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     

荧光定量PCR用于按蚊体内疟原虫子孢子检测的研究

目的 建立一种用于按蚊体内疟原虫子孢子定量检测和虫种鉴别的荧光定量PCR方法。方法 采用针对4种人体疟原虫18S rRNA基因属特异性保守区的1对引物, 以疟原虫18S rRNA基因重组质粒与按蚊DNA的混合物为模板, 进行反应体系和反应条件优化, 验证方法的特异性, 并通过熔解曲线分析进行虫种鉴别。应用阴性按蚊DNA稀释的间日疟原虫18S rRNA基因重组质粒为模板制作标准曲线, 并分别以质粒DNA和实验室子孢子感染阳性的按蚊DNA为模板分析建立的荧光定量PCR方法的敏感性。在反应体系中加入不同部位、 不同用量按蚊DNA, 以探讨按蚊DNA对检测效果的影响。结果 该方法对按蚊、 人血DNA均无扩增, 对4种疟原虫DNA均有特异性扩增且扩增产物的熔解温度 （Tm） 易于区分, 三日疟原虫、 恶性疟原虫、 卵形疟原虫和间日疟原虫的Tm值分别为71.0、 72.7、 73.9 ℃和75.9 ℃。标准曲线中循环阈值（Ct值） 与模板浓度具有良好的相关性 （相关系数r = -0.99）。最低可以检出含50拷贝的质粒DNA或32倍稀释的子孢子阳性按蚊DNA样本。按蚊DNA对荧光定量PCR反应具有抑制作用。荧光定量PCR的Ct值在实验内和实验间均具有良好的重现性。结论 新建立的SYBR Green I染料荧光定量PCR方法具有较高的敏感性和特异性, 可用于按蚊体内疟原虫子孢子的检测, 并可同时对4种人体疟原虫进行鉴别。     

聚合酶链反应检测蚊内恶性疟原虫

根据恶性疟原虫BRK1—14重复序列DNA片段设计的引物(5＇CGC TACA TATGCTAGTTGCCAGAC3＇和5＇CGTGTAC-CATACATCCTACCAAC3＇)供PCR扩增检测蚊体内恶性疟原虫。用此扩增系统检测了经镜检证实子孢子和卵囊阳性的实验室人工感染恶性疟原虫的大劣按蚊及用镜检和     

环介导等温扩增技术检测蚊体内间日疟原虫子孢子的研究

目的建立一种简便、快速和高敏感度的蚊体内间日疟原虫检测的环介导等温扩增方法（LAMP）。方法针对间日疟原虫环子孢子蛋白（CSP）基因种属特异性保守区域6个位点设计2对引物,以感染性按蚊、阴性按蚊、恶性疟原虫及正常人血DNA为模板评价LAMP的特异性。将间日疟原虫CSP基因质粒DNA梯度稀释,并与阴性按蚊DNA按1.0μl加1.3×10^6、1.3×10^5、1.3×10^4、1.3×10^3、1.3×10^2、1.3×10^1、1.3×10^0拷贝混合后为模板进行LAMP反应,观察其检测敏感性;将感染性按蚊与阴性按蚊DNA作1：2、1：4、1：8、1：16、1：32、1：64、1：128、1：256稀释,然后以稀释样本为模板进行LAMP反应,观察批量检测的敏感性。再用此方法与镜检解剖、巢式PCR同时检测同批次人工感染的67只按蚊,评价其应用价值。结果此法检测感染性按蚊阳性,对照组均为阴性;检测不同比例稀释的间日疟原虫CSP基因质粒DNA与按蚊DNA混合物,最低可检测1.3×10^2拷贝的间日疟原虫CSP基因质粒DNA与按蚊DNA的混合物;检测不同比例感染按蚊与阴性按蚊DNA混合样本,最低可检测出在128个按蚊中有1个感染按蚊的混合样本;用此法检测67只同批次人工感染的按蚊,检出率为47.76%,解剖镜检检出率为25.37%（χ^2=7.24,P〈0.01）,巢式PCR检出率为40.30%（χ^2=0.73,P〉0.05）。以镜检解剖作为金标准,LAMP敏感性为100%,巢式PCR敏感性为100%。结论LAMP检测蚊体内间日疟原虫简便、快速、敏感性高,具有广阔的应用前景。     

