PCR检测蚊体内恶性疟原虫子孢子方法的建立

为建立能检测蚊体内恶性疟原虫的聚合酶链反应技术，根据恶性疟原虫ＣＳＰ基因序列，设计合成１对引物，以Ｃｈｅｌｅｘ－１００煮沸法制备ＤＮＡ模板，采用ＰＣＲ方法，恶性疟原虫子孢模板预期被扩增出２４５ｂｐ的ＤＮＡ条带，并将该法与蚊虫解剖镜检子孢方法相比较。结果经人工感染恶性疟原虫的３１只大劣按蚊中１４只ＰＣＲ阳性，被扩增出预期大小的ＤＮＡ片段，其余１７只ＰＣＲ阴性；以该技术检测野外捕捉的９８只按蚊，结果均     

用RAPD—PCR鉴定抗溴氰菊酯淡色库蚊

用ＲＡＰＤ－ＰＣＲ法对淡色库蚊抗溴氰菊酯品系和敏感品系的基因组ＤＮＡ进行了分析鉴别。结果表明：不同引物扩增出的产物数量不等，明显可分辨的带一般在４￣１３条之间，带的大小在０．５￣５．１ｋｂ之间；２２个引物在两品系中各扩增出１７０条可分辨带，绝大部分条带在两品系间是共同的；Ａ，Ｂ，Ｃ，Ｄ，Ｅ，Ｆ，Ｇ７个引物的扩增产物在两者间共显示差异带２１条（２，３，７，４，１，１和３条），其中抗性品系特异带１２条     

用RAPD-PCR鉴定抗溴氰菊酯淡色库蚊

用ＲＡＰＤ－ＰＣＲ法对淡色库蚊抗溴氰菊酯品系和敏感品系的基因组ＤＮＡ进行了分析鉴别。结果表明：不同引物扩增出的产物数量不等，明显可分辨的带一般在４～１３条之间，带的大小在０．５～５．１ｋｂ之间；２２个引物在两品系中各扩增出１７０条可分辨带，绝大部分条带在两品系间是共同的；Ａ，Ｂ，Ｃ，Ｄ，Ｅ，Ｆ，Ｇ７个引物的扩增产物在两者间共显示差异带２１条（２，３，７，４，１，１和３条），其中抗性品系特异带１２条（１，２，５，２，１，０和１条）；敏感品系特异带９条（１，１，２，２，０，１和２条）。这些特异片段可望作为抗性的分子标志用于抗性品系的鉴定以及进一步研究     

含CD44 cDNA片段细胞系的构建及在肝癌中的应用

目的构建含ＣＤ４４ｃＤＮＡ质粒ＰＧＥＸ２Ｔ和ＰＡＺ的单克隆细胞系．并利用此细胞系合成特异的ＣＤ４４ｃＤＮＡ探针，原位检测肝癌中ＣＤ４４ｍＲＮＡ的表达．方法应用常规的质粒转化方法将质粒ＰＧＥＸ２Ｔ和ＰＡＺ转入ＤＨ５菌株中得到单克隆细胞系．并以从此细胞系中提取的质粒为模板，应用ＰＣＲ法合成ＣＤ４４ｃＤＮＡ探针．应用原位杂交法检测肝细胞癌中ＣＤ４４ｖ６ｍＲＮＡ的表达．结果从ＤＨ５１菌株中提取的质粒含ＣＤ４４（２ｖ－１０ｖ）ｃＤＮＡ（１１ｋｂ），从ＤＨ５２菌株中提取的质粒含全长的ＣＤ４４ｃＤＮＡ（１８ｋｂ），ＰＣＲ合成的ＣＤ４４ｖ６ｃＤＮＡ探针长１４０ｂｐ．ＣＤ４４ｖ６ｍＲＮＡ的阳性检出率：转移高危组为８００％（８／１０），转移低危组为２１７％（５／２３），两者相差非常显著（Ｐ＜００１）．结论从单克隆细胞系ＤＨ５１和ＤＨ５２中提取的质粒，经ＰＣＲ扩增可得到特异的ＣＤ４４ｃＤＮＡ探针．肝细胞癌中ＣＤ４４ｖ６ｍＲＮＡ的表达与肿瘤的转移倾向呈正相关．     

