几种常见吸虫的分子系统发生学研究

目的 研究日本血吸虫、卫氏并殖吸虫、华支睾吸虫、布氏姜片吸虫和肝片形吸虫分子系统发生学的关系。方法 利用PCR技术特异性扩增日本血吸虫、卫氏并殖吸虫和华支睾吸虫核糖体小亚基(18SrDNA)的V4区基因并测序。根据Genebank,检索布氏姜片吸虫和肝片形吸虫的相关序列,并与上述3种吸虫的V4区基因序列进行排列比较,计算它们的遗传距离并构建系统发生树。 结果 日本血吸虫与其他吸虫的遗传距离远>其他吸虫之间的遗传距离,在分子系统发生树中,日本血吸虫单独形成一个支系,其他吸虫形成另外一个支系。 结论 日本血吸虫与其他吸虫的亲缘关系较远,它们可能来自不同的祖先。     

华支睾吸虫病患者粪便虫卵核糖体DNA内转录间隔区序列分析

目的 通过PCR方法扩增华支睾吸虫病患者粪便虫卵核糖体DNA （ribosomal DNA,rDNA）内转录间隔区（internal transcribed spacer,ITS）序列进行分析,探讨其用于华支睾吸虫感染检测的价值. 方法 提取2例华支睾吸虫患者粪便中的虫卵DNA作模板,以rDNA ITS基因片段为目的片段进行PCR扩增,对扩增片段进行测序分析. 结果 从2例患者粪便虫卵样本中均扩增出1 123 bp的条带,经序列比对确定患者粪便中的虫卵为华支睾吸虫卵.测序分析发现本研究的两个样本与中国黑龙江分离株（KF740425）相似性为100％. 结论 本研究从2例华支睾吸虫病患者粪便虫卵中成功扩增出rDNA ITS基因,为从分子生物学角度检测华支睾吸虫病提供了资料.     

虫卵ITS-2序列分析鉴别华支睾吸虫和扇棘单睾吸虫的研究

目的建立以分子生物学技术为基础的华支睾吸虫与扇棘单睾吸虫的ITS-2序列分析鉴别方法。方法在华支睾吸虫流行区采集华支睾吸虫和扇棘单睾吸虫成虫,从成虫虫体分离并收集虫卵。以蛔虫、鞭虫、钩虫、蛲虫做对照虫种,按不同虫种和虫卵数分成5个实验组,各组分别提取DNA,并扩增ITS-2序列,对扩增产物进行测序并作同源性分析以确保为目标片段,根据PCR扩增产物电泳获得的条带判定虫种。结果以华支睾吸虫和扇棘单睾吸虫虫卵DNA为模板的PCR产物电泳图谱分别在400bp、500bp左右各显示一条明显条带;在以两种虫卵DNA混合液为模板的PCR产物中,电泳图谱在400bp左右和500bp左右各显示明显条带。将测序得到的两种核苷酸序列进行Blast比对后,显示与GenBank中相应的华支睾吸虫和扇棘单睾吸虫序列高度同源。混入四种肠道线虫虫卵DNA的PCR产物中,除目的条带外,无其他条带。结论该方法可以对华支睾吸虫及扇棘单睾吸虫虫卵进行鉴别,且结果不受常见四种肠道线虫虫卵干扰,敏感性和特异性较高,可作为两虫种的鉴别检测方法。     

华支睾吸虫cDNA文库的构建

目的　构建华支睾吸虫cDNA文库。方法　采集华支睾吸虫阳性鱼 ,分离囊蚴 ,感染实验动物兔 ,解剖兔收集华支睾吸虫成虫虫体。应用“一步法”提取华支睾吸虫总RNA ;经过mRNA纯化、cDNA合成 ,以PcDNA3(Amp +)质粒为载体构建文库。挑取 2 0个克隆进行扩增 ,选择 9个克隆进行DNA序列测定。序列结果与GenBank中相关基因进行比对。结果　获得含有 9 7× 10 5个重组子库容量的华支睾吸虫cDNA文库。其中PC6号克隆基因序列与华支睾吸虫半胱氨酸蛋白酶序列具有 93%的同源性。结论　已构建成华支睾吸虫cDNA文库。     