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。     

老挝占巴塞省按蚊种类及其疟原虫子孢子感染情况调查

目的对老挝占巴塞省按蚊种类及其疟原虫子孢子感染情况进行调查,为当地疟疾防治措施的制定与评估提供参考。方法2017年7月在老挝占巴塞省巴通坡县采用诱蚊灯通宵诱蚊法和通宵人诱法进行捕蚊,对捕获的成蚊进行形态学鉴定,取部分雌性按蚊提取基因组DNA,巢式PCR检测恶性疟原虫和间日疟原虫的18S rRNA基因,计算按蚊疟原虫子孢子阳性率。结果共捕获蚊虫34 687只,分属库蚊、按蚊和伊蚊等8个属的29个种。库蚊属为当地优势属,占捕获蚊虫总数的92.6%(32 110/34 687);其次是按蚊属,占5.6%(1 947/34 687)。迷糊按蚊是按蚊属的优势种,占该属的65.5%(1 275/1 947);其后是可赫按蚊,占12.1%(235/1 947);菲律宾按蚊,占11.9%(232/1 947);中华按蚊,占5.1%(100/1 947)。巢式PCR共检测按蚊336只(可赫按蚊234只、中华按蚊100只和大劣按蚊2只), 8只按蚊间日疟原虫子孢子阳性,阳性率为2.4%(8/336)。其中,可赫按蚊子孢子阳性6只,阳性率为2.6%(6/234);中华按蚊子孢子阳性2只,阳性率为2.0%(2/100);均未检测到恶性疟原虫子孢子。巢式PCR扩增片段长120 bp,其序列与间日疟原虫序列(GenBank登录号为:KT991325、 KT991317、 MG708221、 MF540772、 LT635613、 KU569498、 AF145335、 KT991314和KX007932)的同源性为99%～100%。结论老挝占巴塞省捕获的可赫按蚊和中华按蚊存在间日疟原虫子孢子自然感染,且阳性率均较高。     

试纸条法与PCR法比较:赤道几内亚一个沿海村庄全黑按蚊自然感染的检测

疟疾是赤道几内亚高发病率和死亡率的主要因素之一。以往的调查表明有冈比亚按蚊复合体的5种按蚊分布,冈比亚按蚊复合体和催命按蚊是非洲热带地区主要的传疟媒介。传统方法通过解剖单个按蚊的唾液腺获得按蚊感染疟原虫子孢子率,但这种方法费力费时。以后发展了血清学检测方法和分子检测方法。试纸条法是将针对疟原虫环子孢子蛋白的单抗固定到试纸条,快速简便,按蚊不需要特殊保存条件。近年发展起来的聚合酶链反应(PCR)被用于扩增感染按蚊中恶性疟原虫特异DNA序列,PCR法有极高的敏感性,蚊唾液腺中子孢子少至10个时能被检测到,而试纸条法…     

PCR检测疟疾混合感染的现场应用研究

目的　评价PCR检测技术在间日疟与恶性疟混合感染区诊断疟疾的现场应用价值。　方法　采集海南省疟疾混合感染流行区 3 0 4份滤纸干血滴样本 ,根据红内期疟原虫SSUrRNA基因序列 ,设计合成 3条引物 ,采用PCR技术在同一反应体系中扩增出间日疟原虫和恶性疟原虫不同的DNA片段 ,检测现场所采样本中的间日疟原虫或恶性疟原虫DNA ;同时与镜检法进行比较。　结果　在 3 0 4份样本中 ,PCR法阳性 15份 ,其中间日疟 7份 ,恶性疟 8份 ;镜检法阳性11份 ,其中间日疟 6份 ,恶性疟 5份。镜检阳性的样本PCR均为阳性 ;镜检阴性而PCR阳性的 4份样本 ,其扩增产物经限制性酶切鉴定 ,证实为间日疟原虫或恶性疟原虫DNA。　结论　此PCR检测体系灵敏、特异 ,对诊断或鉴别诊断间日疟原虫和恶性疟原虫混合感染具有实用价值。     

