一种用于检测和定量疟原虫 knowlesi 的 TaqMan 实时 PCR 检测方法。
A TaqMan real-time PCR assay for the detection and quantitation of Plasmodium knowlesi.
机构信息
Malaria Research Centre, University Malaysia Sarawak, Sarawak, Malaysia.
出版信息
Malar J. 2010 Nov 30;9:344. doi: 10.1186/1475-2875-9-344.
BACKGROUND
The misdiagnosis of Plasmodium knowlesi by microscopy has prompted a re-evaluation of the geographic distribution, prevalence and pathogenesis of this species using molecular diagnostic tools. In this report, a specific probe for P. knowlesi, that can be used in a previously described TaqMan real-time PCR assay for detection of Plasmodium spp., and Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale, was designed and validated against clinical samples.
METHODS
A hydrolysis probe for a real-time PCR assay was designed to recognize a specific DNA sequence within the P. knowlesi small subunit ribosomal RNA gene. The sensitivity, linearity and specificity of the assay were determined using plasmids containing P. knowlesi DNA and genomic DNA of P. falciparum, P. knowlesi, P. malariae, P. ovale and P. vivax isolated from clinical samples. DNA samples of the simian malaria parasites Plasmodium cynomolgi and Plasmodium inui that can infect humans under experimental conditions were also examined together with human DNA samples.
RESULTS
Analytical sensitivity of the P. knowlesi-specific assay was 10 copies/μL and quantitation was linear over a range of 10-106 copies. The sensitivity of the assay is equivalent to nested PCR and P. knowlesi DNA was detected from all 40 clinical P. knowlesi specimens, including one from a patient with a parasitaemia of three parasites/μL of blood. No cross-reactivity was observed with 67 Plasmodium DNA samples (31 P. falciparum, 23 P. vivax, six P. ovale, three P. malariae, one P. malariae/P. ovale, one P. falciparum/P. malariae, one P. inui and one P. cynomolgi) and four samples of human DNA.
CONCLUSIONS
This test demonstrated excellent sensitivity and specificity, and adds P. knowlesi to the repertoire of Plasmodium targets for the clinical diagnosis of malaria by real-time PCR assays. Furthermore, quantitation of DNA copy number provides a useful advantage over other molecular assays to investigate the correlation between levels of infection and the spectrum of disease.
背景
由于显微镜对疟原虫 knowlesi 的误诊，促使我们重新评估该物种的地理分布、流行率和发病机制，采用分子诊断工具。在本报告中，我们设计并验证了一种针对疟原虫 knowlesi 的特异性探针，该探针可用于之前描述的用于检测疟原虫 spp.、疟原虫 falciparum、疟原虫 vivax、疟原虫 malariae 和疟原虫 ovale 的 TaqMan 实时 PCR 检测。
方法
针对疟原虫 knowlesi 小亚基核糖体 RNA 基因设计了一种水解探针，用于实时 PCR 检测。使用包含疟原虫 knowlesi DNA 和从临床样本中分离的疟原虫 falciparum、疟原虫 knowlesi、疟原虫 malariae、疟原虫 ovale 和疟原虫 vivax 基因组 DNA 的质粒，确定检测的灵敏度、线性度和特异性。还检测了可在实验条件下感染人类的灵长类疟原虫疟原虫 cynomolgi 和疟原虫 inui 的 DNA 样本，以及人类 DNA 样本。
结果
疟原虫 knowlesi 特异性检测的分析灵敏度为 10 拷贝/μL，定量范围为 10-106 拷贝时呈线性。该检测的灵敏度与巢式 PCR 相当，可从 40 例临床疟原虫 knowlesi 标本中检测到疟原虫 knowlesi DNA，包括一例血样中寄生虫数为三个/μL 的患者。与 67 例疟原虫 DNA 样本（31 例疟原虫 falciparum、23 例疟原虫 vivax、6 例疟原虫 ovale、3 例疟原虫 malariae、1 例疟原虫 malariae/ovale、1 例疟原虫 falciparum/malariae、1 例疟原虫 inui 和 1 例疟原虫 cynomolgi）和 4 例人类 DNA 样本均无交叉反应。
结论
该检测方法具有良好的灵敏度和特异性，可将疟原虫 knowlesi 添加到实时 PCR 检测中用于疟疾临床诊断的疟原虫靶标组合中。此外，DNA 拷贝数定量比其他分子检测具有更大的优势，可用于研究感染水平与疾病谱之间的相关性。
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