巢式PCR检测日本血吸虫感染的研究

本研究通过设计2对巢式引物扩增日本血吸虫高拷贝的Sjα1基因片段,建立检测日本血吸虫感染的巢式PCR技术,并对感染小鼠血清、全血样本以及实验和现场钉螺样本进行检测.建立的巢式PCR方法特异性扩增日本血吸虫420 bp的Sjα1片段,和曼氏血吸虫没有交叉,基因组DNA作为模板时最低检测量为0.1fg.小鼠感染日本血吸虫后2周的血清样本中即能检测出特异性DNA,建立的方法能同时检测血清和全血标本.钉螺实验感染4 h后能检测到日本血吸虫DNA,现场采集钉螺的检测结果显示比传统的镜检方法敏感性高.建立的巢式PCR检测日本血吸虫感染具有较高的敏感性和特异性,为疾病诊断和媒介调查提供了新的分子生物学检测技术. PCR技术,并对感染小鼠血清、全血样本以及实验和现场钉螺样本进行检测.建立的巢式PCR方法特异性扩增日本血吸虫420 bp的sja1片段,和曼氏血吸虫没有交叉,基因组DNA作为模板时最低检测量为0.1fg.小鼠感染日本血吸虫后2周的血清样本中即能检测出特异性DNA,建立的方法能同时检测血清和全血标本.钉螺实验感染4 h后能检测到日本血吸虫DNA,现场采集钉螺的检测结果显示比传统的镜检方法敏感性高.建立的巢式PCR检 日本血吸虫感染具有较高的敏感性和特异性,为疾病诊断和媒介调查提供了新的分子     

实验感染大、小鼠和临床疑似病例肺孢子虫mt LSU rRNA基因检测

为探讨PCR技术对实验感染大、小鼠肺孢子虫肺炎(PCP)和临床疑似病例诊断的可行性。用皮下注射地塞米松法诱导SD大鼠和ICR小鼠PCP并收集临床疑似病例24h深部痰液。受试动物解剖后,制备肺印片,经瑞姬氏复合染色镜检确定肺孢子虫(Pc)感染的阳性率;同时,以针对虫体mtLSUrRNA基因设计的引物,PCR扩增鼠肺组织和支气管灌洗液(BALF),以及痰液样本中的DNA;比较实验动物样本镜检和PCR两种方法的检测结果;测定PCR的敏感性和特异性。结果表明,SD大鼠和ICR小鼠肺印片的阳性率分别为68.8%(1116)和85.7%(1821),肺组织PCR检测的阳性率分别为75%(1216)和80.9%(1721),肺泡灌洗液PCR的阳性率分别为81.2%(1316)和23.8%(521)。肺印片镜检和PCR技术检测结果无显著性差别;可见,PCR对虫体mtLSUrRNA基因的检测具有显著的特异性和敏感性,至少可以检测出0.6pg的肺孢子虫DNA;6例临床疑似病例样本中2例显示肺孢子虫PCR阳性反应。     

套式/多重PCR检测疟疾感染的应用研究

目的了解标签引物-套式/多重PCR技术在检测疟疾感染中的现场应用价值。方法随机采取我省疟疾流行区有发热症状的居民及家庭成员的耳垂血79份,同时制作厚血片和滤纸血样本各1份,用标签引物-套式/多重PCR检测所采集滤纸血样本中的疟原虫,并与镜检法进行比较。结果79份样本中,用标签引物-套式/多重PCR技术检出间日疟原虫阳性6例,镜检初检出10例间日疟原虫阳性,经复查血片后有4例排除了疟疾感染。镜检复核阳性的6例样本PCR均为阳性。以镜检复核为标准,两法阳性和阴性符合率为100%。结论标签引物-套式/多重PCR检测疟疾感染具高度敏感性和特异性,对疟疾鉴别诊断和明确诊断具有重要价值。     

PCR法在小鼠早期念珠菌病诊断中的应用研究

目的 探讨PCR法对早期念珠菌病的诊断价值.方法 采用Biospin真菌基因组DNA提取试剂盒提取感染小鼠全血白色念珠菌DNA,并与血培养和脾脏、肾脏组织病理检查结果比较.结果 白色念珠菌标准菌株和两株临床分离菌株经PCR测定后,均可扩增到分子量大小约为500 bp的特异性条带.在小鼠早期念珠菌病的感染中,运用PCR法较血培养和组织病理检查敏感性高.结论 PCR法是一种快速、灵敏且特异性高的检查手段,为临床早期检测真菌病提供实验依据.     