PCR检测疟疾混合感染的现场应用研究

目的 评价PCR检测技术在间日疟与恶性疟混合感染区诊断疟疾的现场应用价值。方法 采集海南省疟疾混合感染流行区304份滤纸干血滴样本，根据红内期疟原虫SSUrRNA基因序列，设计合成3条引物．采用PCR技术在同一反应体系中扩增出间日疟原虫和恶性疟原虫不同的DNA片段，检测现场所采样本中的间日疟原虫或恶性疟原虫DNA；同时与镜检法进行比较。结果 在304份样本中，PCR法阳性15份，其中间日疟7份．恶性疟8份；镜检法阳性11份，其中间日疟6份，恶性疟5份。镜检阳性的样本PCR均为阳性；镜检阴性而PCR阳性的4份样本，其扩增产物经限制性酶切鉴定，证实为间日疟原虫或恶性疟原虫DNA。结论 此PCR检测体系灵敏、特异．对诊断或鉴别诊断间日疟原虫和恶性疟原虫混合感染具有实用价值。     

PCR鉴别诊断间日疟及恶性疟的研究

目的 :在同一反应体系中建立能鉴别诊断间日疟与恶性疟的 PCR检测方法。方法 :根据红内期疟原虫 SSUr RNA编码基因序列 ,设计合成 3个引物 ,采用聚合酶链反应技术 ,在同一反应体系中 ,间日疟原虫和恶性疟原虫分别预期被扩增出 34 1bp和 4 31bp的 DNA条带。结果 :间日疟原虫和恶性疟原虫模板在同一反应体系中分别被扩增出预期大小的 DNA片段 ,并经限制性酶切证实 ;杜氏利什曼原虫、弓形虫、健康人血及空白对照均无特异性扩增条带 ;以该检测体系至少可检出原虫血症为 2 .56× 10 ^-7间日疟原虫感染和 1.0 8× 10 ^-5恶性疟原虫感染 ;69份镜检阳性的间日疟和 2份恶性疟患者血样 PCR检测均为阳性 ,1例发热待查患者PCR诊断为间日疟 ,与镜检复查结果一致 ,所有疟疾阳性标本中未检出混合感染 ,2 0份健康人血 PCR均为阴性。结论 :该检测体系灵敏、特异 ,对于诊断间日疟和恶性疟以及鉴别间日疟原虫和恶性疟原虫混合感染具有一定的应用价值。     

套式PCR检测蚊体内疟原虫的研究

目的建立一种敏感、特异而快速的检测蚊体内疟原虫的方法。方法采用套式PCR方法,对人工感染间日疟原虫的嗜人按蚊、感染恶性疟和混合感染间日疟与恶性疟的大劣按蚊以及流行季节现场捕获的中华按蚊体内的疟原虫进行检测。结果28批人工感染间日疟原虫的嗜人按蚊、2批人工感染恶性疟的大劣按蚊和1批混合感染间日疟与恶性疟的大劣按蚊的检测结果与镜检结果符合率为100%;589只现场捕获的中华按蚊中,发现间日疟原虫阳性2只,阳性率为0.34%。结论本方法能快速而敏感地检出蚊体内不同种疟原虫。     

Strain specificity in the liver-stage development of Plasmodium falciparum in primary cultures of new world monkey hepatocytes

