聚合酶链反应检测蚊体内马来丝虫幼虫的实验

目的　建立一种特异、灵敏和简捷的聚合酶链反应 (PCR)检测蚊体内马来丝虫幼虫的方法。方法　在应用一种新的模板纯化处理技术 (Microcon 10 0 )基础上 ,采用适应于检测我国马来丝虫的两套DNA扩增引物(P1、P2与P3、P4) ,对实验感染马来丝虫的中华按蚊进行扩增检测。结果　两套引物均可检出蚊体内不同发育期幼丝虫 (L1、L2 和L3) ,其灵敏度达 1只蚊体内 1/ 6 4条L1和 2 0 0只群体蚊中含有 1只感染蚊 (体内有 1条L3)的水平 ,而对犬恶丝虫及未感染蚊却不能扩增出特异条带。结论　初步建立特异、灵敏和简捷的PCR检测蚊体内马来丝虫幼虫的方法。     

应用单克隆抗体检测蚊体内幼丝虫的实验观察

以马来丝虫感染期幼虫为靶抗原制备多株单克隆抗体（ＭｃＡｂ）从中筛选出１Ｇ１、１Ｈ１单克隆抗体，检测人工感染马来丝虫的中华按蚊。当中华按蚊吸食含马来微丝蚴的兔血后，分别解剖，同时以ＥＬＩＳＡ及Ｄｏｔ－ＥＬＩＳＡ检测饲养不同时间的中华按蚊。结果显示，饲养９ｄ的按蚊马来丝虫幼虫阳性率，人工解剖为８６．６％，ＥＬＩＳＡ和Ｄｏｔ－ＥＬＩＳＡ法分别为８２．２％和７７．８％。经统计学处理，人工解剖与单抗检测无显著性差异，其灵敏度为每只蚊０．５条幼虫。实验证明，此ＭｃＡｂ与牛腹腔指状丝虫、猪肺丝虫抗原无交叉反应，显示了一定的特异性。     

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。     

应用单克隆抗体检测蚊体内幼丝虫的实验观察

以马来丝虫感染期幼虫为靶抗原制备多株单克隆抗体从中筛选出１Ｇ１，１Ｈ１单克隆抗体，检测人工感染马来丝虫的中华按蚊。当中华按蚊吸食马来微丝蚴的兔血后，分别解剖同时以ＥＬＩＳＡ及Ｄｏｔ－ＥＬＩＳＡ检测饲养不同时间的中华按蚊。结果显示，饲养９ｄ的按蚊马来丝虫幼虫阳性率，人工解剖为８６．６％，ＥＬＩＳＡ和Ｄｏｔ－ＥＬＳＩＡ法分别为８２．２％和７７．８％。     

中华按蚊传播亚周期型马来丝虫能力的研究

用中华按蚊人工感染亚周期型马来丝虫,观察其摄入的微丝蚴数、胸肌中的幼虫数和感染期幼虫数。以每蚊体内感染期幼虫数与摄入微丝蚴数之比作为传播能力。结果表明,在温度为28±1C,相对湿度75％的条件下,亚周期型马来丝虫在中华按蚊体内约需10～12d发育成熟,传播能力为10％。     

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。     

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。     

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。     

中华按蚊体内马腹腔丝虫各期幼虫形态的观察

为在基本消灭丝虫病地区纵向监测时提供区别蚊体内人、畜丝虫幼虫的科学依据,我们于1984年8～9月,对马腹腔丝虫(Setaria equina)在中华按蚊体内的各期幼虫形态进行了观察。 感染方法 将饲养驯化的中华按蚊蚊笼紧贴在马腹腔丝虫微丝蚴阳性马的腹部,让蚊虫吸血受染。感染后,蚊虫置于25.8～30.8℃和相对湿度85～94％的养蚊室     

Monoclonal antibody to a unique surface epitope of the human filaria Brugia malayi identifies infective larvae in mosquito vectors.

We describe properties of an IgM monoclonal antibody (NEB-D1E5) raised against the human filarial parasite Brugia malayi. The antibody reacts with a stage- and species-specific determinant located on the surface of the infective-stage larva, as determined by indirect immunofluorescence. To use this reagent in epidemiological field studies, we developed an enzyme-linked immunoassay with which B. malayi larvae can be differentiated from other filarial parasites in mosquito vectors, including the morphologically indistinguishable parasite of animals Brugia pahangi. The immunoenzyme assay was 91-94% specific and 90-97% sensitive when performed on infected mosquitoes. In the absence of mosquito tissue, the levels of specificity and sensitivity increased to 100% and 97.5-100%, respectively. Binding of antibody to the surface of living larvae was abrogated by treatment of the worms with the enzymes pronase and proteinase K and with the detergents Triton X-100, octyl beta-D-glucopyranoside, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate (CHAPS). In contrast, treatment with trypsin, endoglycosidase-F, O-Glycanase, N-Glycanase, lipase, various phospholipases, boiling, 2-mercaptoethanol at 37 degrees C, or periodate did not reduce the antigenicity of the larval surface to antibody NEB-D1E5. These results suggest that the species-specific epitope is a peptide domain attached to a hydrophobic anchoring residue.     