重组酶介导的华支睾吸虫特异性核酸等温扩增方法的建立及初步评价

目的　建立一种可用于华支睾吸虫检测的重组酶介导的等温核酸扩增方法（RAA）。方法　以华支睾吸虫18S rRNA基因序列作为靶序列,设计、合成、筛选特异性引物和探针,建立快速检测华支睾吸虫的荧光RAA检测方法。分别以含不同拷贝数DNA片段的重组质粒和不同浓度华支睾吸虫基因组DNA为模板进行荧光RAA扩增,评价其检测敏感性;分别以似蚓蛔线虫、细粒棘球绦虫、日本血吸虫、十二指肠钩虫、曼氏血吸虫基因组DNA为模板进行荧光RAA扩增,评价其检测特异性;通过抽提含华支睾吸虫虫卵的粪便及含囊蚴的淡水鱼肉样本DNA并进行荧光RAA扩增,初步评价其检测现场样本的能力。结果　成功建立了华支睾吸虫检测荧光RAA法,其可在39 ℃ 20 min内实现对华支睾吸虫DNA的特异性扩增。以含不同拷贝数DNA片段的重组质粒为模板,该方法最低检出限为10 拷贝/μL;以不同浓度华支睾吸虫基因组DNA为模板,该方法最低检出限为3 pg/μL;以似蚓蛔线虫、细粒棘球绦虫、日本血吸虫、十二指肠钩蚴及曼氏血吸虫基因组DNA为模板,检测结果均为阴性。该方法可成功检出感染华支睾吸虫的人体、大鼠粪便样本以及麦穗鱼样本,具备较好的现场样本检测能力。结论　成功建立了一种简便、快速、敏感、特异的可用于华支睾吸虫检测的RAA法。     

重组酶介导的华支睾吸虫特异性核酸等温扩增方法的建立及初步评价

目的　建立一种可用于华支睾吸虫检测的重组酶介导的等温核酸扩增方法（RAA）。方法　以华支睾吸虫18S rRNA基因序列作为靶序列，设计、合成、筛选特异性引物和探针，建立快速检测华支睾吸虫的荧光RAA检测方法。分别以含不同拷贝数DNA片段的重组质粒和不同浓度华支睾吸虫基因组DNA为模板进行荧光RAA扩增，评价其检测敏感性；分别以似蚓蛔线虫、细粒棘球绦虫、日本血吸虫、十二指肠钩虫、曼氏血吸虫基因组DNA为模板进行荧光RAA扩增，评价其检测特异性；通过抽提含华支睾吸虫虫卵的粪便及含囊蚴的淡水鱼肉样本DNA并进行荧光RAA扩增，初步评价其检测现场样本的能力。结果　成功建立了华支睾吸虫检测荧光RAA法，其可在39 ℃ 20 min内实现对华支睾吸虫DNA的特异性扩增。以含不同拷贝数DNA片段的重组质粒为模板，该方法最低检出限为10 拷贝/μL；以不同浓度华支睾吸虫基因组DNA为模板，该方法最低检出限为3 pg/μL；以似蚓蛔线虫、细粒棘球绦虫、日本血吸虫、十二指肠钩蚴及曼氏血吸虫基因组DNA为模板，检测结果均为阴性。该方法可成功检出感染华支睾吸虫的人体、大鼠粪便样本以及麦穗鱼样本，具备较好的现场样本检测能力。结论　成功建立了一种简便、快速、敏感、特异的可用于华支睾吸虫检测的RAA法。     

自延吉市自来水分离的棘阿米巴18S rDNA基因型鉴定

目的对吉林延吉市自来水中分离的棘阿米巴分离株Acanthamoeba sp.CJY/W1 18S rDNA基因型鉴定。方法从本地区自来水分离的Acanthamoeba sp.CJY/W1虫株中提取基因组18S rDNA,应用棘阿米巴属特意性引物PCR扩增。将扩增产物测序后用Clustal X和Genedoc软件进行序列分析,与基因库中已有T1至T12型序列进行比较并构建进化树。结果分离的棘阿米巴分离株CJY/W1的18S rDNA全基因为2 252bp,18S rDNA基因分型最接近于T1型,但与T1型之间的基因差异为8%。结论分离的CJY/W1株是不属于T1-T12的新的18S rDNA基因型,接近于T13(Acanthamoeba sp.UWC9,AF132134)。     

华支睾吸虫HMGB1同源分子的识别及其进化研究北大核心CSCD

目的识别华支睾吸虫HMGB1同源分子的CDS及其蛋白序列,并分析其进化特征。方法分别用基于麝猫后睾吸虫HMGB1预测序列的5'和3'RACE以及基于华支睾吸虫HMGB1基因组预测序列的PCR法扩增华支睾吸虫成虫来源的cDNA,对扩增产物进行克隆测序,并进行序列的同源性比较和拼接,分析其ORFs,据此对华支睾吸虫成虫来源的cDNA进行PCR扩增验证其转录;构建进化树,对识别出的HMGB1同源分子进行进化分析。结果识别出两个华支睾吸虫HMGB1同源蛋白CDS,大小分别为381和477 bp,二者均在华支睾吸虫成虫转录,产物均一;推测其编码蛋白分别为127和159个氨基酸,其分子序列与血吸虫同源性较高,而与人及其他脊椎动物同源性低。结论成功识别出华支睾吸虫HMGB1同源蛋白CDS序列,为研究其与肝胆管癌发生的关联性和机制奠定了基础。     

