南京地区日本血吸虫病的流行病学调查及其基于血清差异多肽检测方法的建立与应用
发布时间：2018-07-17 02:31
【摘要】：血吸虫病是对人与动物危害最严重的寄生虫病之一,是世界卫生组织认定的分布范围最广、经水传播疾病(Water-borne diseases)感染率最高的寄生虫病。至2015年底,江苏省除南京市四个区县(栖霞、六合、浦口、江宁)与镇江市3个区县(丹徒、扬中、润州)尚处于血吸虫病控制阶段,其余各市(县、区)均已达血吸虫病传播阻断标准。然而,长江中上游部分省市仍然处于疫情控制阶段,其阳性钉螺与感染病人病畜仍时有发现,部分达标地区存在钉螺复燃情况,且钉螺(Oncomelania hupensis)可随水系扩散与人群流动频繁等因素,对江苏血防构成较大的威胁。因此,对当前低流行态势下的重点区域血吸虫病流行情况做一全面调查,对血防策略的调整具有重要意义。血防监测包括对血吸虫中间宿主(钉螺)分布、终宿主(人与易感动物)的流行病学调查、应用“哨鼠”开展早期预警、晚期血吸虫病人的诊断与治疗。在当前低流行态势下,以抗体ELISA检测、粪便查虫卵为主的方法,已满足不了早期监测的要求,亟需发展快速、准确的检测新方法：寄生虫学剖检查成虫的哨鼠检测方法也存在时效性差、耗时耗力、准确性不高等缺点,迫切需要发展更为准确、快速、灵敏的方法；再者,针对新发展晚血病人与现症病人的鉴别,在血吸虫病低流行态势下,需建立新的辅助诊断方法。本研究在南京地区开展了血吸虫病流行病学调查,并基于血清差异多肽建立诊断新方法以及应用于实验小鼠、预警“哨鼠”及晚血与现症病人的鉴别诊断。1.钉螺及其感染尾蚴与人畜感染日本血吸虫抽样调查2013-2014年在栖霞区、2015年在高淳区的芜申运河水阳江段开展了钉螺密度与人畜感染情况调查。方法：根据《血吸虫病控制与消除标准GB15976-2015》,机环结合法开展钉螺调查,DDIA血清学与尼龙绢袋集卵孵化粪检法开展人群调查,塑料杯顶管粪便孵化法检测动物血吸虫感染；并结合往年报表数据分析。结果：栖霞区与高淳区的水阳江段均未查到阳性钉螺：但水阳江段活螺密度显著高于其他地区,提示水阳江段具备血吸虫病传播风险。人群调查显示,栖霞区2个行政村的血清学检查阳性率均低于1%,高淳区3个行政村血检阳性率2.48%,粪检均未查出阳性。家畜粪检未检测到感染。结论：综合2004-2014江苏省血吸虫病防治工作报表分析,江苏省内有螺面积逐年减少,活螺数与活螺密度维持在相对稳定的较低水平；血吸虫病呈低度流行态势,近年无血吸虫急性感染病例报道；高淳区水阳江段血检阳性率高于1%的血吸虫病传播控制标准,也高于全省其他地区的血清阳性率,提示该地区具有较高风险,需加强疫情监测。2.实验兔血清肽谱分析及基于血血清差异多肽检测法的建立分析血吸虫急性感染实验兔的血清肽质量指纹图谱,并根据血清差异多肽建立诊断方法。方法：通过血清采集、磁珠富集血清多肽、MALDI-TOF/TOF质谱检测与ClinProTools分析血清肽质量指纹图谱,建立人工感染兔的血清差异多肽诊断方法(CPT方法),并进行敏感性与特异性分析,比较多种方法检测不同感染时期的准确性。结果：Mr 1787蛋白分子在兔感染后第6d有显著上调(P0.05)；在感染后第9 d,Mr 2834蛋白分子有极显著上调(P0.01),Mr 3483呈显著上调(P0.05),Mr 4018有显著下调(P0.05)。在感染后第12 d,Mr 3151蛋白分子有显著下调(P0.05),而Mr 4018则出现极显著下调(P0.01),Mr 3530呈显著上调(P0.05),两者趋势一直持续至感染后第30 d。经二级质谱鉴定并搜库比对,预测Mr 1787蛋白为α烯醇化酶的片段,Mr 3530预测为热休克蛋白90或热休克蛋白47片段,提示此两种蛋白可能为血吸虫感染兔的早期疾病标志物。基于感染第5周与健康对照组血清差异多肽指纹图谱,利用ClinProTools软件建立感染组与健康对照组的区别诊断模型,盲测样本显示,感染1、2、3、4 w的家兔血清鉴定为阳性的占30%、55%、75%、80%,而同期ELISA检测结果分别为0,0,20%和50%,利用弓形虫感染血清验证,结果显示特异性好。结论：基于血清差异表达多肽建立的诊断方法,敏感性要优于常规的ELISA法,有望应用于血吸虫病早期检测并筛选具有诊断价值的血清多肽(蛋白)标志物。3.血血清多肽检测方法的应用3.1血清差异多肽检测法在实验小鼠模型上的应用小鼠动物模型在血吸虫感染实验及相关研究中应用广泛,发展对其快速、准确、简便的诊断方法意义重大。方法：采用血清富集并磁珠纯化多肽、MALDI-TOF/TOF质谱检测与ClinProTools分析肽质量指纹图谱,根据急性感染第5 w与健康对照组血清差异表达多肽,建立检测方法(CPT方法),并比较ELISA、寄生虫学剖检等多种方法的检测敏感性,并验证急性感染不同时期、不同感染度小鼠的准确性。结果：与传统的ELISA方法比较,CPT方法在第1w即检测到急性感染小鼠5%(3/60)的阳性率,在第2w至第5w呈逐渐上升趋势,分别为35%(21/60),75%(45/60),87.93%(51/58)与98.15%(53/54),而ELISA方法在感染后第3w才检测到阳性,其3-5 w的感染率分别为65%(39/60),77.59%(45/58)与94.44%(51/54),均低于CPT方法；寄生虫学剖检在第3、4 w有70%(7/10)的小鼠被检出阳性,低于CPT方法同期检出率。针对不同感染度小鼠的诊断,在感染14,18,22条尾蚴的小鼠中,所有小鼠均被预测为感染,准确性达100%,感染4、6、10条尾蚴组的小鼠预测准确率分别为40%(4/10),50%(5/10)与80%(8/10)。结论：血清差异多肽检测法较血清抗体ELISA检测法至少提前2-3 w,较寄生虫学剖检极大的降低了工作量,可以用于低度感染状态下的实验小鼠检测和样本初筛,有望为“哨鼠”检测提供新的方法。3.2血清差异多肽检测法在血防“哨鼠”预警上的应用利用“哨鼠”监测长江水体血吸虫感染风险,是我省血防预警工作的重要手段,寄生虫学剖检查成虫与肝脏虫卵结节是判断“哨鼠”感染与否的主要手段,但时效性较差,且耗时耗力,需要发展新的快速检测方法。方法：剖检2015年现场试验的660只哨鼠(含栖霞与高淳60只、宁镇扬600只)并采集血清、5例本室保存的经解剖查成虫确诊的阳性“哨鼠”与50例阴性“哨鼠”血清,经血清多肽富集与质谱检测,获取多肽指纹图谱,并导入已建立的血清多肽检测方法(CPT法)验证。结果：5例阳性“哨鼠”与50例阴性“哨鼠”均可以被准确诊断,2015年现场试验的660只哨鼠经CPT验证为未感染,与寄生虫学剖检结果一致。结论：利用血清差异表达多肽检测方法,较常规“哨鼠”解剖查成虫方法,节省人力物力,并可以提早1-2w对“哨鼠”进行初筛,缩短了检测血吸虫感染的“窗口期”可与常规病原学检测方法联合应用于“哨鼠”早期预警。3.3血清差异多肽检测法在鉴别晚血病人上的应用晚期血吸虫病人的诊断及治疗是血防工作重点,尤其在低流行态势下,对血吸虫病地方考核更为重要。病原学检查与抗体检测,很难区别现症与既往病人。方法：采集晚血、新发展晚血、晚血治愈、健康对照四组人群,血清多肽指纹图谱检测并应用ClinProTools分析,建立基于新发晚血组与健康对照组血清差异多肽的检测方法。结果：晚血与新发晚血、新发晚血组中切脾与否在血清肽谱无显著差异。与健康对照组相比,晚血组在Mr 2662,2991,3241,5337与5906处均显著下调,在Mr 2082与4282处均显著上调；而有史治愈组与健康对照组比较发现,在Mr 2662,4209,5337与5906处显著下调,2082处显著上调。新发晚血组与有史对照组相比,在Mr 3241处显著下调。提示Mr 3241显著下调、4282显著上调可以作为判定新发晚血的指征,二级质谱鉴定并搜库,推测该两种多肽(蛋白)均为免疫球蛋白重链可变区。根据新发晚血组与健康对照组的差异表达多肽建立的诊断方法,盲测试验显示,晚血组、有史治愈组、无血吸虫感染组均与晚血病人具有一致的血清肽谱特征,而粪检阳性病人血清有73.3%(11/15)无法被验证,不能归于新发晚血或健康对照组,水阳江段流行病学调查获取的血清抗体阳性但粪检阴性样本有86.2%(25/29)被验证为晚血组。结论：血清差异多肽检测法可以间接将晚血病例与现症病例区分,但后者可能包括既往感染或反复感染,故有部分仍然被验证为晚血组,提示感染者有进一步发展为晚血病人的可能；水阳江段流行病学调查人群可能为既往感染,非现症病人；血清差异多肽检测联合常规寄生虫学检查,可以对血吸虫流行病学调查进行初筛。