中华按蚊对马来丝虫微丝蚴易感性的观察

目的：研究中华按蚊对不同密度马来丝虫微丝蚴以及贵州和浙江两地虫源的易感性。方法：观察中华按蚊感染不同密度马来丝虫微丝蚴（ｍｆ）后蚊虫的存活率、蚊虫体内微丝蚴发育至感染期蚴（Ｌ３）率和感染蚊体内Ｌ３平均条数，同时观察浙江虫源（６代）和贵州虫源（３１代），微丝蚴发育至Ｌ３率。结果：感染微丝蚴密度为３２．５ｍｆ／μｌ和１４１． ５ｍｆ／μｌ组的微丝蚴发育至Ｌ３率分别为３６．２％和８．７％，感染蚊体内Ｌ３平均条数分别为８．２４和０．３条。中华按蚊感染浙江虫源（６代）和贵州虫源（３１代），微丝蚴发育至Ｌ３率分别是４５．１％和２６．８％。结论：中华按蚊对感染密度为３２．５ｍｆ／μｌ－６６．４ｍｆ／μｌ时较易感，其对浙江虫源（６代）较贵州虫源（３１代）易感。     

马来丝虫微丝蚴定量注射感染中华按蚊和致倦库蚊的实验观察

本文报告采用改进的微量注射技术和较为精细的计数方法,以2—10条微丝蚴/蚊,定量注射感染中华按蚊和致倦库蚊,找出了这两种蚊虫的微丝蚴感染量与感染期幼丝虫阳性率、幼丝虫平均发育成熟率之间的定量关系;比较了在注射感染条件下两种蚊虫对马来丝虫易感性的差异。     

马来丝虫微丝蚴定量注射感染中华按蚊和致倦库蚊的...

With an improved technique, quantitative inoculation of Brugia malayi microfilariae (mff) into Anopheles sinensis and Culex quinquefasciatus was carried out in our laboratory in 1988. The development of B. malayi mff in these two species of mosquitoes was observed. In the system of B. malayi and An. sinensis, a well-fitted linear correlation appeared between the number of mff inoculated and the infective larva (L3) positive rate (r = 0.9910; P less than 0.01). The regression equation with the regression coefficient of 8.490 suggested a high susceptibility of this species of mosquito to B. malayi even under the condition of experimental inoculation. The quantitative relationship between the number of mff inoculated and the average filarial maturity rate was also exhibited. At the same dosages of 4 and 10 mff/mosquito, the L3 positive rates and the average filarial maturity rates in An. sinensis were much higher than those in Cx. quinquefasciatus (P less than 0.01), indicating significant difference occurred inside the bodies of these two species of mosquitoes.     

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.     

肾移植患者巨细胞病毒感染检测方法的比较

目的 :采用酶联免疫吸附法 (ELISA)、定性及定量聚合酶链反应法 (PCR)监测肾移植术后巨细胞病毒(Cytomegalovirus,CMV)活动性感染 ,探讨不同检测方法在肾移植术后CMV活动性感染防治中的价值。　 方法 :于移植前、后抽取 1 0 8例肾移植受者的外周静脉血 ,采用ELISA法、定性及定量PCR法检测血液中CMV IgM及CMV DNA ;对于CMV活动性感染者应用丙氧鸟苷治疗。　 结果 :术后 58例患者发生CMV活动性感染。诊断CMV活动性感染的灵敏性和特异性 :ELISA法为 1 9%和 1 0 0 % ;定性PCR法为 71 %和 58% ;定量PCR法为 93 %和 1 0 0 %。在CMV活动性感染临床症状出现前 ,定性及定量PCR法检测CMV DNA的阳性率分别为 2 2 %和 86 % ,而ELISA法检测CMV IgM无一例阳性 ;用丙氧鸟苷治疗后 ,55例有效 ,3例无效。　 结论 :与ELISA法及定性PCR法比较 ,定量PCR法能够更灵敏、特异的监测肾移植术后CMV活动性感染 ,并可用于评价丙氧鸟苷的抗病毒疗效 ,指导CMV活动性感染的治疗     

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.