Primary cultures of Aotus and Saimiri monkey hepatocytes were infected with sporozoites of the Plasmodium falciparum NF 54 strain from mosquitoes fed on gametocyte cultures, and with sporozoites of the P. falciparum Santa Lucia strain from mosquitoes fed on an infected Aotus monkey. After 4-8 days, one exoerythrocytic (EE) parasite per 30,000 sporozoites was detected in one of three experiments performed with the P. falciparum NF54 strain. However, numerous EE parasites were detected in Aotus and Saimiri cells infected with sporozoites of the P. falciparum Santa Lucia strain. At day 6, most of the parasites contained several hundred nuclei, and were morphologically similar to those previously described in vivo using light or electron microscopy. A monoclonal antibody directed against the repeat region of the circumsporozoite protein of P. falciparum labeled the plasma and parasitophorous vacuole membrane of five-day-old EE parasites by immunoelectron microscopy, thus supporting previous observations by immunofluorescence indicating that the CS protein persists throughout the EE development of P. falciparum. These results demonstrate that liver stages of P. falciparum can be obtained in Aotus and Saimiri monkey cells, they also suggest a parasite strain specificity for hepatocytes.     

Investigation of malaria sporozoites by immunoradiometric assay

Two-site immunoradiometric assay (IRMA) (Zavala et al., 1982) using monoclonal antibodies to P. falciparum and P. vivax was applied to detect sporozoites in laboratory-maintained An. dirus and also mosquitoes collected from endemic areas of malaria in Thailand. Study in P. falciparum infected mosquitoes revealed that the circumsporozoite (CS) antigen was first found in the abdominal portion on day 10 post-infection, while it could be observed in the salivary glands from day 15 onwards. The head-thorax portion of wild-caught mosquitoes were investigated by IRMA compared with the dissection technique. The results showed that none of the mosquitoes collected from Phrae was positive for malaria. The mosquitoes collected from Chantaburi showed 4 out of 1243 An. dirus that were positive for P. falciparum by IRMA, with sporozoites ranging from 207 to 3875. Among 3123 An. minimus collected from Kanchanaburi, 3 were positive by IRMA, 2 for P. falciparum and one P. vivax with sporozoites found in head-thorax portion were 1880, 2380 and 1026 respectively. Not a single sporozoite was found in the mosquitoes collected from these areas by the dissection technique. However 7 out of 1219 An. minimus from Kanchanaburi were found to possess undeveloped oocysts in the stomach wall. It is evident that the IRMA is efficient, convenient and suitable for the investigation of sporozoites in this region. The application of this technique in further epidemiological study of malaria is in progress.     

Identification of Plasmodium falciparum-infected mosquitoes by a double antibody enzyme-linked immunosorbent assay

A double antibody micro enzyme-linked immunosorbent assay (ELISA) for identifying Plasmodium falciparum sporozoites in mosquitoes is described. Using monoclonal antibodies made against South American P. falciparum sporozoites, the ELISA was able to detect and identify sporozoite antigens of South American and Asian origins in extracts of dried infected mosquitoes.     

The biotin-streptavidin system in a two-site ELISA for the detection of plasmodial sporozoite antigen in mosquitoes

A two-site ELISA has been designed for the detection of sporozoite antigen in mosquitoes. Biotin-labelled monoclonal antibodies against sporozoites and a streptavidin-biotin-peroxidase complex were used to visualize the antigen. Evaluation of the sensitivity and specificity of the procedure was carried out and background levels of reactivity on the basis of negative mosquitoes were calculated. The test has been deliberately kept as simple as possible for use in the tropics and was designed using Anopheles stephensi infected with in vitro cultivated Plasmodium falciparum gametocytes. A minimum of about 100-350 sporozoites could be detected in mature salivary gland infections; in addition sporozoite antigen was detected in mosquitoes several days before the entry of sporozoites into the salivary glands. No reaction was demonstrable either with bloodstage or ookinete antigens of P. falciparum, or with mosquitoes carrying sporozoites of other plasmodial species. The number of sporozoites in positive mosquitoes and the generating capacity of a single oocyst could be assessed by the use of a calibration curve based on dilution data of a known sporozoite suspension. It was found that a single oocyst can produce about 10,000 sporozoite equivalents.     