[Abstract]:Schistosomiasis is one of the most serious parasitic diseases that have the most serious harm to people and animals. It is the most widely distributed WHO identified by the WHO. By the end of 2015, there are four districts and counties in Nanjing (Qixia, Liuhe, Pukou, Jiangning) and 3 districts and counties of Zhenjiang (Dantu, Yangzhong). However, some provinces and cities in the upper and middle reaches of the Yangtze River are still in the stage of epidemic control. However, the positive snails and infected animals are still found in the upper and middle reaches of the Yangtze River, and the reburning of Oncomelania snails in some of the standard areas and the Oncomelania snails (Oncomelania hupensis) can be found. Such factors as the diffusion of water system and the frequent flow of people constitute a great threat to the schistosomiasis in Jiangsu. Therefore, a comprehensive survey of the epidemic situation of schistosomiasis in the key areas under the current low epidemic situation is of great significance to the adjustment of the strategy of schistosomiasis. The epidemiological investigation of animals, the application of the "sentinel" early warning, the diagnosis and treatment of advanced schistosomiasis patients. Under the current low epidemic situation, the method of detecting the eggs with antibody ELISA and fecal egg detection has not met the requirements of early monitoring. It is urgent to develop a fast and accurate new method for detection of adult worms. The detection methods of sentinel mice also have the disadvantages of poor timeliness, time consuming, and low accuracy. It is urgent to develop a more accurate, rapid and sensitive method. Furthermore, a new method of auxiliary diagnosis should be established for the identification of the newly developed late blood patients and the present patients in the low epidemic situation of schistosomiasis. This study has carried out blood in the Nanjing area. The epidemiological investigation of the parasitics and the establishment of a new diagnostic method based on the serum differential polypeptide and the application in the experimental mice. The differential diagnosis of the early warning "sentinel" and the late blood and the present disease patients.1. snail and its infected cercariae and human and animal infection of Schistosoma japonicum in Qixia District in 2013-2014 years, in 2015 in Gaochun District Wushen canal water Yangjiang A survey on the density of Oncomelania snails and human and animal infection was carried out in the section. Methods: according to the control and elimination standard of schistosomiasis and elimination standard GB15976-2015>, the investigation of Oncomelania snails, DDIA serology and nylon spun hatching fecal test were carried out to conduct a population survey, and the hatching method of plastic cup pipe jacking excrement was used to detect the infection of Schistosoma japonicum. Data analysis. Results: there were no positive Oncomelania snails in the water Yangjiang section of