PCR及PCR-ELISA法检测蚊体内马来丝虫幼虫

目的：建立一种检测蚊体内马来丝虫幼虫的灵敏、快速、特异的方法。方法：选择应用PCR及PCR－ELISA法检测马来丝虫幼虫DNA的最佳反应条件，并在该条件下分别以马来丝虫Ⅰ期、Ⅱ期、Ⅲ期幼虫各1条作模板，测定检测的灵敏度及检测实验室人工感染中华按蚊体内的马来丝虫幼虫。结果：PCR及PCR－ELISA法均能检测出1条Ⅰ期幼虫（L1），而PCR的检测下限为1／10条L1，PCR－ELISA检测下限为1／100条L1；将分离的感染期幼虫加阴性蚊媒进行粗提、扩增及电泳，结果未见明显的扩增条带，扩增产物作ELISA检测，全部为阴 性；个体解剖人工感染的中华按蚊120只，分别收集113只阳性蚊体内的幼丝虫，用两种方法检测，结果均为阳性。结论：初步建立了PCR及PCR－ELISA法检测蚊体内马来丝虫幼虫的方法。     

应用DNA探针检测蚊体内丝虫幼虫的研究

本文用人工合成的寡核苷酸探针，经斑点杂交法检测蚊体内马来丝虫幼虫。可测到丝虫和微丝蚴２ｎｇ的ＤＮＡ量，不与其它动物丝虫标本发生交叉反应。将单个蚊直接压于硝酸纤维素膜上进行检测，感染蚊中含有１条感染期幼虫就可出现阳性反应。将一组蚊虫在裂解液中研磨集体检测时，在２０只蚊虫中有１只感染蚊即可检出。显示该探针可用于马来丝虫地区的蚊媒监测。     

应用DNA探针检测蚊体内丝虫幼虫的研究

本文用人工合成的寡核苷酸探针，经斑点杂交法检测蚊体内马来丝虫幼虫。可测到丝虫和微丝蚴２ｎｇ的ＤＮＡ量，不与其它动物丝虫标本发生交叉反应。将单个蚊直接压于硝酸纤维素膜上进行检测，感染蚊中含有１条感染期幼虫不可出现了性反应。将一组蚊虫在裂液中研磨集体检测时，在２０只蚊虫中有１只感染蚊即可检出。显示该探针可用于马来丝虫地区的蚊媒监测。     

马来丝虫非放射性寡核苷酸探针的制备及现场应用研究

目的　寻找一种高度特异、敏感的马来丝虫的检测方法 ,并将其用于现场检测。　方法　根据马来丝虫 Hha 重复序列 ,合成马来丝虫特异性引物 1对和寡核苷酸探针 1条 ,采用地高辛加尾标记试剂盒对人工合成寡核苷酸探针进行非放射性标记 ,并对标记后的探针进行标记效率检测 ;将血液标本和蚊虫标本分别用蛋白酶 K等处理液和 Chelex- 10 0处理后 ,结合 PCR技术 ,对该探针的特异性和敏感性进行检测 ;并将此探针用于平远县马来丝虫病的监测。　结果　经测定 ,标记后探针的有效浓度为 1.5～ 2 fmol/μl;结合 PCR技术 ,该探针可检出 10 0 μl阳性血样中 1条微丝蚴 ;5 0只蚊中含有 1只只感染 1条幼丝虫阳性蚊亦能准确检出。班氏丝虫等其它寄生虫标本的检测结果均为阴性 ;采自平远县的 2 4 0份血液标本和 10 0 8只中华按蚊标本均为阴性。　结论　该探针杂交检测方法特异性强 ,敏感性高 ,可作为一种新的马来丝虫病监测方法在现场中使用。     

用种特异的DNA探针鉴别蚊媒体内的马来丝虫

对蚊媒体内的丝虫感染性幼虫进行定量,是评价丝虫病防治效果的最好方法。然而,在同一地区的人丝虫和动物丝虫,可通过同种蚊媒传播,若以生化学或形态学方法区分这些感染性幼虫,又往往不大可能。为此,作者采用种特异的马来丝虫DNA探针鉴别马来丝虫。作者分离了马来丝虫的8个地理株及帝汶丝虫、彭亨丝虫、匐行恶丝虫(D. repens)、部利丝虫(Breinlia booliti)、加里曼丹吴策线虫(W. Kalimantani)和心脏丝虫(Cardiofilaria Spp.),保种子沙鼠、猫、叶猴、猕猴、大鼠等动物体内,以东乡伊蚊叮咬这些动     

