Real-time PCR for detection and identification of Plasmodium spp.
Real-time PCR for detection and identification of Plasmodium spp.
复制标题
DOI：
10.1128/jcm.43.5.2435-2440.2005
复制
发表时间：
2005-05-01
影响因子：
9.4
通讯作者：
Kaul, KL
中科院分区：
文献类型：
--
作者：
Mangold, KA;Manson, RU;Kaul, KL
需要快速准确地检测血液中的疟原虫以制定适当的治疗方法。我们开发并使用实时 PCR 检测方法来检测和区分四种疟原虫属。通过使用单一扩增反应和熔解曲线分析导致人类疾病。使用共有引物来扩增多拷贝 18S rRNA 基因的物种特异性区域，并使用 SYBR Green 在 LightCycler 仪器中进行检测。检测到感染寄生虫血症密度为 0.01% 至 0.02% 的患者标本，分析灵敏度估计为每个反应 0.2 个基因组当量。基于扩增子内核苷酸变化的熔解曲线分析为准确区分恶性疟原虫、间日疟原虫、卵形疟原虫和三日疟原虫提供了基础。为了进行测定验证，对来自新加坡国立大学医院和伊利诺伊州埃文斯顿西北医疗中心的 358 份患者血液样本进行了分析。在 76 份双盲患者样本中，显微镜诊断为恶性疟原虫、间日疟原虫或卵形疟原虫感染，其中 74 份 (97.4%) 通过实时 PCR 检测到，其中包括 3 份含有混合恶性疟原虫-疟原虫的样本。间日感染。在送去疟疾检测的 82 份样本中，没有任何一份疟原虫 DNA 被扩增，但显微镜下疟原虫感染呈阴性。此外，出于疟疾检测以外的原因采集血液的 200 份患者的血液样本也被实时 PCR 确定为阴性。具有熔解曲线分析的实时 PCR 可以作为吉姆萨染色血涂片检查的快速、客观的补充，并且在进一步验证后可以取代显微镜检查。
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 Plasinodium 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.