Qixia and Gaochun areas, but the density of living snail was significantly higher in the Shui Yang section than in other areas, suggesting the risk of schistosomiasis transmission in the Shui Yang section. The survey showed that the positive rate of the serological examination of the 2 administrative villages in Qixia was lower than 1%, and the blood test of 3 administrative villages in Gaochun area was positive. The sex rate was 2.48%, the fecal examination was not detected. Conclusion: comprehensive 2004-2014 Jiangsu province schistosomiasis prevention and control work report analysis, the snail area in Jiangsu province is decreasing year by year, the number of living snails and living snail density maintain relatively low level relatively stable; schistosomiasis is low epidemic situation, and in recent years there is no schistosomiasis sensual sex. The infection cases reported that the positive rate of blood test in the Gaochun district was higher than 1% of the standard of schistosomiasis transmission control. It was also higher than the positive rate in other regions of the province, suggesting that the region has high risk. It is necessary to strengthen the analysis of the serum peptide spectrum of the.2. experimental rabbit and the analysis of the blood serum differential polypeptide detection method based on the detection of blood serum differential polypeptide. The quality fingerprint of the serum peptide of the experimental rabbits was dyed and the diagnostic method was established according to the serum polypeptide. Methods: the serum peptide was enriched by the serum collection, the magnetic beads were enriched and the quality fingerprint of the serum peptide was detected by MALDI-TOF/TOF mass spectrometry and ClinProTools analysis, and the diagnostic method of serum differential polypeptide (CPT method) was established. A variety of methods were used to detect the accuracy of different infection periods. Results: Mr 1787 protein molecules were significantly up-regulated (P0.05) in 6D after infection, and at ninth d after infection, Mr 2834 protein molecules were significantly up-regulated (P0.01), Mr 3483 was significantly up (P0.05), Mr 4018 was significantly down (P0.05). Twelfth d after infection, Mr 31 The 51 protein molecules had a significant downregulation (P0.05), while Mr 4018 had a very significant downregulation (P0.01), and Mr 3530 was significantly up-regulated (P0.05). The two trends continued until the infection thirtieth D. were identified by two mass spectrometry and searched the ratio. The Mr 1787 protein was predicted to be an alpha enolase fragment, Mr 3530 was predicted as a heat shock protein 90 or a heat shock protein 47 fragment. The two proteins may be an early disease marker of rabbits infected by Schistosoma japonicum. Based on the differential peptide fingerprint of the serum of the healthy control group for fifth weeks and the healthy control group, the differential diagnosis model of the infection group and the healthy control group was established by ClinProTools software. The blind samples showed that the sera of the rabbits infected with 1,2,3,4 w accounted for 30%, 55%, 75%, respectively. 