检测滤纸干血滴中间日疟原虫(英文)

从滤纸干血滴上用Chelex处理洗脱下的疟原虫DNA,经套式PCR扩增间日疟原虫SSUrRNA基因特异性121bp片段,分析该方法的敏感性和特异性。37例血样检测结果全部阳性,当原虫密度低至25个原虫/uL血时仍可成功检测到该特异条带,且其它三种人疟原虫(恶性疟原虫,三日疟原虫和卵形疟原虫)血样均为阴性。提示滤纸干血滴与PCR扩增技术相结合,是疟疾诊断或流行病学调查的实用工具。     

间日疟原虫SSUrDNA特定片段的体外扩增及鉴定

根据已知序列，设计出一对含克隆位点的寡聚核苷酸引物，用于间日疟原虫ＳＳＵｒＤＮＡ特定片段扩增；采用双温度点ＰＣＲ法，从我国间日疟患者血样ＤＮＡ抽提物中，直接扩增出预期大小片段（约６４０ｂｐ），而培养红内期恶性疟原虫株、人源利什曼原虫株，人源弓形虫株及正常人ＤＮＡ样本均无此扩增带出现。扩增片段进一步经限制性酶切和Ｎｏｒｔｈｅｒｎ印迹杂交分析，证实确为间日疟原虫ＳＳＵｒＤＮＡ目的片段。     

PCR与单克隆抗体ELISA夹心法快速诊断结核病的研究

应用聚合酶链反应（ＰＣＲ）技术建立的扩增结核杆菌复合体特异重复序列ＩＳ９８６基因的方法和用单克隆抗体ＴＢ１５－Ｃ３经ＥＬＩＳＡ夹心法检测结核杆菌特异性抗原决定簇，对１０种抗酸杆菌，２种普通菌进行了检测。ＰＣＲ仅对结核杆菌复合体扩增出２４５ｂｐ特异性条带，ＥＬＩＳＡ检测除人型结核杆菌、ＢＣＧ阳性外，还与鸟型及瘰疬分枝杆菌反应阳性。ＰＣＲ检测人型结核杆菌的敏感性为１ｐｇ，相当于１３个左右细菌，ＥＬＩＳ     

急性白血病患儿脑脊液PCR扩增HCMV－DNA的研究

应用多聚酶链式反应（ＰＣＲ）扩增和洋地黄探针杂交的方法，对５３例急性白血病（ＡＬ）患儿脑脊液（ＣＳＦ）样本进行了检测。经过ＰＣＲ扩增，可见明显的人巨细胞病毒ＤＮＡ（ＨＣＭＶ－ＤＮＡ）４００ｂｐ扩增带；经洋地黄探针特异性杂交鉴定，ＡＬ患儿ＣＳＦ标本ＰＣＲ扩增产物的ＨＣＭＶ－ＤＮＡ阳性检出率为８３．０２％。本实验为ＡＬ病人ＣＳＦ的ＨＣＭＶ检测提供了一个简便方法。     

THE POTENTIAL USES OF GENOTYPING OF PLASMODIUMFALCIPARUMISOLATESBYTHENESTEDPOLYMERASECHAINREACTION

巢式ＰＣＲ方法被应用于泰国疟区（Ｂｏｒａｉ）恶性疟原虫的基因分型。应用特异的等位基因引物扩增了恶性疟原虫裂殖子表面蛋白１（ＭＳＰ１）基因的１、４变异多态区。以凝胶电泳分析扩增的目的基因片段。结果表明：共分别检测出恶性疟原虫６个ＭＡＤ２０等位基因型、４个Ｋ１和１个ＲＯ３３等位基因型。在９个月的流行季节，上述虫株的等位基因频率没有明显的改变，关存在较高程度的恶性疟原虫不同等位基因株的混合感染。本文进一步阐明了巢式ＰＣＲ技术在疟疾流行病学上的应用前景及优点。     

急性白血病患儿脑脊液PCR扩增HCMV—DNA的研究

应用多聚酶链式反应（ＰＣＲ）扩增和洋地黄深针杂交的方法，对５３例急性白血病（ＡＬ）患儿脑脊液（ＣＳＦ）样本进行了检测，经过ＰＣＲ扩增，可见明显的人巨细胞病毒ＤＮＡ（ＨＣＭＶ－ＤＮＡ）４００ｂｐ扩增带，经洋地黄探针特异性杂交鉴定，ＡＬ患儿ＣＳＦ标本ＰＣＲ扩增产生的ＨＣＭＶ－ＤＮＡ阳性检出率为８３．０２％。本实验为ＡＬ病人ＣＳＦ的ＨＣＭＶ检测提供了一个简便方法。     