新孢子虫核酸检测方法的建立及应用评价

以新孢子虫Nc-5基因为目的基因,建立检测新孢子虫的PCR方法并初步应用。根据Gen Bank发布的新孢子虫Nc-5特异性基因序列设计引物,以新孢子虫基因组DNA为模板,利用梯度PCR法优化反应条件,建立PCR检测方法。以弓形虫、疟原虫、牛环形泰勒虫、包拉米虫和马尔太虫为模板进行扩增以验证建立方法的特异性。采用紫外分光光度计测定新孢子虫基因组DNA浓度和纯度,倍比稀释后的DNA进行PCR扩增,产物电泳分析确定建立方法的敏感性;选择高浓度和低浓度的新孢子虫基因组DNA重复检测3次,分析建立方法的重复性和稳定性。利用该方法对实验室建立的小鼠感染模型进行初步应用,评价建立方法的检测效果。结果成功建立新孢子虫的核酸扩增检测方法,扩增序列与Gen Bank(LN714476.1)中Nc-5基因序列一致性为100%,建立的新孢子虫核酸扩增检测方法与弓形虫、疟原虫、牛环形泰勒虫、包拉米虫和马尔太虫均无交叉反应,最低能检测新孢子虫DNA浓度为0.5788 pg/μL,应用该方法检测感染新孢子虫的BALB/c小鼠模型,可在肺和脑组织中检测到新孢子虫的感染。结果表明,建立的新孢子虫核酸扩增检测方法有效,并为后续研究奠定基础。     

用于我国新生儿HIV-1感染早期诊断的单管降落巢式三重PCR检测方法的建立

目的 我国面临着艾滋病母婴垂直传播的挑战.发展经济、有效的新生儿HIV-1感染早期诊断方法至关重要.本研究拟结合降落PCR、巢式PCR、多重PCR技术,以期建立一种能够在同一个PCR反应体系中同时扩增Env、Gag、Pol三个基因区的单管降落巢式三重PCR方法用于我国新生儿滤纸干血斑样本(DBS)中的HIV-1前病毒DNA检测,并对所建立的方法进行初步评价.方法 样本采自云南、新疆、广西、河南四省HIV-1阳性母亲所生婴儿.用18个DBS样本进行方法优化(感染婴儿及未感染婴儿样本分别9个).采用由重组质粒及8E5细胞制备的涵盖HIV-1各亚型的DNA稀释盘确定方法的亚型适用性及检测下限.采用134个DBS样本进行临床评价(含25个感染婴儿的64个DBS样本及30个未感染婴儿的70个DBS样本).结果 所建方法对HIV-1 A、AE、AG、B、BC、C、D、F、H亚型均可适用,检测下限为3拷贝/PCR反应体系；其临床特异度100％,阳性检出率在3月龄时接近95％、6月龄时可达到100％.结论 本研究建立的方法能够检测出我国新生儿DBS样本中各亚型的HIV-1前病毒DNA,且省时、省力、节约成本,在我国新生儿HIV-1感染的早期诊断领域具备应用潜力.     

抗锥由病新药──锥46治疗水牛伊氏锥虫病的研究

为证实锥４６对水牛伊氏锥虫病的疗效，进行了人工感染治疗试验和田间试验。人工感染伊氏锥虫病的治疗试验结果显示：１．５ｍｇ／ｋｇ锥４６对锥虫有根治作用并无毒副作用，１ｍｇ×２／ｋｇ和１ｍｇ／ｋｇ对锥虫有即时疗效而不能根治。１．５ｍｇ／ｋｇ，１ｍｇ／ｋｇ和０．５ｍｇ／ｋｇ锥４６对自然感染伊氏锥虫病水牛的治愈率分别为１００％，７１．４％和８５．７％。田间试验证实１．５ｍｇ／ｋｇ锥４６对水牛伊氏锥虫病的总治愈率为９４．１％。研究结果提示１．５ｍｇ／ｋｇ锥４６可作为治疗水牛伊氏锥虫病的推荐剂量。     

胶乳凝集试验对家畜伊氏锥虫感染早期诊断的观察

<正> 前文报道用化学交联方法将DEAE52柱层析纯化的伊氏锥虫抗原和羧化聚苯乙烯胶乳相连,制备锥虫胶乳试剂,可快速诊断家畜伊氏锥虫感染。其阳性率大于95％,假阳性率低于2％,此结果远优于一般常用的血凝和补体结合试验。本文结合动物实验并扩大现场试验,对胶乳凝集试验的早期诊断及实际应用价值作了进一步的观察。     