不同蚊种对亚周期型马来丝虫的易感性研究

本文用中华按蚊、东乡伊蚊、淡色库蚊分别人工感染亚周期型马来丝虫,通过观察其微丝蚴摄人数、幼虫穿壁率、幼虫发育成熟率及感染24h 幼虫的黑化率等指标探讨了蚊种对亚周期马来丝虫的易感性。结果表明,中华按蚊、东乡伊蚊对亚周期型马来丝虫易感,淡色库蚊不易感。黑化反应在三蚊种体内均可发生,从多方面影响幼虫在淡色库蚊体内的发育。     

人房内中华按蚊,嗜人按蚊自然感染牛腹腔丝虫的纵向观察

中华接蚊、嗜人按蚊均可感染牛腹腔丝虫和马来丝虫。在马来丝虫病流行区进行蚊媒调查，特别是在马来丝虫病防治后期的蚊媒监测中，蚊体内人、畜丝虫幼虫的鉴别尤为重要[1]。为了排除牛腹腔丝虫幼虫对马来丝虫病防治后期蚊媒监测的干扰，1987～1992年，1995～1996年，我们选定已无微丝蚴血症者的蔡金镇两个原马来丝虫病流行村，总人口1963人为观察区、先后共8年观察人房内中华接蚊和嗜人按蚊自然感染牛腹腔丝虫的情况。材料与方法1．牛腹腔丝虫自然传染原调查：上午抽采观察区村民饲养的水牛耳背静脉血各6大滴（约120μl），涂制成1．5×3．0…     

马来丝虫非放射性寡核苷酸探针的制备及现场应用研究

目的 寻找一种高度特异，敏感的马来丝虫的检测方法，并将其用于现场检测。方法 根据马来丝虫HhaⅠ重复序列，合成马来丝虫特异性引物1对和寡核苷酸探针1条，采用地高辛加尾标记试剂盒对人工合成寡核苷酸探针进行非放射性标记，并对标记后的探针进行标记效率检测，将血液标本和蚊虫标本分别用蛋白酶K等处理液和Chelex-100处理后，结合PCR技术，对该探针的特异性和敏感展出一进行检测；并将此探针用于平远县马来丝虫病的监测。结果 经测定，标记后探针的有效浓度为1.5-2fmol/μl；结合PCR技术，该探针可检出100μl阳性血样中1条微丝蚴；50只蚊中含有1只只感染1条幼丝虫阳性蚊亦能准确检出，班氏丝虫等其它寄生虫标本的检测结果均为阴性；采自平远县的240份血液标本和1008只中华按蚊标本均为阴性。结论 该探针杂交检测方法特异性强，敏感性高，可作为一种新的马来丝虫病监测方法在现场中使用。     

丝虫病研究Ⅴ.彭亨丝虫和马来丝虫微丝蚴在埃及伊蚊、东乡伊蚊、尖音库蚊骚扰亚种及骚扰阿蚊体内的脱鞘

长期以来,有人认为班氏丝虫、马来丝虫、彭亨丝虫及罗阿丝虫的微丝蚴(mf)被蚊摄入后,立即在胃内脱鞘。也有人报告:彭亨丝虫mf被四斑按蚊摄入后,立即在血块中脱鞘。Yamamoto等(1983)报道,彭亨丝虫在骚扰阿蚊的胃及腹部血腔内脱鞘。从而认为,鞘可能起着保护幼虫的作用。为了了解mf脱鞘是否常在蚊血腔中进行,作者采用感染丝虫的长爪沙鼠作蚊血源进行实验。感染彭亨丝虫的鼠血mf平均密度为73.5条/cmm和2.6条/cmm;感染马来丝虫的为1.5条/cmm,均以40mg/kg戊     

