95.7%(89/93)。普通PCR法对高中低度病人血清的阳性检出率分别为100%(4/4),84.6%(11/13),86.0%(80/93),nested-PCR法则分别为100%(4/4),100%(13/13)和87.1%(81/93),ELISA分别为100%,84.6%和83.9%,IHA分别为100%,92.3%和91.4%,三种核酸检测法与两种免疫学检测法的阳性检出率均与感染度相关。LAMP法对47例高、中、低感染度病人血清治疗后九个月的阴转率分别为100%,100%和79.5%,普通PCR法分别为100%,100%和87.2%,nested-PCR法分别为100%,100%和82.1%,两种免疫学检测法(ELISA和IHA)均维持在较低水平,阴转率分别在0%~28.2%和0%~33.3%之间,与核酸检测法的阴转率存在显著差异,P<0.05。LAMP法的敏感性与nested-PCR相当或稍高,显著高于普通PCR法,在对疗效考核的评价中,与nested-PCR法和普通PCR法具有较好的一致性,kappa值分别为0.604和0.735。但由于其操作简便、反应快速、结果直观、成本低廉,无需昂贵仪器,仅需一口水浴锅即可完成扩增,特别适宜于疫区现场应用。更多还原

【Abstract】 In our previous studies, nucleic acid detection assays have been proved a potential tool for the early detection and therapy evaluation of schistosomiasis japonica in rabbit models with moderate and heavy intensity infection. In this study, LAMP method was successfully established, and four groups of light-infection rabbit models, which was more consistent with the current epidemiological situation were used to evaluate the utility of LAMP for early detection and therapy evaluation of schistosomiasis. Meanwhile, LAMP assay was optimized. Then three established methods, such as common PCR, nested-PCR and LAMP were compared together to evaluate the ability of LAMP assay for detedction of S. japonicum infection and efficacy of chemotherapy. This study includs five parts:PartⅠDetection of S. japonicum DNA in serum of rabbit model by established LAMP assayOn the basis of PCR assay, LAMP method was established. The detectin limit was 1.02 copies, which was 10 times more sensitive than nested-PCR, and 100 times more sensitive than common PCR. No cross-reactivity was observed with S. mansoni, C. sinensis and T. spiralis. For early detection and therapy evaluation in a rabbit model infected with 500 S. japonicum cercariae, LAMP could also amplify the specific DNA fragment from the serum at the 3rd day post-infection, and the DNA detection became negative at the 17th week post-treatment, which was consistent with nested-PCR, but was 1 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 for field diagnosis in endemic area. PartⅡOptimization of LAMP reaction system and reaction coditionThe optimum reaction system of LAMP: In a total volum of 25μL, contains 1×Bst-DNA polymerase buffer, 4mmol/L MgSO4, 1.0mmol/L dNTP, 0.24mmol/L out primers, 0.96mmol/L inner primers, 0.8mol/L betaine, 8 U Bst-DNA polymerase, and 4μL template DNA. Traditional phenol-chloroform method was selected to extract DNA from serum samples, compared with other three DNA extraction kits. Observing the color change(from orange to green) is more accurate to identify the amplification by addition of SYBR GreenⅠafter incubation.PartⅢLAMP assay for early detection and therapy evaluation of light-infection schistosomiasis japonicumFour grades of light-infection rabbit models infected with 200, 100, 50 and 30 S. japonicum cercariae were established, to evaluate the utility of nucleic acid amplification methods for field diagnosis, especially for LAMP assay. The results showed that all the three amplification methods, including common PCR, nested PCR and LAMP assay, could detect S. japonicum DNA at the 3rd day post-infection in four different groups of light-infected rabbit model, especially for the rabbit infected with 30 S. japonicum cercariae which EPG was 14. But LAMP assay was the most rapid method, and the result was easier to observe than the other to PCR methods. For the therapy evaluation, LAMP detection became negative at 14 weeks post-treatment (21 weeks post-infection) in groupⅠand groupⅡ, which was consistent with nested-PCR assay, but was 2 weeks later than common PCR. In groupⅢand groupⅣ, LAMP and nested-PCR detection became negative at 10 weeks post-treatment (17 weeks post-infection), which was one