PCR与单克隆抗体ELESA夹心法快速诊断结核病的研究

应用聚合酶性反应（ＰＣＲ）技术建立的扩增结核杆菌复合体特异重复序列ＩＳ９８６基因的方法和用单克隆抗体ＴＢ１５－Ｃ３经ＥＬＩＳＡ夹心法检测结核杆菌特异性抗原决定簇，对１０种抗酸杆菌，２种普通菌进行了检测．ＰＣＲ仅对结核杆菌复合体扩增出２４５ｈｐ特异性条带，ＥＬＩＳＡ检测除人型结核杆菌、ＢＣＧ阳性外，还与鸟型及瘰疬分枝杆菌反应阳性．ＰＣＲ检测人型结核杆菌的敏感性为１ｐｇ，相当于１３个左右细菌，ＥＬＩＳＡ检测的被感性为１５ｎｇ／ｍｌ．应用ＰＣＲ及ＥＬＩＳＡ检测了９６份结核临床标本．ＰＣＲ的检出率高于抗酸染色涂片的阳性率（Ｐ＜０．０５），ＰＣＲ的检出率虽高于细菌培养的阳性率，但相差不显著（Ｐ＞０．０５）．ＥＬＩＳＡ检测阳性率明显高于抗酸染色涂片、细菌培养和ＰＣＲ的阳性率（Ｐ＜０．００１）．ＥＬＩＳＡ的假阳性率高于ＰＣＲ的假阳性率，但相差不显著（Ｐ＞０．０５）．研究表明，ＰＣＲ及ＥＬＩＳＡ均是特异、敏感、快速的诊断结核病的方法．但在使用中又各自有其特点．     

蚊体内间日疟原虫SSUrDNA特定片段的克隆与鉴定

利用套式ＰＣＲ技术体外扩增蚊体内间日疟原虫ＳＳＵｒＤＮＡ长１２１ｂｐ的特定片段，将此片段克隆到载体ＰＵＣ１８中，转化受体菌株ＤＨ５α，测序结果表明，扩增ＤＮＡ序列与背景序列完全一致，证实为间日疟原虫ＳＳＵｒＤＮＡ目的片段。     

PCR－ELISA在检测血清中丙型肝炎病毒上的应用

ＰＣＲ－ＥＬＩＳＡ在检测血清中丙型肝炎病毒上的应用中国科学院上海生物工程研究中心建立了一个替代目前使用的套式ＰＣＲ的血清中丙型肝炎病毒ＲＮＡ检测的新方法。该方法只需经过一次ＰＣＲ扩增，即可通过对掺入标记物的ＰＣＲ产物的ＥＬＩＳＡ检测得到与套式ＰＣＲ相...     

恶性疟原虫疫苗的研究──Ⅰ．环子孢子蛋白基因中央保守区的体外扩增

恶性疟原虫环子孢子蛋白基因中央保守区编码产物具有３７个ＮＡＮＰ串联重复序列，并认为（ＮＡＮＰ）ｎ具有保护性免疫作用。本文通过酚／氯仿抽提乙醇沉淀方法提取恶性疟原虫全基因组ＤＮＡ，首次采用ＰＣＲ方法获得环子孢子蛋白ＣＳＰ基因中央保守区片段。我们用地高辛标记的ＰＣＲ产物作ＤＮＡ探针，杂交试验表明具有高度的敏感性和特异性。同时，本文还探讨了ＰＣＲ技术在病原学检测、疫苗研制、流行病学调查、疗效考核等方面的潜在应用前景。     

Detection of four Plasmodium species by genus- and species-specific loop-mediated isothermal amplification for clinical diagnosis

Loop-mediated isothermal amplification (LAMP), a novel nucleic acid amplification method, was developed for the clinical detection of four species of human malaria parasites: Plasmodium falciparum, P. vivax, P. malariae, and P. ovale. We evaluated the sensitivity and specificity of LAMP in comparison with the results of microscopic examination and nested PCR. LAMP showed a detection limit (analytical sensitivity) of 10 copies of the target 18S rRNA genes for P. malariae and P. ovale and 100 copies for the genus Plasmodium, P. falciparum, and P. vivax. LAMP detected malaria parasites in 67 of 68 microscopically positive blood samples (sensitivity, 98.5%) and 3 of 53 microscopically negative samples (specificity, 94.3%), in good agreement with the results of nested PCR. The LAMP reactions yielded results within about 26 min, on average, for detection of the genus Plasmodium, 32 min for P. falciparum, 31 min for P. vivax, 35 min for P. malariae, and 36 min for P. ovale. Accordingly, in comparison to the results obtained by microscopy, LAMP had a similar sensitivity and a greater specificity and LAMP yielded results similar to those of nested PCR in a shorter turnaround time. Because it can be performed with a simple technology, i.e., with heat-treated blood as the template, reaction in a water bath, and inspection of the results by the naked eye because of the use of a fluorescent dye, LAMP may provide a simple and reliable test for routine screening for malaria parasites in both clinical laboratories and malaria clinics in areas where malaria is endemic.     