马来丝虫贵州株传代的实验观察

目的 观察马来微丝蚴贵州虫株传代现象。方法 分别用周期型马来丝虫贵州虫株f31和f25微丝蚴人工感染中华按蚊，在温度、相对湿度相同的条件下，观察幼丝虫在蚊体内发育情况。结果 贵州虫株f31在感染中华按蚊后24h解剖，仅在按蚊腹部和胃内可见脱鞘和未脱鞘的微丝蚴，第2～9d未发现各期幼丝虫，经6批次实验，结果相同。贵州虫株f25感染中华按蚊后24h解剖，蚊胃内未发现微丝蚴，在胸部可见早期腊肠期(L1)幼丝虫，第2～9d解剖可见各期发育中的幼丝虫。结论 贵州虫株f31马来微丝蚴经6批次传代失败，可能与同一地方单一虫株的多次传代发生遗传突变有关。     

Combined detection of Brugia malayi and Wuchereria bancrofti using single PCR

A single step PCR method has been developed for the combined detection of the human filarial parasites, Brugia malayi and Wuchereria bancrofti. Parasites' DNA were isolated from filaria positive blood samples that were collected from endemic areas. The primers used were Hha1 and Ssp I, which amplified the DNA fragments of 322 bp and 188 bp specific to B. malayi and W. bancrofti, respectively. The sensitivity of the assay was tested with blood and mosquito samples having one W. bancrofti in a pool of 10 B. malayi. The assay was further evaluated on field collected blood and mosquito samples. Use of this assay as a diagnostic tool for the detection of filariasis being the most promising aspect of this study, offers scope for detection of both the parasites even at low levels of infection.     

A rapid and simplified method of DNA extraction for the detection of Brugia malayi infection in mosquitoes by PCR assay.

Currently used protocols for the extraction of filarial parasite DNA from mosquito samples are tedious and involve extensive use of expensive and hazardous chemicals. Therefore, in order to arrive at a simple procedure, four different methods (A, B, C and D) were tried for the extraction of DNA from mosquitoes infected with filarial parasite, Brugia malayi. Method D was found to be as efficient as the current procedure for the extraction of DNA from a single microfilaria in pools of 25 mosquitoes and the DNA was suitable for polymerase chain reaction (PCR) amplification, yielding a band of 322 base pairs with primers specific for B. malayi. Method D involved drying and crushing the mosquitoes to a powder, which was homogenized in 100 microl TE buffer, vortexed, boiled for 10 min, centrifuged at 14000 r.p.m. for 10 min, and the supernatant used for the PCR assay. Dot-blot hybridization confirmed the specificity of the PCR amplified fragment. The DNA extracted by this method was stable for about 1 year. When comparing with the standard method, the cost of a single PCR reaction, inclusive of DNA extraction, was reduced by 50% and the hands on time was minimized fivefold. Hence, this simple TE-based method is rapid, safe and also cost-effective in assessing the B. malayi infection in pools of vector mosquitoes.     

A polymerase chain reaction assay for the survey of bancroftian filariasis

A polymerase chain reaction (PCR) assay based on a highly repeated DNA sequence found in Wuchereria bancrofti (SspI repeat) has been modified for the survey of bancroftian filariasis in expatriate workers (Myamese, Karen and Mon) from Myanmar where human filariasis is endemic. The PCR was very sensitive with the ability to detect the presence of as little as 10 pg of parasite DNA. The primers used in this PCR also showed highly specific amplification of parasite DNA without the presence of non-specific and non-target PCR products such as Brugia malayi, Plasmodium falciparum and human DNA. The primers were used to investigate filariasis in four provinces in the central and western Thailand, Samut Songkram, Ratchaburi, Nakhon Pathom and Tak during 1997-2001. Among them, Tak and Ratchaburi are the only endemic areas of bancroftian filariasis. In this field study, 1,299 human blood samples (501 from Samut Songkram, 510 from Ratchaburi, 109 from Nakhon Pathom, and 179 from Tak) were collected and screened by PCR. The result showed that 1, 2, 3, and 33 patients from Samut Songkram, Ratchaburi, Nakhon Pathom, and Tak respectively were infected with W. bancrofti. These numbers were corresponded to the prevalence rate of infection of 0.2, 0.4, 2.8, and 18.5%, respectively. The PCR was able to detect the third-stage infectious larvae (L3) from Culex quinquefasciatus, mosquito vector of the W. bancrofti, that was experimentally fed to infected patient. The PCR screening of each of field mosquito pools from two endemic areas, Ratchaburi and Tak, showed that no L3 of W. bancrofti was detected.     