80%, and the results of ELISA detection in the same period were 0,0,20% and 50% respectively. The results showed that the specificity of Toxoplasma infection was good. Conclusion: the diagnostic method based on the establishment of serum differential expression polypeptide is better than the conventional ELISA method. It is expected to be applied to the early detection of schistosomiasis and screening the diagnostic value of serum polypeptide (protein). Application of marker.3. blood serum polypeptide detection method 3.1 The Application of serum differential polypeptide detection method in experimental mice model is widely used in the experiment of Schistosoma infection and related research. It is of great significance to develop a rapid, accurate and simple diagnostic method for its rapid, accurate and simple diagnostic methods. TOF/TOF mass spectrometry detection and ClinProTools analysis peptide mass fingerprint, according to the acute infection fifth W and the healthy control group serum differential expression polypeptide, establish the detection method (CPT method), and compare the ELISA, the Parasitology examination and so on various methods detection sensitivity, and verify the acute infection different period, the different infection degree mice accuracy. Result: compared with the traditional ELISA method, the CPT method detected the positive rate of 5% (3/60) in acute infection mice at 1W, and gradually increased in 2W to 5W, 35% (21/60), 75% (45/60), 87.93% (51/58) and 98.15% (53/54), while ELISA method was positive in the post infected 3W. The 3-5 infection rate was 65% (77.59%), 77.59% respectively. (45/58) and 94.44% (51/54) were lower than the CPT method; parasitology was detected in 70% (7/10) mice in 3,4 W, lower than the CPT method in the same period. In mice infected with different infection degrees, all mice infected with cercariae were predicted to be infected with the accuracy of 100%, infected with the small cercariae group. The accuracy rate of rat prediction was 40% (4/10), 50% (5/10) and 80% (8/10). Conclusion: the serum differential polypeptide detection method is at least 2-3 w ahead of the serum antibody ELISA detection method, which can greatly reduce the workload and can be used for the detection of experimental rats and the initial screening of samples under the condition of low degree infection. It is expected to provide a new method for the detection of "sentinel". Methods the application of.3.2 serum differential polypeptide detection method in the early warning of schistosomiasis "sentinel" was used to monitor the risk of Schistosoma infection in the Yangtze River. It is an important means for the early warning of schistosomiasis in the Yangtze River. The Parasitology examination of adult and liver egg nodules is the main means to judge whether the "sentinel" is infected or not, but the timeliness is poor, and A new rapid detection method is needed to develop a new rapid detection method. Methods: 660 sentinel mice (including 60 Qixia and Gaochun, Ningzhenyang, Ningzhenyang) in 2015 were examined and serum was collected. 