Identification of four species of human malaria parasites by fast PCR

Malaria is one of the most prevalent infectious diseases in the world. Accurate identification of four species of human malaria parasite is essential for appropriate treatment. Here, we developed a simple and rapid method of identifying Plasmodium species using a fast polymerase chain reaction (PCR) assay. Based on the previous literature, we amplified small subunit ribosomal RNA genes of four human malaria parasites. To establish a minimum detection limit, a blood sample with a known number of P. falciparum parasites (parasitemia: 3%) was diluted serially(from 0.03% to 0.000003%). We compared the detection limits between single (one-step) PCR and nested (two-step) PCR. Other clinical blood samples, which were infected with P. falciparum (parasitemia: 2.8%), P. vivax (parasitemia: 0.13%), P. ovale (parasitemia: 0.04%), respectively, were also tested by our PCR system. The PCR findings were compared to those of blood film Giemsa staining and rapid diagnostic tests (RDT). The sensitivity of our method is less than one parasite in 1 microl of blood(estimated parasitemia: 0.000003%) for both single PCR and nested PCR, though an increased number of cycles (40 cycles) was required for single PCR. Using clinical samples, it was proven that amplified products by single PCR could clearly distinguish between P. falciparum, P. vivax, and P. ovale. To detect P. vivax and P. ovale, the PCR system was more sensitive than RDT. The total required time for our method was within three to four hours from DNA extraction to PCR detection. Taken together, our method is easier and faster than the previously reported PCR-based malaria parasite identification systems, and is also useful for cases in which diagnosis by Giemsa staining and RDT is difficult.     

Malaria diagnosis: standardization of a polymerase chain reaction for the detection of Plasmodium falciparum parasites in individuals with low-grade parasitemia

 In Brazil, no study has been done concerning the detection of malaria parasites by polymerase chain reaction (PCR) related to the diagnosis of Plasmodium falciparum malaria. In the present report we describe a highly sensitive methodology for malaria diagnosis using a nested PCR method based on amplification of the p126 P. falciparum gene detected by simple ethidium bromide staining. The P. falciparum Palo Alto strain (culture samples) was serially diluted in blood from an uninfected donor to a final level of parasitemia corresponding to 10^–8% and was processed for PCR amplification. In each of these dilutions a parasitological examination was performed to compare the sensitivity with that of PCR amplification. Blood samples (field samples) were obtained from 51 malarious patients with positive thick blood smears (TBS) who were living in endemic regions of the Brazilian Amazon. They corresponded to 42 P. falciparum and 9 P. vivax cases, with parasitemia levels ranging from only 16 to 20,200 parasites/μl for P. falciparum disease and from 114 to 11,000 parasites/μl for P. vivax malaria. We demonstrate that the use of nested PCR allows the detection of 0.005 parasites/μl without the use of radioactive material. The use of a 1-ml sample volume and the organic DNA extraction method should be suitable in blood banks and for the evaluation of patients during and after drug treatment. Received: 10 January 1996 / Accepted: 10 April 1996     

Real-time PCR for detection and identification of Plasmodium spp

Rapid and accurate detection of malaria parasites in blood is needed to institute proper therapy. We developed and used a real-time PCR assay to detect and distinguish four Plasmodium spp. that cause human disease by using a single amplification reaction and melting curve analysis. Consensus primers were used to amplify a species-specific region of the multicopy 18S rRNA gene, and SYBR Green was used for detection in a LightCycler instrument. Patient specimens infected at 0.01 to 0.02% parasitemia densities were detected, and analytical sensitivity was estimated to be 0.2 genome equivalent per reaction. Melting curve analysis based on nucleotide variations within the amplicons provided a basis for accurate differentiation of Plasmodium falciparum, P. vivax, P. ovale, and P. malariae. For assay validation, 358 patient blood samples from the National University Hospital in Singapore and Evanston Northwestern Healthcare in Illinois were analyzed. Of 76 blinded patient samples with a microscopic diagnosis of P. falciparum, P. vivax, or P. ovale infection, 74 (97.4%) were detected by real-time PCR, including three specimens containing mixed P. falciparum-P. vivax infections. No Plasmodium DNA was amplified in any of the 82 specimens sent for malaria testing but that were microscopically negative for Plasmodium infection. In addition, 200 blood samples from patients whose blood was collected for reasons other than malaria testing were also determined to be negative by real-time PCR. Real-time PCR with melting curve analysis could be a rapid and objective supplement to the examination of Giemsa-stained blood smears and may replace microscopy following further validation.     