广东省平远县鱼源性吸虫混合感染调查及DNA(RAPD)分析

目的　调查广东省平远县棘隙吸虫及其他鱼源性吸虫混合感染情况。方法　对鱼类宿主取鱼鳃和鱼肉等组织 ,镜检囊蚴 ;用水洗过筛沉淀法粪检虫卵 ,部分虫卵阳性动物解剖检查。采用DNA -RAPD技术 ,比较福建棘隙吸虫广东株与福建株。结果　查出华支睾吸虫 (Clonorchissinensis)、东方次睾吸虫 (Metorchisorientalis)、福建棘隙吸虫 (Echinochasmusfujianensis)、日本棘隙吸虫 (E japonicus)、抱茎棘隙吸虫 (E perfoliatus)和钩棘单睾吸虫 (Haplorchispumilio) 6种。第二中间宿主为麦穗鱼等 11种 ,感染率 48 3% (182 / 377) ,有 92 4%的阳性鱼混合感染 2种以上吸虫囊蚴。保虫宿主狗、猫感染率 79 2 % (19/ 2 4) ,每只阳性动物均有 2种以上吸虫混合感染。 15个引物在福建棘隙吸虫广东株与福建株两者共获DNA片段 185个 ,共享度 (F) =0 984,遗传距离 (D) =0 0 16。结论　当地为人兽共患的多种鱼源性吸虫混合感染区。形态学观察和分子生物学检测结果相印证 ,福建棘隙吸虫广东株与福建棘隙吸虫福建株为同一虫种。     

淡水鱼华支睾吸虫囊蚴 LAMP 检测方法的建立

目的 建立一种高效灵敏的环介导等温扩增(LAMP)方法检测淡水鱼中华支睾吸虫囊蚴。 方法 以华支睾吸虫 ITS2 基因序列为靶序列,合成 Cs-1、Cs-2、Cs-3 三组 LAMP 引物体系。 以华支睾吸虫囊蚴 DNA 样本为模板,日本血吸虫成虫、带绦虫成虫、蛔虫虫卵及其他吸虫囊蚴 DNA 样本为参考,比较三组引物体系的特异度;以不同浓度的华支睾吸虫成虫基因组 DNA 为模板,比较三组引物体系的灵敏度;用 LAMP 法及 PCR 法平行检测 16 份华支睾吸虫囊蚴样本和 23 份其他吸虫囊蚴样本,评价 LAMP 法的敏感性和特异性。 结果 三组引物体系的灵敏度及特异度以 Cs-3 为最优,以其建立的 LAMP 法反应体系,检测华支睾吸虫成虫 DNA 的最低检出浓度可达到 3. 33×10^-4ng / μL,敏感性和特异性与 PCR 法一致,均为 100%。 结论 成功建立了一种可用于检测淡水鱼中华支睾吸虫囊蚴的 LAMP 方法,为淡水鱼寄生虫病监测工作提供了便捷高效的检测手段。     

Geographical variation of the liver fluke,Clonorchis sinensis,from Korea and China based on the karyotypes,zymodeme and DNA sequences

Genetic characterization was carried out in order to reveal the geographical variations of the oriental liver fluke, Clonorchis sinensis (Trematoda: Opisthorchiidae), collected in Korea and China. The chromosome number was 2n=56 in both Korean (Kimhae) and Chinese (Liaoning) flukes, and chromosomes were divided into two groups based on their sizes; consisting of 8 pairs of large and 20 pairs of small chromosomes. However, the karyotypes showed some differences between Korean and Chinese flukes. Isozyme analysis showed that two loci from each enzyme of aconitase and esterase (alpha-Na and beta-Na); only one locus each from six enzymes, glucose-6-phosphate dehydrogenase (G6PD), alpha-glycerophosphate dehydrogenase (GPD), 3-hydroxybutyrate dehydrogenase (HBDH), malate dehydrogenase (MDH), phosphoglucose isomerase (PGl) and phosphoglucomutase (PGM). Most of loci in two populations of C. sinensis showed homozygous monomorphic banding patterns and one of them, GPD was specific as genetic markers between two different populations. Two populations were very closely clustered within the range of genetic identity value of 0.998-1.0. We also compared patterns of intraspecific polymorphism of two markers with contrasted modes of evolution, nuclear ribosomal DNA (rDNA) and mitochondrial DNA (mtDNA) of the liver fluke from Kimhae, Guangxi and Liaoning. They showed a high homology. In conclusion, three populations of C. sinensis from Korea and China showed high homogeneity in the nucleotide sequences of the 18S rDNA, ITS2 and mtCOI gene.     