5 cases of positive "sentinel" and 50 negative "sentinel" serums, which were confirmed by anatomic adults, were preserved in this room, and were detected by serum polypeptide enrichment and mass spectrometry. The peptide fingerprint was used and the established serum polypeptide detection method (CPT method) was introduced. Results: 5 cases of positive "sentinel" and 50 negative "sentinel" could be accurately diagnosed. In 2015, 660 sentinel mice were tested by CPT as uninfected and consistent with the results of parasitic examination. The method, compared with the conventional "sentinel" dissection method, saves manpower and material resources, and can early screen the "sentinel" by 1-2W, shortening the "window period" of detecting schistosomiasis infection, which can be applied to the early warning of "sentinel" in the early warning of "sentinel".3.3 serum differential polypeptide detection method in the identification of late blood patients. The diagnosis and treatment of patients with advanced Schistosoma is the focus of schistosomiasis, especially in the low epidemic situation, it is more important for the local examination of schistosomiasis. It is difficult to distinguish between the present and the past patients by the pathogenic examination and the antibody test. Methods: collecting late blood, new development of late blood, late blood cure, healthy control four groups, serum polypeptide fingerprint atlas inspection ClinProTools analysis was used to establish a method for the detection of serum differential peptide in the new late blood group and the healthy control group. The results showed that there was no significant difference in the serum peptide spectrum between the late blood and the new late blood and the new late blood group. Compared with the healthy control group, the late blood group decreased significantly at Mr 2662299132415337 and 5906, at Mr 2082. Compared with the healthy control group, there was a significant decrease in Mr 266242095337 and 5906, and the 2082 up regulated significantly in Mr 266242095337 and 5906. The new late blood group was significantly lower in the Mr 3241 than in the history control group, suggesting that the Mr 3241 was significantly down, and the 4282 up up could be used as a indication of the new late blood and two grade. The two peptides (proteins) were all the variable regions of the immunoglobulin heavy chain. According to the diagnostic method of the differential expression of polypeptide in the new late blood group and the healthy control group, the blind test showed that the late blood group had a history cure group and the non schistosomiasis infection group had the same serum peptide spectrum characteristics with the late blood patients, and the fecal test was positive. 73.3% (11/15) of the sera of the sex patients could not be verified, and could not be attributed to the new late blood or the healthy control group. The serum antibody positive of the water Yang river section was positive but 86.2% (25/29) of the negative samples of the feces was confirmed as the late blood group. Conclusion: the serum differential polypeptide detection method can be used to distinguish the late blood cases from the present cases indirectly, but the latter is the latter. It may include previous infection or recurrent infection, and some of them are still proved to be late blood groups, suggesting the possibility of further development of late blood patients; the epidemiologic survey of the Shui Yang section may be a previous infection, not a present, and a serum differential polypeptide detection combined with a regular parasitic examination can be used for the epidemic of schistosomiasis. A preliminary screening of the study is carried out.