滤纸干血滴抽提恶性疟原虫DNA用于PCR扩增

以ＳＴＥ－蛋白酶Ｋ－ＳＤＳ、甲醇－蛋白酶Ｋ－ＳＤＳ、Ｃｈｅｌｅｘ－１００加热法和Ｃｈｅｌｅｘ－１００蛋白酶Ｋ等四种方法对恶性疟患者滤纸干血滴样品进行ＤＮＡ抽提，供ＰＣＲ扩增特异性ＡＭＡ－１ＤＮＡ片段。结果显示，除ＳＴＥ－蛋白酶Ｋ－ＳＤＳ法抽提ＤＮＡ进行ＰＣＲ试验未能获得扩增产物外，其他３种方法抽提ＤＮＡ后进行ＰＣＲ试验均获得特异性约９００ｂｐＤＮＡ片段，可供进一步试验。     

套式PCR检测蚊体内疟原虫的敏感性与特异性

目的：分析套式聚合酶链反应技术检测蚊体内疟原虫的敏感性与特异性。方法：采用２对针对间日疟原虫小亚单位核糖体核糖核酸基因（ＳＳＵｒＤＮＡ）的特异引物，利用套式ＰＣＲ技术，从蚊体ＤＮＡ样本中，扩增间日疟原虫ＳＳＵｒＤＮＡ片段，进行间日疟原虫的检测。结果：扩增产物经琼脂糖凝胶电泳分析，可见扩增出特异的约１２１ｂｐ大小的ＤＮＡ片段，估测每份蚊虫ＤＮＡ样本中含有３个以上子孢子或１００只蚊中含有１只阳性蚊即可得此片段，而对恶性疟原虫、食蟹猴疟原虫、约氏疟原虫及正常蚊虫ＤＮＡ不能扩增出此片段。结论：此法具有高度的敏感性和特异性。     

聚合酶链反应和DNA探针联合检测临床标本中结核杆菌的研究

目的为提高聚合酶链反应（ＰＣＲ）检测未培养临床标本中结核杆菌的敏感性和特异性，方法，首先通过琼脂糖凝胶电泳检测ＰＣＲ扩增产物，然后用Ｓｏｕｔｈｅｒｎ法转移到至膜上，与地高辛标记的人型结核杆菌ＤＮＡ探针杂交。结果ＰＣＲ电泳检测的灵敏度为１ｐｇ而ＤＮＡ探针检测将灵敏度提高到１００ｆｇ。２４种受试菌株中，只有结核分支杆菌复合体和蟾分杆菌有２４５ｂｐ扩增带，其中牛型结核分支杆菌与１８８ｂｐ探针不杂交，对２     

聚合酶链反应用于鉴别恶性疟原虫不同分离株的研究

用恶性疟原虫主要裂殖子表面抗原基因序列（ＭＳＡ－１）为引物的聚合酶链反应（ＰＣＲ）技术检测恶性疟原虫感染。用此方法扩增实验室体外培养的ＦＣＣ１／ＨＮ株恶性疟原虫，显示１条３００ｂｐ的ＤＮＡ片断，而对实验室培养的巴布亚新内内亚ＦＣＱ－２７株则显示１条４４０ｂｐＤＮＡ条带。用此方法检测１５份采自云南缅边界的恶性疟血样均能扩增出ＤＮＡ条带，但不同血样的ＤＮＡ条带的分子量不同，部分血样还存在１条以上的ＤＮ     

PCR结合索氏转移杂交在结核病诊断中的应用

利用Ｈｅｒｍａｎｓ引物扩增插入序列ＩＳ９８６所得之２４５ｂｐ片断是一结核分支杆菌复合体特异性片断，采用随机引物法将地戈辛标记该片断。将纯化结核分支杆菌ＤＮＡ经聚合酶链反应（ＰＣＲ）扩增，索氏转移后与该探针杂交，检测灵敏度可达１ｆｇＤＮＡ。通过对７９例痰标本、１４例结核性胸腔积液及２６例结核性关节腔积液的ＰＣＲ和ＤＮＡ索氏杂交试验比较，认为采用２４５ｂｐ探针，将ＤＮＡ索氏转移杂交与ＰＣＲ体外扩增相结合，不仅提高了结核病基因诊断的敏感性，同时也提高了诊断的特异性。     

用rDNA研究海南省大劣按蚊的种型分类地位

目的 :为了进一步确定海南省大劣按蚊的种型分类地位。方法 :以采自海南省的大劣按蚊实验室品系、野外现场捕蚊及采自泰国的大劣按蚊 A种 ( AFRIMS实验室品系 )为材料 ,用 PCR扩增比较海南省与泰国大劣按蚊的 r DNA第二内转录间隔区 ( ITS2 )序列。结果 :测出序列 84 1bp,包括 ITS2及两侧的 5.8S和 28S编码基因的一小部分序列 ,ITS2长 716bp,海南省与泰国大劣按蚊序列相同 ,未发现种内或种群内变异。结论 :证实海南省存在大劣按蚊 A种 ,与以往染色体核型分析及蚊卵扫描电镜的观察结果一致。     