2株间日疟原虫18SrDNA的克隆及其同源性分析

目的克隆间日疟原虫河南分离株与湖北分离株红内期18SrDNA,并进行同源性分析。方法采用PCR方法从间日疟患者血样DNA中扩增间日疟原虫18SrDNA,纯化后与pGEM．Teasy质粒连接,转化大肠埃希氏菌JMl09;阳性克隆质粒经双酶切鉴定后,进行序列测定,采用BLAST和MEGA4生物软件分析同源性。结果间日疟原虫18SrDNA扩增片段大小为998bp：阳性克隆重组质粒经双酶切鉴定,与预期结果相符;序列测定结果显示,河南、湖北2分离株间日疟原虫18SrDNA序列完全相同．与GenBank中报道的12株间日疟原虫相同序列进行比对,其同源性均大于99％：用邻位连接法（neigh—bor-joining,NJ）和非加权组平均法（UPGMA）2种方法构建系统发生树发现,河南分离株、湖北分离株与间日疟原虫X13926．1株遗传距离小．同属一个分支。结论克隆了间日疟原虫河南与湖北分离株红内期18SrDNA,该基因序列在不同地理株间遗传稳定。     

中华按蚊实验室种群中肠内细菌菌群分析

目的为获取实验室饲养的3个发育期中华按蚊中肠内的革兰氏阴性菌菌株。方法取实验室饲养的20只中华按蚊吸血成蚊的中肠,研磨稀释后涂板培养,挑取单个菌落,培养扩增。以各菌株的DNA为模板,对16SrRNA基因的V3高变区进行PCR扩增、测序和Blast分析。对经鉴定的各类细菌进行革兰氏染色。以中华按蚊的Ⅳ龄幼虫(10只)、刚羽化雌成蚊(50只)和吸血雌成蚊(20只)等3个发育期的中肠基因组DNA为模板,用变性梯度凝胶电泳技术(DGGE)分析3个发育期中肠细菌16SrDNAV3区序列;建立在3个发育期均存在的16SrDNAV3区的克隆文库,并进行测序分析。将两种方法获得的16SrDNAV3区序列进行亲缘关系分析,确定同种的革兰氏阴性菌。结果从吸血成蚊的中肠分离到28个菌株,经分类鉴定为5种细菌,除短芽孢杆菌革兰氏染色呈阳性外,气单孢菌、丛毛单胞菌、金黄杆菌和A4菌均为革兰氏阴性菌。DGGE分析显示,共检测到4组条带在3个发育期均存在,经序列分析来源于5种细菌,分别为气单孢菌、A4菌、假单胞菌、无色杆菌和某未知菌种,其中气单孢菌(GQ301543)和A4菌(FJ8701127)来源的16SrDNAV3区与分离得到的该两种菌株的序列一致性均为100%。结论获得2种在中华按蚊3个发育期中肠内均存在的革兰氏阴性菌株,分别为气单孢菌和A4菌。     

Infection by Clonorchis sinensis in Asian immigrants in Brazil. Treatment with praziquantel

Fifteen adult patients with assymptomatic infection due to Clonorchis sinensis, diagnosed by coprological examination, were studied. They all came from Asia (twelve from Taiwan, two from South Korea and one from Hong Kong) and were examined at the Adolfo Lutz Institute and the Department of Infectious Diseases, School of Medicine, University of S?o Paulo, in S?o Paulo, Brazil. Six patients were women and nine men. All studied patients were admitted to hospital and treated with praziquantel (60 mg/kg). Previous to treatment and on the 15th, 30th and 60th days after praziquantel administration, patients were submitted to quantitative stool examinations, according to Kato-Katz's technique and to hematological and biochemical serum analysis. After a 60 day follow-up nine patients (60%) were negative for C. sinensis eggs in stools. Those not cured after praziquantel administration (six patients, 40%) revealed a sharp decline in faecal elimination of C. sinensis eggs.     