【学位授予单位】：扬州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R532.21
本文编号：2128620
[Abstract]:Schistosomiasis is one of the most serious parasitic diseases that have the most serious harm to people and animals. It is the most widely distributed WHO identified by the WHO. By the end of 2015, there are four districts and counties in Nanjing (Qixia, Liuhe, Pukou, Jiangning) and 3 districts and counties of Zhenjiang (Dantu, Yangzhong). However, some provinces and cities in the upper and middle reaches of the Yangtze River are still in the stage of epidemic control. However, the positive snails and infected animals are still found in the upper and middle reaches of the Yangtze River, and the reburning of Oncomelania snails in some of the standard areas and the Oncomelania snails (Oncomelania hupensis) can be found. Such factors as the diffusion of water system and the frequent flow of people constitute a great threat to the schistosomiasis in Jiangsu. Therefore, a comprehensive survey of the epidemic situation of schistosomiasis in the key areas under the current low epidemic situation is of great significance to the adjustment of the strategy of schistosomiasis. The epidemiological investigation of animals, the application of the "sentinel" early warning, the diagnosis and treatment of advanced schistosomiasis patients. Under the current low epidemic situation, the method of detecting the eggs with antibody ELISA and fecal egg detection has not met the requirements of early monitoring. It is urgent to develop a fast and accurate new method for detection of adult worms. The detection methods of sentinel mice also have the disadvantages of poor timeliness, time consuming, and low accuracy. It is urgent to develop a more accurate, rapid and sensitive method. Furthermore, a new method of auxiliary diagnosis should be established for the identification of the newly developed late blood patients and the present patients in the low epidemic situation of schistosomiasis. This study has carried out blood in the Nanjing area. The epidemiological investigation of the parasitics and the establishment of a new diagnostic method based on the serum differential polypeptide and the application in the experimental mice. The differential diagnosis of the early warning "sentinel" and the late blood and the present disease patients.1. snail and its infected cercariae and human and animal infection of Schistosoma japonicum in Qixia District in 2013-2014 years, in 2015 in Gaochun District Wushen canal water Yangjiang A survey on the density of Oncomelania snails and human and animal infection was carried out in the section. Methods: according to the control and elimination standard of schistosomiasis and elimination standard GB15976-2015>, the investigation of Oncomelania snails, DDIA serology and nylon spun hatching fecal test were carried out to conduct a population survey, and the hatching method of plastic cup pipe jacking excrement was used to detect the infection of Schistosoma japonicum. Data analysis. Results: there were no positive Oncomelania snails in the water Yangjiang section of Qixia and Gaochun areas, but the density of living snail was significantly higher in the Shui Yang section than in other areas, suggesting the risk of schistosomiasis transmission in the Shui Yang section. The survey showed that the positive rate of the serological examination of the 2 administrative villages in Qixia was lower than 1%, and the blood test of 3 administrative villages in Gaochun area was positive. The sex rate was 2.48%, the fecal examination was not detected. Conclusion: comprehensive 2004-2014 Jiangsu province schistosomiasis prevention and control work report analysis, the snail area in Jiangsu province is decreasing year by year, the number of living snails and living snail density maintain relatively low level relatively stable; schistosomiasis is low epidemic situation, and in recent years there is no schistosomiasis sensual sex. The infection cases reported that the positive rate of blood test in the Gaochun district was higher than 1% of the standard of schistosomiasis transmission control. It was also higher than the positive rate in other regions of the province, suggesting that the region has high risk. It is necessary to strengthen the analysis of the serum peptide spectrum of the.2. experimental rabbit and the analysis of the blood serum differential polypeptide detection method based on the detection of