Genetic variation of Anopheles dirus A and D (Diptera:Culicidae) in China: inferred by mtDNA-CO I gene sequences]

OBJECTIVE: To interpret genetic variation and population structure of Anopheles dirus A and D from China by molecular marker. METHODS: Samples included An. dirus A of Hainan laboratory colony (n=13), and field specimen from Mengla (n=17) and Jiangcheng (n=17) in Yunnan Province. The specimens were identified by PCR assay before study, mtDNA-CO I region was amplified and sequenced. Genetic variation and population structure was estimated according to sequence data. RESULTS: The mtDNA-CO I gene with a length of 959 bp was analyzed. There were three haplotypes in An. dirus A and six haplotypes in An. dirus D. The above haplotypes distributed in three populations uniformly. The average number of pairwise differences within Mengla population (7.4412) was greater than that of Jiangcheng (1.2794) and Hainan (1.0513) populations, which suggested that the level of genetic divergence was the highest within Mengla population. The result of hierarchical AMOVA estimation showed a limited geneflow (Fst=0.799 9), therefore the variation level in a population (20.01%) was smaller than among the populations (79.99%). CONCLUSION: The inter-specific genetic variation between An. dirus A and D in China was small and the level of divergence among individuals was high.     

A rapid polymerase chain reaction based method for identification of the Anopheles dirus sibling species

A simple polymerase chain reaction (PCR) based method was developed to differentiate the Anopheles dirus, species A, B, C and D in Thailand using specific primers designed from species specific sequences. The PCR protocol was optimized to obtain products of 120 bp, 75 bp, 60 bp and 172 bp for species A, B, C and D, respectively. This method used a cocktail of four primer sets to identify these An. dirus sibling species. The method is very sensitive as only a small portion of mosquito was required allowing the rest of the mosquito to be used for other analyses. Specimens also kept for up to 14 years could be analyzed unambiguously from either larvae or adult. This method is advantageous over other PCR-based methods for identification of malaria vectors because it does not require any specific DNA extraction. A mosquito specimen was homogenized in 1x PCR buffer, then the supernatant directly used for PCR identification, allowing a large number of samples to be processed at the same time. It provides a simple and rapid practical method for screening An. dirus species, which is essential in malaria vector epidemiological studies in Southeast Asia.     

不同地区微小按蚊rDNA-ITS2序列差异

目的　比较我国云南、海南省、广西壮族自治区及泰国等不同地区微小按蚊核糖体 DNA第 2内转录间隔区 ITS2 )序列差异。　方法　取单只雌蚊蚊腿消化提取 DNA,PCR特异扩增 r DNA- ITS2片段 ,并对其扩增产物纯化、测序及分析。　结果　发现 2种不同的 ITS2序列 Gen Bank登录号 :AF4 16 783,AF4 16 784 ) ,分别与微小按蚊 A和 C同源 ,ITS2序列长度及 GC含量分别为 4 81bp,5 4 .0 4 %和 4 83bp,5 4 .2 3% ,两者无显著性差异 ;但 2 2个固定位点存在碱基置换和插入 /缺失 ,序列差异为 5 .8%。　结论　研究区内微小按蚊存在 A和 C两个不同的亲缘种     

Species-diagnostic differences in a ribosomal DNA internal transcribed spacer from the sibling species Anopheles freeborni and Anopheles hermsi (Diptera:Culicidae)

Approximately 460 base pairs (bp) of DNA sequence that included the second internal transcribed spacer (ITS2) and some flanking 5.8S and 28S ribosomal RNA coding regions were compared between the two closely related and morphologically indistinguishable mosquito species Anopheles freeborni and A. hermsi and a third related species, A. occidentalis. Sequences were determined from 14 clones of polymerase chain reaction (PCR)-amplified DNA obtained from four colonies of A. freeborni, two colonies of A. hermsi, and one individual A. occidentalis. Four clones showed independent single bp differences from the consensus for the relevant species. Eleven sites differed between the consensus sequences of A. hermsi and A. freeborni; 28 sites differed between A. hermsi and A. occidentalis. With the exception of a single bp mismatch in the 5.8S and two single bp mismatches near the undetermined junction of the ITS2 and 28S regions, all differences were confined to the ITS2 region. A PCR-based species-diagnostic assay for the cryptic species A. hermsi and A. freeborni was developed; it uses four synthetic oligonucleotides, two derived from areas of interspecies sequence difference in the ITS2, and two derived from highly conserved regions in the flanking coding sequences. Small amounts of mosquito DNA amplified in the presence of these four primers produce fragments of diagnostic size for each species: 900 bp for A. freeborni, 350 bp for A. hermsi, and approximately 1.2-1.4 kb for various other Anopheles species tested. We believe that this general approach to the development of species-diagnostic assays can be extended easily to other complexes of closely related, morphologically indistinguishable species.     