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。     

Susceptibility of Mansonia uniformis to Brugia malayi microfilariae from infected domestic cat

Microfilariae of Brugia malayi is transmitted to man and other susceptible hosts via mosquito. The transmission of B. malayi from cat to man by Ma. uniformis bite has never been reported. The Ma. uniformis mosquito is the normal vector for Wuchereria bancrofti but has never been reported as a vector for B. malayi, or a susceptible host for the growth and development of the microfilariae of B. malayi. The purpose of this study was to examine the development of B. malayi in Mansonia uniformis after feeding on the blood of an infected cat in the laboratory. The B. malayi infected cat was identified using PCR with the primers Bm-1/Bm-2 on DNA (at 10 ng/50 microl) extracted from the WBC of the cat. W. bancrofti was employed as a negative control. The sensitivity of the B. malayi DNA detection by PCR was 0.0001 ng. Adult Ma. uniformis mosquitos at the ages of 5, 10, and 15 days, 100 mosquitos in each group, were fed on the infected cat blood. Recovery of third stage microfilariae was found to be the highest in the 5-day old mosquito group (48%), followed by the 10- and 15-day old mosquito groups (32% and 18%, respectively). The mean number of B. malayi microfilariae found in thorax, head, and abdomen of the mosquitos were composed. The 5-day old (40.3%) and 10-day old (41.9%) mosquitos were significantly more susceptible to microfilariae than the 15-day old mosquitos (17.8%) (p-values using the Scheffe method: 0.027 and 0.039, respectively). There was no significant difference in the mean number of microfilariae in the thorax (p = 0.482) by age, but the mean numbers of microfilariae in the heads, and abdomens were significantly different by age between the 5- and10-, and the 15-day old mosquitos (p < 0.001 and p = 0.004, respectively).     

PCR检测恶性疟原虫感染蚊方法的建立

目的 建立一种简便的可应用于现场蚊感染恶性疟原虫的PCR检测方法。方法 采用针对恶性疟原虫小亚单位核糖体DNA（SSUrDNA）的特异引物，利用PCR技术，从实验室感染及现场采集的感染恶性疟原虫的蚊基因组DNA中，扩增恶性疟原虫SSUrDNA片段，进行恶性疟原虫的检测。结果 扩增产物经琼脂糖凝胶电泳分析，可检测出特异的约188bp大小的DNA片段，其基因序列与预计序列完全一致。估测每份蚊DNA样本中含有10个以上子孢子即可获得此片段，而对间日疟原虫及未感染蚊DNA不能扩增出此片段。结论 该PCR检测方法可应用于现场恶性疟原虫感染蚊的检测。     

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.     

Detection and differentiation of filarial parasites by universal primers and polymerase chain reaction-restriction fragment length polymorphism analysis

Filarial nematode parasites are a serious cause of morbidity in humans and animals. Identification of filarial infection using traditional morphologic criteria can be difficult and lead to misdiagnosis. We report on a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)-based method to detect and differentiate a broad range of filarial species in a single PCR. The first internal transcribed spacer 1 (ITS1) along with the flanking 18S and 5.8S ribosomal DNA (rDNA) were isolated and cloned from Wuchereria bancrofti, Brugia malayi, and Brugia pahangi. Sequence analysis identified conserved sites in the 18S and 5.8S rDNA sequence that could be used as universal priming sites to generate ITS1-distinctive PCR products that were useful for distinguishing filariae at the genus level. The addition of a digestion of the ITS1 PCR product with the restriction endonuclease Ase I generated a fragment profile that allowed differentiation down to the species level for W. bancrofti, B. malayi, B. pahangi, Dirofilaria immitis, and D. repens. The PCR-RFLP of ITS1 rDNA will be useful in diagnosing and differentiating filarial parasites in human, animal reservoir hosts, and mosquito vectors in disease-endemic areas.