blood serum differential polypeptide. The quality fingerprint of the serum peptide of the experimental rabbits was dyed and the diagnostic method was established according to the serum polypeptide. Methods: the serum peptide was enriched by the serum collection, the magnetic beads were enriched and the quality fingerprint of the serum peptide was detected by MALDI-TOF/TOF mass spectrometry and ClinProTools analysis, and the diagnostic method of serum differential polypeptide (CPT method) was established. A variety of methods were used to detect the accuracy of different infection periods. Results: Mr 1787 protein molecules were significantly up-regulated (P0.05) in 6D after infection, and at ninth d after infection, Mr 2834 protein molecules were significantly up-regulated (P0.01), Mr 3483 was significantly up (P0.05), Mr 4018 was significantly down (P0.05). Twelfth d after infection, Mr 31 The 51 protein molecules had a significant downregulation (P0.05), while Mr 4018 had a very significant downregulation (P0.01), and Mr 3530 was significantly up-regulated (P0.05). The two trends continued until the infection thirtieth D. were identified by two mass spectrometry and searched the ratio. The Mr 1787 protein was predicted to be an alpha enolase fragment, Mr 3530 was predicted as a heat shock protein 90 or a heat shock protein 47 fragment. The two proteins may be an early disease marker of rabbits infected by Schistosoma japonicum. Based on the differential peptide fingerprint of the serum of the healthy control group for fifth weeks and the healthy control group, the differential diagnosis model of the infection group and the healthy control group was established by ClinProTools software. The blind samples showed that the sera of the rabbits infected with 1,2,3,4 w accounted for 30%, 55%, 75%, respectively. 80%, and the results of ELISA detection in the same period were 0,0,20% and 50% respectively. The results showed that the specificity of Toxoplasma infection was good. Conclusion: the diagnostic method based on the establishment of serum differential expression polypeptide is better than the conventional ELISA method. It is expected to be applied to the early detection of schistosomiasis and screening the diagnostic value of serum polypeptide (protein). Application of marker.3. blood serum polypeptide detection method 3.1 The Application of serum differential polypeptide detection method in experimental mice model is widely used in the experiment of Schistosoma infection and related research. It is of great significance to develop a rapid, accurate and simple diagnostic method for its rapid, accurate and simple diagnostic methods. TOF/TOF mass spectrometry detection and ClinProTools analysis peptide mass fingerprint, according to the acute infection fifth W and the healthy control group serum differential expression polypeptide, establish the detection method (CPT method), and compare the ELISA, the Parasitology examination and so on various methods detection sensitivity, and verify the acute infection different period, the different infection degree mice accuracy. Result: compared with the traditional ELISA method, the CPT method detected the positive rate of 5% (3/60) in acute infection mice at 1W, and gradually increased in 2W to 5W, 35% (21/60), 75% (45/60), 87.93% (51/58) and 98.15% (53/54), while ELISA method was positive in the post infected 3W. The 3-5 infection rate was 65% (77.59%), 77.59% respectively. (45/58) and 94.44% (51/54) were lower than the CPT method; parasitology was detected in 70% (7/10) mice in 3,4 W, lower than the CPT method in the same period. In mice infected with different infection degrees, all mice infected with cercariae were predicted to be infected with the accuracy of 100%, infected with the small cercariae group. The accuracy rate of rat prediction was 40% (4/10), 50% (5/10) and 80% (8/10). Conclusion: the serum differential polypeptide detection method is at least 2-3 w ahead of the serum antibody ELISA detection method, which can greatly reduce the workload and can be used for the detection of experimental rats and the initial screening of samples under the condition of low degree infection. It is expected to provide a new method for the detection of "sentinel". Methods the application of.3.2 serum differential polypeptide detection method in the early warning of schistosomiasis "sentinel" was used to monitor the risk of Schistosoma infection in the Yangtze River. It is an important means for the early warning of schistosomiasis in the Yangtze River. The Parasitology examination of adult and liver egg nodules is the main means to judge whether the "sentinel" is infected or not, but the timeliness is poor, and A new rapid detection method is needed to develop a new rapid detection method. Methods: 660 sentinel mice (including 60 Qixia and Gaochun, Ningzhenyang, Ningzhenyang) in 2015 were examined and serum was collected. 