合成的寡核苷酸探针用于诊断恶性疟原虫感染的评价

采用简化方法抽提体外培养的FCB1株或NF54株恶性疟原虫基因组DNA,用含0.1%皂苷的氯化钠、柠檬酸钠溶液溶解红细胞,游离的疟原虫用4% sarcosyl和1mg/ml蛋白酶K溶解,获得的原虫DNA经CsCl密度梯度纯化,然后在Tris和EDFA中透析,用分光光度计测定DNA含量。DNA样品印渍于硝酸纤维膜上,用0.5N NaOH降解DNA,硝酸纤维膜经Tris-NaCl溶液洗涤后真空干燥备用。血液标本点于浸湿的硝酸纤维膜上,干后直接在硝酸纤维膜上按上述方法溶解红细胞和疟原虫,然后降解DNA,洗涤后烘干备用。杂交试验的探针为一种末端标记γ-~(82)PAFP的合成的存在于恶性疟原虫基因组中的21个核苷酸重复片断(5'-AGGTCTTAACTTGACTAACAT-3′)。结果表明该同位素标记的寡核苷酸能与微量恶性疟原虫DNA特异地杂交。放射自显影2小时,该探针能检测出1μg的纯化DNA,如果显影过夜,能测出100pg的DNA,而与1μg的人DNA无交叉杂交。对不同感染率的体     

恶性疟原虫特异的基因克隆

本文用鸟枪法将恶性疟原虫基因组 DNA 片段克隆至载体 pBR_(322)质粒中,利用抗性遗传标志和琼脂糖凝胶电泳筛选重组克隆,克隆 pBF_8,pBF_(13),pBF_(23),pBF_(24)用 HindⅢ酶解后,均显示单一插入片段,分子大小分别为1.8,2.3,0.94和6.0kb。经 Southern Blot 和 Dot Blot 分析表明,克隆 pBF_(13)对恶性疟原虫基因组 DNA 特异,与人 DNA 无交叉性杂交,可用作诊断用探针。     

质粒PF.rep20探针检测我国恶性疟原虫的初步研究

用~(33)P中标记的质粒PF rep20探针以DNA-DNA斑点杂交检测5种疟原虫,仅恶性疟原虫P.f.呈现阳性反应,间日疟原虫、约氏疟原虫、食蟹猴疟原虫及诺氏疟原虫不发生杂交;~(32)P标记的P.f.海南株基因组DNA探针与上述诸疟原虫均可发生杂交。基因诊断海南省和云南省恶性疟患者血样本39份的杂交阳性率,质粒探针和基因探针分别为94.9％和97.4％。检测人工培养的海南株、云南株和安徽株P.f.敏感度,质粒探针均为0.001％原虫血症和10Pg原虫DNA,基因组探针对海南株原虫为0.0001％和1Pg。提示质粒PF rep20探针种的特异性强,敏感性较高,似可用于我国恶性疟原虫检测。     

用[α-~(32)P]—dATP标记恶性疟原虫DNA探针诊断恶性疟疾的研究

本文报告从培养的恶性疟原虫提取基因组DNA,以[α-~(32)p]—dATP标记,作为探针,按DNA斑点杂交法检查取自海南省恶性疟患者的血样23份,结果全部呈杂交阳性反应,镜检阳性样本DNA探针检出率为100%,与10例间日疟患者血样全部出现交叉杂交。该探针可检测出恶性疟原虫密度为0.0001%的原虫血症水平,与鼠疟原虫、正常人血和人白细胞无交叉杂交。     

用DNA杂交技术检出血内恶性疟原虫

用光学显微镜诊断疟疾已不能适应大规模流行病学研究的需要。最近研究进展表明可以用载有恶性疟原虫DNA重复序列的DNA探针检出疟原虫。本文作者报导的这种DNA-DNA杂交检出法,只需病人指尖血就能测定,并且可获得半定量的结果。恶性疟原虫DNA是从体外培养中分离获得,用α-~(32)P-dATP和α-~(32)P-dTTP标记     

恶性疟原虫var基因家族与抗原变异研究进展

恶性疟原虫变异抗原基因(var基因)家族编码的恶性疟原虫红细胞膜蛋白1(PfEMP1)是介导恶性疟原虫抗原变异和红细胞黏附微血管的媒介。本文从恶性疟原虫抗原变异和致病性、感染红细胞表面变异分子的表达、var基因家族的基因结构、PfEMP1蛋白的黏附特性以及var基因家族的变异调控等方面对恶性疟原虫var基因家族与抗原变异的研究进行综述。     

现场应用DNA杂交试验检测恶性疟原虫

本文报道在冈比亚利用从坦桑尼亚恶性疟原虫株中得到的并在pBR322中克隆的21个碱基对重复排列的1.7Kb片段,用~(32)P进行放射性标记的DNA探针,对3组疟区10岁以下儿童检测恶性疟的研究结果。将采集的血标本放入加有肝素的试管内,离心弃上清,取50μl压积红细胞与100μl含1％ SDS和     