大劣按蚊和斯氏按蚊对约氏疟原虫不同易感性的遗传多态性研究

目的 比较分析对约氏疟原虫易感的斯氏按蚊和不易感的大劣按蚊的基因组DNA的多态性，探讨媒介按蚊与疟原虫遗传基因间的相互关系。方法 设计5条随机引物，应用随机扩增多态性DNA（random amplified polymorphic DNA，RAPD）技术对斯氏按蚊、大劣按蚊成蚊（未感染蚊和感染蚊）及其幼虫的基因组DNA进行随机扩增，扩增产物经琼脂糖凝胶电泳后分析。结果 5条随机引物对2种按蚊扩增的条带数不同，其中P5对斯氏按蚊扩增的效果较好，共16～17条带型，长度为200～1800bp；P4对大劣按蚊扩增效果较好，出现17条带型，长度为180～1500bp；P1对大劣按蚊无扩增产物。用P3对2种按蚊扩增，感染前后的扩增产物电泳条带数相同，但亮度不同；成蚊与幼虫带型数量不同，斯氏按蚊成虫与幼虫有3条相同带型（400、650、850bp），大劣按蚊成虫与幼虫有4条相同带型（350、500、750、800bp）；大劣和斯氏按蚊成蚊带型分别为10和13条，其中相同带型6条（300、350、400、550、600、800bp）。结论 斯氏按蚊和大劣按蚊之间存在基因差异。同种未感染蚊和感染蚊基因带型仅存在亮度差异，而幼虫与成虫的基因差异较大。     

应用气相色谱法鉴别海南省大劣按蚊

大劣按蚊是海南省疟疾的主要传播媒介。本文应用气相色谱法,分析海南省不同地区、不同季节大劣按蚊的皮质烃类。经统计学分析,它们的成分无显著性差异。应用聚类分析法,它们的距离系数很接近。提示海南省的大劣按蚊很可能是同一种。     

用RAPD技术分析中国嗜人按蚊种型

目的　鉴别中国不同地区嗜人按蚊 ,特别是海南岛与内陆嗜人按蚊间有无种型差异。　方法　海南、四川、江苏三地收集嗜人按蚊标本 ,通过对蚊基因组 DNA的提取纯化及 RAPD- PCR扩增和产物检测 ,最终筛选出 3条引物并制定出稳定的 RAPD图谱。　结果　三地嗜人按蚊绝大部分谱带完全相同 ,但又有明显不同的谱带存在。　结论　中国不同地区嗜人按蚊存在一定的种型差异 ,并为解释海南岛与内陆嗜人按蚊在生态习性和传疟作用方面的不同提供了科学依据。     

Diagnostic restriction fragment patterns of DNA from the four isomorphic species of Anopheles dirus

Total DNA from isofemale lines of the four isomorphic species of the Anopheles dirus were screened against twenty restriction endonucleases. Seven enzymes (Ava II, Alu I, Bgl II, Hae III, Hinf I, Mbo I and Sau3A I) produced some unique DNA fragments for each of the lines. Seven other enzymes (BamH I, BstN I, Cfo I, EcoR I, Kpn I, Nru I and Pst I) produced unique fragments in two of the lines. The remaining six enzymes (Hha I, Mnl I, Msp I, Nae I, Rsa I and Taq I) gave vague patterns which might result from either biological or technical causes. The results demonstrated that restriction fragment length of the DNA could be used as a means to distinguish isomorphic species of the Anopheles dirus.     

Identification of isomorphic malaria vectors using a DNA probe

About 7,000 recombinant clones, derived from chromosomally-identified families of wild-caught females of Anopheles dirus species D, were screened. The most promising clone was totally specific to species D when tested against single F1 females of all four species of the complex. In fresh specimens the clone was positive for DNA levels 150 times less than the normal DNA content of single individuals. Fresh adult males and females, larvae, and dried specimens have been successfully identified. The clone was sequenced; it is 124 bp long and appears to be repeated in the genome about 1.8 x 10(4) times.