5 cases of positive "sentinel" and 50 negative "sentinel" serums, which were confirmed by anatomic adults, were preserved in this room, and were detected by serum polypeptide enrichment and mass spectrometry. The peptide fingerprint was used and the established serum polypeptide detection method (CPT method) was introduced. Results: 5 cases of positive "sentinel" and 50 negative "sentinel" could be accurately diagnosed. In 2015, 660 sentinel mice were tested by CPT as uninfected and consistent with the results of parasitic examination. The method, compared with the conventional "sentinel" dissection method, saves manpower and material resources, and can early screen the "sentinel" by 1-2W, shortening the "window period" of detecting schistosomiasis infection, which can be applied to the early warning of "sentinel" in the early warning of "sentinel".3.3 serum differential polypeptide detection method in the identification of late blood patients. The diagnosis and treatment of patients with advanced Schistosoma is the focus of schistosomiasis, especially in the low epidemic situation, it is more important for the local examination of schistosomiasis. It is difficult to distinguish between the present and the past patients by the pathogenic examination and the antibody test. Methods: collecting late blood, new development of late blood, late blood cure, healthy control four groups, serum polypeptide fingerprint atlas inspection ClinProTools analysis was used to establish a method for the detection of serum differential peptide in the new late blood group and the healthy control group. The results showed that there was no significant difference in the serum peptide spectrum between the late blood and the new late blood and the new late blood group. Compared with the healthy control group, the late blood group decreased significantly at Mr 2662299132415337 and 5906, at Mr 2082. Compared with the healthy control group, there was a significant decrease in Mr 266242095337 and 5906, and the 2082 up regulated significantly in Mr 266242095337 and 5906. The new late blood group was significantly lower in the Mr 3241 than in the history control group, suggesting that the Mr 3241 was significantly down, and the 4282 up up could be used as a indication of the new late blood and two grade. The two peptides (proteins) were all the variable regions of the immunoglobulin heavy chain. According to the diagnostic method of the differential expression of polypeptide in the new late blood group and the healthy control group, the blind test showed that the late blood group had a history cure group and the non schistosomiasis infection group had the same serum peptide spectrum characteristics with the late blood patients, and the fecal test was positive. 73.3% (11/15) of the sera of the sex patients could not be verified, and could not be attributed to the new late blood or the healthy control group. The serum antibody positive of the water Yang river section was positive but 86.2% (25/29) of the negative samples of the feces was confirmed as the late blood group. Conclusion: the serum differential polypeptide detection method can be used to distinguish the late blood cases from the present cases indirectly, but the latter is the latter. It may include previous infection or recurrent infection, and some of them are still proved to be late blood groups, suggesting the possibility of further development of late blood patients; the epidemiologic survey of the Shui Yang section may be a previous infection, not a present, and a serum differential polypeptide detection combined with a regular parasitic examination can be used for the epidemic of schistosomiasis. A preliminary screening of the study is carried out.
【学位授予单位】：扬州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R532.21
【参考文献】
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1 仇志琴;黄玉政;陶永辉;张英;虞丰;;血清差异表达多肽谱用于人卵巢癌诊断的研究[J];南京医科大学学报(自然科学版);2012年07期
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