重组酶聚合酶扩增结合横向流体试纸条快速检测人腺病毒CSCD

目的 建立一种快速、敏感的人腺病毒等温核酸扩增检测方法.方法 针对人腺病毒hexon基因保守区序列设计特异性重组酶聚合酶扩增（RPA）引物及探针、优化反应时间和温度、用横向流体试纸条（LFD）和毛细管电泳检测扩增产物,建立人腺病毒RPA-LFD快速检测方法,评价该方法的敏感度和特异性并与Real-time PCR法比较.结果 RPA-LFD方法检测人腺病毒的最低检出限为2拷贝DNA分子/反应,且与其他呼吸道病原无交叉反应,临床样本检测结果与Real-time PCR法一致性为100％.结论 建立的RPA-LFD方法具有敏感性、特异性高、快速且不需要昂贵的仪器设备等优点,为人腺病毒快速检测提供了新工具.     

伊氏锥虫微管结合蛋白p15基因的分子克隆与表达

根据报道的布氏锥虫(Trypanosoma brucei)微管结合蛋白p15(Tb-MAPp15)基因及其3′非翻译区保守序列设计引物,采用PCR法从伊氏锥虫云南水牛株(Trypanosoma evansi stock YNB)基因组DNA中克隆得到伊氏锥虫微管结合蛋白p15(Te-MAP p15)基因。克隆片段长273bp,编码90个氨基酸,预测分子量9.0kDa。经同源性比较,所得基因与Tb-MAP p15基因同源率达到94%。抗原性分析Te-MAP p15基因表达的氨基酸序列比T.brucei微管结合蛋白p15多5个,均参与组成其中1个抗原决定簇,并且此抗原决定簇序列形成在蛋白中常作为识别位点的α螺旋。将该基因亚克隆到pGEX-6P-1原核表达载体,转化大肠杆菌E.coli RS21宿主菌,经IPTG诱导,可成功表达。重组融合蛋白大小为35kDa,与预期大小一致,经SDS-PAGE和Western-blot鉴定为重组伊氏锥虫微管结合p15蛋白。     

Detection of Trypanosoma congolense and T. brucei subspecies in cattle in Zambia by polymerase chain reaction from blood collected on a filter paper

To facilitate epidemiology studies of African trypanosomiasis in cattle in Zambia, we adapted a polymerase chain reaction (PCR) method using blood spotted on filter papers. For easy preparation of template DNA from the dried blood, we adapted a simple DNA extraction method using Chelex-100, an anion-exchange resin. Using primers directed for repetitive nuclear DNA sequences, species-specific DNA amplifications were detected from the blood of rats infected with Zambian isolates of T. congolense and T. brucei subspecies. The method was sensitive enough to detect a single trypanosome for both species. In the Eastern Province of Zambia, 240 cattle were examined for motile flagellates in the buffy coat by the microhematocrit method, and 100 of them were positive for the test. These 100 animals were further examined by thin blood smears and PCR for species identification. The thin blood smear revealed 62 and 14 animals with T. congolense and T. brucei subspecies infection, respectively, whereas the PCR detected 73 of the former and 38 of the latter species. These results indicate that dried blood spots on filter papers are a useful source of DNA for detection of African trypanosomes by PCR.     

Loop-mediated isothermal amplification for detection of African trypanosomes

While PCR is a method of choice for the detection of African trypanosomes in both humans and animals, the expense of this method negates its use as a diagnostic method for the detection of endemic trypanosomiasis in African countries. The loop-mediated isothermal amplification (LAMP) reaction is a method that amplifies DNA with high specificity, efficiency, and rapidity under isothermal conditions with only simple incubators. An added advantage of LAMP over PCR-based methods is that DNA amplification can be monitored spectrophotometrically and/or with the naked eye without the use of dyes. Here we report our conditions for a highly sensitive, specific, and easy diagnostic assay based on LAMP technology for the detection of parasites in the Trypanosoma brucei group (including T. brucei brucei, T. brucei gambiense, T. brucei rhodesiense, and T. evansi) and T. congolense. We show that the sensitivity of the LAMP-based method for detection of trypanosomes in vitro is up to 100 times higher than that of PCR-based methods. In vivo studies in mice infected with human-infective T. brucei gambiense further highlight the potential clinical importance of LAMP as a diagnostic tool for the identification of African trypanosomiasis.     

Detection of Trypanosoma brucei spp. in human blood by a nonradioactive branched DNA-based technique.