不同DNA探针检测疟原虫的灵敏度比较

本文通过分子杂交技术,以恶性疟原虫为靶DNA,对从恶性疟基因文库中筛出的含有高度重复顺序的pBF_4DNA探针和含单考贝基因的pBF_(13)DNA探针以及恶性疟原虫全基因组DNA探针进行了杂交比较,结果pBF_(13)DNA探针比全基因组DNA探针敏感度低10倍,比pBF_4DNA探针敏感度低100倍,全基因组DNA探针可检到的DNA含量为100pg。     

免疫筛选恶性疟原虫cDNA克隆

目的 :获取恶性疟原虫 (海南株 )抗原的 c DNA克隆并进行初步鉴定。方法 :采用 dot-EL ISA,以兔免疫血清对恶性疟原虫 c DNA表达文库约 80万个重组噬斑进行筛选 ,并用 2 0株单克隆抗体和恶性疟患者血清对强阳性克隆进行再筛选。 PCR初步鉴定 17个强阳性克隆。结果 :兔免疫血清确定了 17个强阳性克隆, 患者血清检测到 11个阳性克隆 (含 8个强阳性 ) ,11株单抗与 9个 c DNA克隆呈阳性反应。 17个强阳性克隆均能扩增出大小在 30 0 bp- 2 .5kb左右的条带。结论 :已筛选到能与抗恶性疟原虫兔血清、单克隆抗体及患者血清产生特异性免疫反应的 c DNA克隆。     

用重复DNA克隆作为种特异性探针检测恶性疟原虫DNA

用恶性疟原虫重复顺序克隆进行杂交的方法可提高检测系统的重现性和敏感性。分离疟原虫DNA并用CsCl放线菌素D离心去除人DNA杂质。Tanzania株Ⅰ用Sau 3AI部分酶切并克隆到有Bam HI切点的噬菌体M13mp 18中,用标记的全部恶性疟原虫基因组DNA探针进行噬菌斑杂交选出带有重复顺序的克隆株。用标记的恶性疟基因组DNA探针与人     

Detection of Plasmodium falciparum in blood using DNA hybridization

A rapid and simple assay for detecting Plasmodium falciparum in human blood was developed. The assay is based on DNA-DNA spot hybridization, using radiolabeled P. falciparum DNA as a probe and finger prick blood as the assay sample. It is very sensitive, able to detect parasitemia levels of 0.0001% in 10 microliter of blood. The assay can be quantified and used to estimate parasitemia levels. Several hundred blood samples can be processed simultaneously, and the entire procedure is completed within 24 hr. This assay can be useful for epidemiological surveys, for screening of blood by blood banks and for health authorities examining immigrants and tourists coming from malaria infested areas.     

环介导等温扩增技术检测恶性疟原虫的研究

目的环介导等温扩增(LAMP)技术检测恶性疟原虫。方法酚氯仿法提取恶性疟原虫基因组DNA,设计四条扩增恶性疟原虫环子孢子蛋白基因的LAMP引物,以间日疟原虫、弓形虫、卡氏肺孢子虫、人全血DNA为对照,进行LAMP反应。LAMP产物经显色、电泳鉴定。将原虫血症为1.5%的恶性疟原虫血样用正常人血按1∶10倍比稀释为1.5×10-3、1.5×10-4、1.5×10-5、1.5×10-6、1.5×10-7、1.5×10-86个浓度后进行LAMP,检测其敏感性。结果恶性疟原虫检测管经显色后呈绿色(阳性),对照组均呈棕色(阴性)。恶性疟原虫LAMP产物经电泳后呈LAMP特征性梯状条带,对照组均无扩增产物。LAMP可检测恶性疟原虫的敏感度为1.5个疟原虫/107RBC。结论检测恶性疟原虫的LAMP方法特异、敏感及简便。     

复式PCR检测间日疟原虫和恶性疟原虫的现场应用

目的探讨复式PCR方法在疟疾诊断中的现场应用价值。方法根据疟原虫18 S rRNA基因序列,合成疟原虫属特异性上游引物和间日疟原虫、恶性疟原虫种特异性下游引物,优化PCR反应体系,建立在同一PCR反应体系中同时检测间日疟原虫、恶性疟原虫基因组特异性DNA片段的疟疾诊断方法,并评价其现场应用价值。结果间日疟原虫和恶性疟原虫基因组DNA经过复式PCR后,分别扩增出1 451 bp和833 bp的特异性条带,而伯氏疟原虫、食蟹猴疟原虫及健康人血样均无扩增带出现,用该反应体系可检出原虫血症为1.1×10-6和5.6×10-7的间日疟原虫和恶性疟原虫感染。与镜检法相比,复式PCR检测119份现场样本,112份与镜检结果相同,阳性率为54%,漏诊率为0.8%,误诊率为0,而镜检法依次分别为53%、1.7%和3.4%。结论复式PCR方法检测疟原虫具有简便、快速、特异、敏感等优点,在疑似病例的鉴别诊断和分子流行病学调查中具有良好的应用价值。     