Molecular test for vivax malaria with loop-mediated isothermal amplification method in central China

Vivax malaria has the highest incidence in central China. High-throughput and cost-effective testing methods are essential for vivax malaria diagnosis in this area. Loop-mediated isothermal amplification (LAMP) is an established nucleic acid amplification method, which provides a promising platform for the molecular detection of malaria parasites in development countries. This study was performed to compare the LAMP method, the nested PCR-based method, and microscopic examinations in diagnosing vivax malaria. LAMP detected vivax malaria parasites in 160 of 164 microscopically positive samples (sensitivity, 97.6%), whereas nested PCR detected Plasmodium vivax in 162 of 164 samples (sensitivity, 98.8%). No false-positive results were obtained by LAMP or nested PCR among fever-positive and healthy samples. The sensitivities and specificities of the two molecular tests were consistently high. In addition, the reproducibility of the LAMP assays using water bath, which reduced the cost substantially. LAMP method is an accurate, rapid, simple, and cost-effective method that may be useful for diagnosis in field diagnoses instead of nested PCR. This method is feasible to diagnose vivax malaria parasite in endemic areas of central China.     

Detection and species determination of malaria parasites by PCR: comparison with microscopy and with ParaSight-F and ICT malaria Pf tests in a clinical environment

A rapid procedure for the diagnosis of malaria infections directly from dried blood spots by PCR amplification was evaluated with samples from 52 patients. Plasmodium infections were identified with a genus-specific primer set, and species differentiation between Plasmodium falciparum and Plasmodium vivax was analyzed by multiplex PCR. The PCR test with any of the three primer sets was able to detect as few as four parasites per microliter by gel electrophoresis or by nonisotopic paper hybridization chromatography. The diagnoses obtained by PCR correlated closely with those obtained by Giemsa staining except for two samples observed to have mixed P. falciparum-P. vivax infections. These were initially missed by microscopic analysis. In comparison with antigen-capture assays for P. falciparum, the PCR assays were able to detect three infections that were missed by the ParaSight-F test. The PCR test was negative for nine ParaSight-F-positive samples and one ICT Malaria Pf-positive sample, and these were confirmed to be false-positive results. The PCR thus gave no false-negative or false-positive results. Patients undergoing antimalarial therapy were also monitored by the PCR assay. Four of seven patients who were PCR positive for P. vivax at the time of discharge were later readmitted to the hospital with a recurrence of P. vivax infection. We would like to propose that PCR is a sensitive and easy method that can serve as a useful addition to microscopy for the diagnosis and the clinical monitoring of treatment of malaria.     

Applied genomics: data mining reveals species-specific malaria diagnostic targets more sensitive than 18S rRNA

Accurate and rapid diagnosis of malaria infections is crucial for implementing species-appropriate treatment and saving lives. Molecular diagnostic tools are the most accurate and sensitive method of detecting Plasmodium, differentiating between Plasmodium species, and detecting subclinical infections. Despite available whole-genome sequence data for Plasmodium falciparum and P. vivax, the majority of PCR-based methods still rely on the 18S rRNA gene targets. Historically, this gene has served as the best target for diagnostic assays. However, it is limited in its ability to detect mixed infections in multiplex assay platforms without the use of nested PCR. New diagnostic targets are needed. Ideal targets will be species specific, highly sensitive, and amenable to both single-step and multiplex PCRs. We have mined the genomes of P. falciparum and P. vivax to identify species-specific, repetitive sequences that serve as new PCR targets for the detection of malaria. We show that these targets (Pvr47 and Pfr364) exist in 14 to 41 copies and are more sensitive than 18S rRNA when utilized in a single-step PCR. Parasites are routinely detected at levels of 1 to 10 parasites/μl. The reaction can be multiplexed to detect both species in a single reaction. We have examined 7 P. falciparum strains and 91 P. falciparum clinical isolates from Tanzania and 10 P. vivax strains and 96 P. vivax clinical isolates from Venezuela, and we have verified a sensitivity and specificity of ～100% for both targets compared with a nested 18S rRNA approach. We show that bioinformatics approaches can be successfully applied to identify novel diagnostic targets and improve molecular methods for pathogen detection. These novel targets provide a powerful alternative molecular diagnostic method for the detection of P. falciparum and P. vivax in conventional or multiplex PCR platforms.     

Novel nested direct PCR technique for malaria diagnosis using filter paper samples

The use of direct nested PCR enables the detection of Plasmodium spp. from blood samples collected on filter papers without requiring the time-consuming procedures associated with DNA extraction. Direct PCR provides a rapid, highly sensitive, and cost-effective alternative to diagnosing malaria using filter paper samples and standard nested PCR.