We have developed a nonradioactive branched DNA (bDNA)-based assay for the diagnosis of the African trypanosomiases in simple buffy coat preparations of human blood. Two repetitive DNA sequences specific to the Trypanosoma brucei complex were chosen as targets of the bDNA assay, a technique which amplifies the signal from a target molecule rather than the target itself. Comparable sensitivities were observed with cloned target sequences, purified T. brucei DNA, procyclic trypanosomes, and bloodstream trypomastigotes. The results of bDNA analysis of human blood samples from Côte d'Ivoire (n = 50) showed excellent agreement with those of buffy coat microscopy. The bDNA technology offers certain advantages over alternative molecular biological techniques, including the simplicity of sample preparation and of the procedure itself, the stability of the reagents, the ability to process large numbers of samples simultaneously, and freedom from crosscontamination artifacts. We have successfully applied the bDNA technique to the detection of T. brucei in clinical samples from regions where T. brucei infection is endemic; to our knowledge, this is the first report of the molecular detection of T. brucei in human blood.     

Molecular dipstick test for diagnosis of sleeping sickness

Human African trypanosomiasis (HAT) or sleeping sickness is a neglected disease that affects poor rural populations across sub-Saharan Africa. Confirmation of diagnosis is based on detection of parasites in either blood or lymph by microscopy. Here we present the development and the first-phase evaluation of a simple and rapid test (HAT-PCR-OC [human African trypanosomiasis-PCR-oligochromatography]) for detection of amplified Trypanosoma brucei DNA. PCR products are visualized on a dipstick through hybridization with a gold-conjugated probe (oligochromatography). Visualization is straightforward and takes only 5 min. Controls both for the PCR and for DNA migration are incorporated into the assay. The lower detection limit of the test is 5 fg of pure T. brucei DNA. One parasite in 180 microl of blood is still detectable. Sensitivity and specificity for T. brucei were calculated at 100% when tested on blood samples from 26 confirmed sleeping sickness patients, 18 negative controls (nonendemic region), and 50 negative control blood samples from an endemic region. HAT-PCR-OC is a promising new tool for diagnosis of sleeping sickness in laboratory settings, and the diagnostic format described here may have wider application for other infectious diseases.     

Futile import of tRNAs and proteins into the mitochondrion of Trypanosoma brucei evansi

Trypanosoma brucei brucei has two distinct developmental stages, the procyclic stage in the insect and the bloodstream stage in the mammalian host. The significance of each developmental stage is punctuated by specific changes in metabolism. In the insect, T. b. brucei is strictly dependent on mitochondrial function and thus respiration to generate the bulk of its ATP, whereas in the mammalian host it relies heavily on glycolysis. These observations have raised questions about the importance of mitochondrial function in the bloodstream stage. Peculiarly, akinetoplastic strains of Trypanosoma brucei evansi that lack mitochondrial DNA do exist in the wild and are developmentally locked in the glycolysis-dependent bloodstream stage. Using RNAi we show that two mitochondrion-imported proteins, mitochondrial RNA polymerase and guide RNA associated protein 1, are still imported into the nucleic acids-lacking organelle of T. b. evansi, making the need for these proteins futile. We also show that, like in the T. b. brucei procyclic stage, the mitochondria of both bloodstream stage of T. b. brucei and T. b. evansi import various tRNAs, including those that undergo thiolation. However, we were unable to detect mitochondrial thiolation in the akinetoplastic organelle. Taken together, these data suggest a lack of connection between nuclear and mitochondrial communication in strains of T. b. evansi that lost mitochondrial genome and that do not required an insect vector for survival.     

Immunization with recombinant actin from Trypanosoma evansi induces protective immunity against T. evansi, T. equiperdum and T. b. brucei infection

Actin gene of Trypanosoma evansi (STIB 806) was cloned and expressed in Escherichia coli. The predicted amino acid sequence of T. evansi actin shows 100%, 98.7%, and 93.1%, homology with Trypanosoma equiperdum, Trypanosoma brucei brucei, and Trypanosoma cruzi. Recombinant actin was expressed as inclusion bodies in E. coli. It was purified and renatured for immunological studies. Mice immunized with the renatured recombinant actin were protected from lethal challenge with T. evansi STIB 806, T. equiperdum STIB 818, and T. b. brucei STIB 940, showing 63.3%, 56.7%, and 53.3% protection, respectively. Serum collected from the rabbit immunized with recombinant actin inhibited the growth of T. evansi, T. equiperdum, and T. b. brucei in vitro cultivation. Serum from mice and rabbits immunized with recombinant actin only recognized T. evansi actin but not mouse actin. The results of this study suggest that the recombinant T. evansi actin induces protective immunity against T. evansi, T. equiperdum, and T. b. brucei infection and may be useful in the development of a vaccine with other cytoskeletal proteins to prevent animal trypanosomiasis caused by these three trypanosome species.     

Cytomegalovirus DNA detection of an immediate early protein gene with nested primer oligonucleotides.