重组质粒DNA探针用于间日疟诊断的研究

用~(32)P标记含间日疟原虫DNA片段的重组质粒pVA1作为探针,通过DNA打点杂交试验检测红内期间日疟原虫。该探针检测间日疟原虫基因组DNA的敏感度达1ng;与现场采集的25份间日疟患者血样(原虫血症为0.003％～0.7％)的杂交阳性率为72％,与6例恶性疟和3例食蟹猴疟血样中各1例杂交阳性;与3例约氏疟和6例正常人血样均为阴性。     

多重PCR快速检测恶性疟和间日疟的研究

目的 建立一种简便快速、能同时检测恶性疟和间日疟的核酸检测方法。方法 针对两种疟原虫18S rRNA基因设计2对(3条引物),优化引物浓度与退火温度,建立可扩增出两种疟原虫基因片段的多重PCR。并进行最低检测限确定和临床标本检测,以镜检法为金标准分析灵敏度和特异度等指标。结果 该方法可扩增出431 bp(恶性疟原虫)和341 bp(间日疟原虫)基因片段,最低检测限为10²copies/反应,检测临床标本的结果与镜检法无差别(P＞0.05),敏感度为93.55%,特异度为70.83%,阳性预测值为89.23%,阴性预测值为80.95%。结论 所建立的多重PCR方法可快速检测疟疾感染并鉴别分型,灵敏度高,值得推广。     

Molecular cloning of Plasmodium falciparum blood stage antigens and application of the recombinant proteins in serodiagnosis.

A Plasmodium falciparum genomic DNA library was established in the expression vector lambda gt11, cloned in Escherichia coli. The library was screened with human hyperimmune sera by in situ hybridization. Twenty clones expressing P. falciparum sequences as polypeptides fused to beta-galactosidase were identified. One, CD3A/9025/60, reacted with all immune sera and expressed polypeptides that were larger than beta-galactosidase as well as reacting with antibodies to beta-galactosidase and to P. falciparum. When the fusion proteins were used as target antigens to diagnose malaria antibodies, a result was obtained which correlated well with indirect fluorescence assay.     

Use of a DNA hybridization assay for the detection of Plasmodium falciparum in field trials

A DNA probe consisting of 21 base pair repeats obtained from a Tanzanian isolate of Plasmodium falciparum, cloned in pBR322 and labeled with 32P by nick translation was used to detect malaria parasitemia in samples obtained during a malaria survey undertaken in The Gambia. In an initial trial the hybridization assay had a specificity for P. falciparum of 100% and a sensitivity of 68%. False negative results were obtained only on samples with low parasitemia. Assay of red cells collected during an earlier malaria survey which had been stored for 1 year at -20 degrees C gave a higher level of sensitivity (85%), suggesting a beneficial effect from freezing and thawing. This was confirmed by examining in the same assay red cells processed immediately after collection and after 2 weeks of storage at -20 degrees C. Freezing and thawing gave a 21% increase in positivity, and a sensitivity of 100% was achieved with the frozen samples. Quantitation of autoradiographs by visual inspection and by scintillation counting gave a reasonable correlation with parasite counts. The DNA hybridization assay has considerable promise as an epidemiological tool.     

Assessment of a synthetic DNA probe for Plasmodium falciparum in African blood specimens

Synthetic DNA oligomers homologous to 21-base long repetitive sequences of Plasmodium falciparum DNA were labeled with 32P using T4 kinase, and were hybridized with purified DNA and with processed blood samples from Africa. The sequence PFR1, its antiparallel oligomer PFR1R, and PFR1 covalently attached to biotin hybridized similarly to P. falciparum DNA. One-microliter aliquots of blood from Zaire spotted on prewet nylon filters and hybridized with PFR1 gave detectable autoradiogram signals from samples with parasitemias as low as 1,000 parasites/mm3. Blood lysis and protein digestion followed by alkylation allowed dot-blot processing of larger aliquots of blood. After hybridization with PFR 1 and autoradiography, 26 samples were scored positive visually, compared with 34 scored positive by microscopy. The effective sensitivity for processed 10-microliter samples was about 500 parasites/mm3. Signals from hybridized probes were quantitated by liquid scintillation counting and densitometry, and were proportional to the amounts of purified P. falciparum DNA applied to the filter. Autoradiogram signals also were roughly proportional (correlation coefficient, r = 0.77) to the number of parasites/mm3 of blood from field samples as determined by microscopic examination.     

恶性疟原虫重组乳酸脱氢酶胶体金免疫层析法检测试纸条的研制

目的 用氯金酸标记抗恶性疟原虫乳酸脱氢酶（LDH）单克隆抗体（McAb），研制用于诊断恶性疟原虫和间日疟原虫感染的胶体金免疫层析（GICA）试纸。方法 采用柠檬酸盐还原法制备胶体金颗粒、标记抗恶性疟原虫重组乳酸脱氢酶单克隆抗体，制成免疫层析检测试纸条。用该试纸条检测已知疟疾病人及健康人血样，鉴定方法的敏感性和特异性。结果 检测重组LDH抗原的最小量为5ng／ml；对30例恶性疟病人血样及40例间日疟病人血样进行检测，敏感性分别为83．33％和57．50％，检测100例健康者血样特异性为98．00％。结论 初步建立了同时诊断恶性疟原虫和间日疟原虫感染的GICA检测法。