Molecular genetic profiles among individual Clonorchis sinensis adults collected from cats in two geographic regions of China revealed by RAPD and MGE-PCR methods

Clonorchis sinensis causes the important food-borne zoonosis, clonorchiasis, which is endemic in East Asia, especially in China mainly in Guangdong, Guangxi and Heilongjiang provinces and Korea. Although comparisons on isoenzymes and some molecular profiles of C. sinensis collected from different parts of China and Korea have been studied, few works on the genetic variation among the individuals from different regions of China has been reported. In the present study, individual adults of C. sinensis were collected from cats in two geographic locations (Guangdong province in the South and Heilongjiang province in the North) of China and 44 of them were examined by using random amplified polymorphic DNA (RAPD)-PCR and mobile genetic elements (MGEs)-PCR techniques to assess the individual genetic variability within and between the two groups of this parasite. Six arbitrary primers and two pairs of MGE primers were employed in the genomic DNA amplification. The molecular patterns showed significant polymorphism among the individuals. The RAPD data displayed that the similarity coefficient (SC) of the individuals within Heilongjiang group was much higher than that of the Guangdong group, which was further confirmed by MGE-PCR results. Individuals from Heilongjiang were found genetically closer with lesser polymorphisms than those collected from Guangdong province. These results demonstrated that RAPD and MGE-PCR techniques, particularly RAPD method, could be useful for investigating genetic variations among C. sinensis individuals. They may also indicate that the genetic variation of C. sinensis occurs in the subtropical region--Guangdong--faster than that in the cold-region--Heilongjiang province--due to more generations (life cycle) occurred.     

基于18S rDNA基因序列探讨几种食源性吸虫系统发生关系

目的 研究几种食源性吸虫间的系统发生关系.方法 PCR扩增华支睾吸虫和东方次睾吸虫18S rDNA片段并测序,从GenBank检索其他食源性吸虫和日本血吸虫的18S rDNA序列,利用CLUSTAL X软件比对后,用MEGA 5.0软件计算食源性吸虫间的遗传距离,并用邻接法、最大似然法和最大简约法构建系统发生树.结果 共获得12种食源性吸虫序列,12种吸虫间遗传距离从0.001到0.083,系统发生树显示后睾科与异形科先聚在一起,然后与并殖科和双腔科聚成一支,片形科单独聚成一支.结论 后睾科与异形科有较近的亲缘关系.

Abstract：

Objective To discuss the phylogeny of several food-borne trematodes.Methods The 18S rDNA partial sequences of Clonorchis sinensis and Metorchis orientalis were specifically amplified by PCR and then sequenced,the corresponding sequences of other food-borne trematodes were obtained from GenBank.After aligned by CLUSTAL X program all sequences were used to calculate the genetic distance and construct the phylogenic trees using neighbor-joining method,maximum parsimony method and maximum likelihood method in MEGA5.0 program.Results The 18S rDNA panial sequences of 12 species of food-bome trematode were obtained.The genetic distances among 12 trematodes were from 0.001 to 0.083.phylogenic trees showed that Opisthorchiidae and Hererophyidae polymerize first,then form a clader with Paragonimidae and Dicrocoeliidae,Fasciolidae form another clader.Conclusion Opisthorchiidae is closer to Hererophyidae in evolution.     

新疆维吾尔自治区喀什地区亚洲璃眼蜱携带病原菌研究

目的 采用16S rDNA V3-V4区高通量测序,并结合特异基因的PCR检测,研究新疆维吾尔自治区喀什地区亚洲璃眼蜱体内菌群特征及常见病原菌携带情况,为当地蜱传疾病防控提供理论依据。方法 于2021年5月在喀什地区巴楚县采用布旗法采集游离蜱342只,经形态学和特异性16S rDNA检测鉴定蜱种;除228只用于分离培养外,其余114只提取全基因组DNA,采用Illumina Novaseq测序平台进行16S rDNA V3-V4区高通量测序,分析喀什地区亚洲璃眼蜱携带菌的种类及其相对丰度水平;根据新疆地区既往报道常见蜱媒病原菌分布情况及蜱种带菌特征,选取6种常见病原菌对其特异性基因进行PCR检测,分析各病原菌的携带情况,并与高通量测序结果进行比较分析。结果 16S rDNA V3-V4区高通量测序分析显示1个样本携带贝纳柯克斯体;所有样本在属水平均检出立克次体属,16个样本检出无形体属,但缺少种水平注释;所有样本均未检出疏螺旋体。PCR法检测表明14只蜱携带伯氏疏螺旋体,7只蜱携带米氏疏螺旋体,3只蜱携带嗜吞噬细胞无形体,2只蜱携带贝纳柯克斯体,斑点热立克次体及查菲埃立克体未检出。结论...