A rapid and sensitive polymerase chain reaction (PCR) was developed to detect conserved sequences from the immediate early gene of human cytomegalovirus (HCMV). The primers sequences were from EcoRI J fragment of Ad169. The first primer set was selected to amplify a 242 bp fragment and the next primer set was nested within the first and amplified a 146 bp fragment. With the single PCR system it was possible to detect 100 fg HCMV DNA but with double PCR 5-10 fg were detectable. Specific amplification was seen in urines from patients with HCMV infections. 20 urine samples were analysed by single PCR, double PCR and virus cultivation. The double PCR was the most sensitive method. Urines from healthy seropositive persons and cells infected with other members of the herpes virus family were negative with all three methods. This suggests that specific amplification by double PCR is sensitive and can be used for rapid detection of HCMV DNA in cases with activated infection.     

Kinetoplast DNA of Trypanosoma evansi

We show here that the kinetoplast DNA (kDNA) networks from six Trypanosoma evansi strains differ from those of T. brucei by their lack of maxi-circles and absence of mini-circle sequence heterogeneity. The lack of maxi-circles is sufficient to account for the inability of T. evansi to multiply in tsetse flies, since this requires functional mitochondria containing maxi-circle gene products. Judged by restriction enzyme analysis, five of the six T. evansi strains contain mini-circles that differ less than 4% in sequence. This type A mini-circle is found in strains from East Africa, West Africa and South America. Another strain from East Africa contains a very different mini-circle (type B), which shows about the same degree of hybridization to type A mini-circles as to a mini-circle from T. brucei. We propose that the pronounced sequence heterogeneity of the mini-circles of T. brucei has arisen by recombination of strains that had diverged for long periods of time in reproductive isolation. We further propose that the homogeneous mini-circles of T. evansi (and T. equiperdum) reflect the inability of species to mate. This proposal implies that mini-circle heterogeneity indicates (infrequent) genetic exchange and that all kinetoplastid flagellates with heterogeneous mini-circles exchange DNA.     

Kinetoplast DNA analysis of four Trypanosoma evansi strains.

Kinetoplast DNA (kDNA), the mitochondrial DNA of trypanosomes, is a network of thousands of topologically interlocked DNA minicircles and about 50 maxicircles. In this study, we have analysed the kDNA molecules of 6 strains of Trypanosoma evansi from different geographical areas. 2 strains were found to be dyskinetoplastic mutants and other 4 kinetoplastic strains absent of maxicircles. The electrophoretic analysis of the minicircles digested with various restriction endonucleases clearly shows that all of the kinetoplastic strains lack profound minicircle heterogeneity typical of T. brucei. However, a slight restriction fragment length polymorphism could be observed with 2 enzymes (Dde I and HinfI) within the minicircle population of each cloned strain. We propose that this sequence diversity is the result of point mutations. Further analysis of the minicircles by nucleotide sequencing revealed that the 4 minicircles of T. evansi strains share extensive regions of homology with each other but only about 50% homology with other species. This homogeneity of T. evansi minicircle sequences may provide a useful tool for classification and identification.     

Evidence for kinetoplast and nuclear DNA homogeneity in Trypanosoma evansi isolates

The kinetoplast DNA minicircles from 13 stocks of trypanosomes designated as Trypanosoma evansi were digested with various restriction enzymes. We also examined the distribution of restriction site polymorphisms in the nuclear DNA of 9 of these stocks, using 7 different variable surface glycoprotein (VSG) and non-VSG probes. Restricted kinetoplast DNA (kDNA) fragments of some of these strains were cloned into M13 or PUC 18 vectors and sequenced. The restriction and sequence mapping showed that most of T. evansi isolates belonged to the A1 and A2 types of Borst and to two new closely related types A3 and A4. A notable exception was RoTat 4/1 derived from a Sudanese stock which was found to display a characteristic brucei-like minicircle heterogeneity. The T. evansi minicircles analysed are not only homogeneous in sequence but also the region similar to the conserved region in Trypanosoma brucei and Trypanosoma equiperdum is flanked on its 5' end by a palindromic repeat of part of the conserved region. The highly conserved sequence GGGCGGT which appears to correspond to the initiation of synthesis of one of the Okazaki fragments contains an additional G and is located as in T. brucei and T. equiperdum about 73 bp 5' from the ORI. The nuclear DNA analysis confirms the kDNA study in that all the T. evansi stocks are members of a very homogeneous group in terms of sequence divergence. Moreover, our analysis also confirms that T. evansi is more closely related to the West African T. b. brucei and T. b. gambiense than to other African trypanosomes.