week longer than common PCR, indicating a higher sensitivity of LAMP than the PCR method. The results showed chemotheraputic efficacy was correlated with the infection intensity. LAMP assay had a higher sensitivity than common PCR, and was more rapid than nested-PCR, had a potential for field diagnosis in endemic area.PartⅣApplication of LAMP for clinical diagnosis of schistosomiasis japonicum and for evaluating chemotheraputic efficacyAfter getting the encouraging results from animal model, LAMP was used to detect 110 sera samples of patient, the results showed that the positive rate(95.5%) of LAMP was higher than common PCR(87.3%) and nested-PCR(89.1%). For 45 sera of healthy persons from non-endemic area, no positive result was found by all the three nucleic acid amplification methods. The specificity of the three assays was 100%. The negative predictive value(NPV) of common PCR, nested-PCR and LAMP was 87.5%, 88.9%, 95%, respectively. While the positive predictive value(PPV) of the three methods was 93.2%, 91.6%, 91.3%, respectively. And for 47 sera of patient after 3 months, 6months and 9 months post-treatment, the negative rate of LAMP was 23.4%, 61.7%, 83.0%, respectively. And the negative rate of PCR was 31.2%, 68.1%, 89.4%, respectively, and was 27.7%, 76.6%, 85.1% for nested- PCR. For the two immunologic methods(ELISA and IHA), the negative rate was 17.0%, 19.1%, 25.5% and 23.4%, 42.5%, 31.9%, respectively. The difference between nucleic acid detection methods and immunological assays was significant, P<0.05.PartⅤEvaluation of LAMP for detection of different intensity infection of schistosomiasis in patient and for evaluation of chematheraputic efficacyTo evaluate the clinical utility of LAMP for diagnosis of patients and for therapy evaluation, 110 sera of patinets with chronic disease and 47 sera of patients post-treatment were classfied according to the level of EPG. The detection results showed that the possitive detection rate of LAMP for heavy intensity infection patients was 100%(4/4), for moderate intensity infection patients was 92.3%(12/13), and for light intensity infection patients was 95.7%(89/93). The positive rate of common PCR was100%(4/4), 84.6%(11/13), 86.0%(80/93), respectively. And was 100%(4/4), 100%(13/13), 87.1%(81/93) for nested-PCR. While for two immunological methods(ELISA and IHA), the positive rate was 100%, 84.6%, 83.9% and 100%, 92.3%, 91.4%, respectively. The detection rate of three nuleic acid amplification method and two immunological methods was correlated with the intensity of infection. For therapy evaluation, the negative rate for heavr, moderate and light intensity infection of LAMP in sera at 9 months post-treatment was 100%,100% and 79.5%, respectively. Common PCR was 100%, 100% and 87.2%, respectively. And was 100%, 100%, 82.1% for nested-PCR. The negative rate of ELISA and IHA maintained at a low level, ranged from 0%~28.2% and 0%~33.3%, respectively. The difference between nucleic acid detection methods and immunological assays was significant, P<0.05. The negative rate was correlated with infection intensity and the duration post-treatment.LAMP assay had a similar sensitivity with nested-PCR, and was higher than common PCR assay. The consistency of LAMP for therapy evaluation with nested-PCR and common PCR was well,kappa index was 0.604 and0.735, respectively. But LAMP has several advantages over conventional PCR and nested-PCR assays. LAMP is more rapid and simple, does not require sophisticated equipment, only a water bath can accomplish the whole reaction process, and the amplification can be visually observed, which is of great value for applications in poorly equipped laboratories or in large-scale epidemiological studies conducted in isolated areas.更多还原