A rapid sporozoite ELISA using 3,3',5,5'-tetramethylbenzidine as the substrate chromogen

A modified version of the standard 2-site sporozoite enzyme-linked immunosorbent assay (ELISA) using 3,3',5,5'-tetramethylbenzidine (TMB) as the substrate chromogen solution was adapted for rapid detection and identification of Plasmodium falciparum and P. vivax circumsporozoite (CS) proteins. The TMB-ELISA was evaluated using sporozoites from experimentally infected mosquitoes and laboratory colonized uninfected mosquitoes. Our data indicate comparable sensitivity levels between the TMB-ELISA and the standard ELISA, i.e., 50 P. falciparum or P. vivax sporozoites/50 microliters of test solution. Reactions inherent to the method were specific and background reactivity was minimal. The TMB-ELISA is rapid (1 hr), simple, uses a minimal amount of monoclonal antibodies, and is suitable for use in a wide range of laboratories.     

Monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for detection of Plasmodium malariae sporozoites in mosquitoes

A monoclonal antibody specific for a repeated epitope of the circumsporozoite protein of Plasmodium malariae sporozoites has been used to develop a two-site, single antibody-based enzyme-linked immunosorbent assay that can detect P. malariae sporozoites in mosquitoes. The assay uses a purified monoclonal antibody produced against sporozoites of the Uganda I/CDC strain of P. malariae to capture the antigen and the same monoclonal antibody labeled with horseradish peroxidase as the detector. Sporozoites have been detected in laboratory-infected mosquitoes stored at room temperature in the presence of a desiccant for as long as 18 months. The detection limit of the assay is approximately 50 P. malariae sporozoites per test well. Cross-reaction has not been observed with mosquitoes infected with P. falciparum, P. vivax, or P. ovale sporozoites.     

Immunoenzymatic labeling of multiple plasmodial salivary gland sporozoites in a single test.

A direct, double- and triple-staining immunoenzymatic method detected and differentiated sporozoites by color in Anopheles stephensi salivary glands and in mixed sporozoite slide preparations. A double-staining method used beta-galactosidase- and alkaline phosphatase-labeled monoclonal antibodies to the circumsporozoite (CS) proteins of Plasmodium berghei and P. falciparum in mosquito salivary glands. The CS proteins were distinguished clearly by the blue-green and red substrate products of beta-galactosidase and alkaline phosphatase, respectively. A triple-staining method differentiated by color among a mixture of P. falciparum and two strains of P. vivax sporozoites. Monoclonal antibodies to the CS proteins conjugated to beta-galactosidase (P. falciparum), alkaline phosphatase (P. vivax variant), and horseradish peroxidase (P. vivax predominant) readily color differentiated sporozoites by the blue-green, purple-blue, and orange-brown substrate products, respectively. This assay may have potential use in malaria transmission studies, genetic crosses of variant strains of plasmodia to determine assortment of CS antigen alleles, and as a technique to determine the fate of the CS antigen in infected mosquitoes.     

Immunodot assay of Plasmodium falciparum sporozoites in mosquitoes using a direct binding membrane system

We describe a membrane based immunodot assay for the detection of Plasmodium falciparum sporozoites mixed with mosquitoes. A crude sodium dodecyl sulfate extract of mosquitoes and sporozoites is passed through a bi-layered membrane system, the top layer being a polyvinyldiene difluoride hydrophilic pre-filter which screens out debris but allows the passage of antigen. Sporozoite, as well as mosquito, proteins are bound to the hydrophobic membrane below. This membrane was probed with a monoclonal antibody to the repeat region of the P. falciparum circumsporozoite protein, a peroxidase labeled second antibody and a tetramethyl-benzidine substrate. The method detects as few as 10 sporozoites/mosquito or 100 sporozoites in a pool of 10.     

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。