采用多重巢式 RT-PCR 同步检测临床分离株中的间日疟原虫和恶性疟原虫配子体。
Simultaneous detection of Plasmodium vivax and Plasmodium falciparum gametocytes in clinical isolates by multiplex-nested RT-PCR.
机构信息
Molecular Biology of Malaria and Opportunistic Parasites Research Unit, Department of Parasitology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand.
出版信息
Malar J. 2012 Jun 10;11:190. doi: 10.1186/1475-2875-11-190.
BACKGROUND
Gametocyte carriage is essential for malaria transmission and endemicity of disease; thereby it is a target for malaria control strategies. Malaria-infected individuals may harbour gametocytes below the microscopic detection threshold that can be detected by reverse transcription polymerase chain reaction (RT-PCR) targeting gametocyte-specific mRNA. To date, RT-PCR has mainly been applied to the diagnosis of Plasmodium falciparum gametocytes but very limited for that of Plasmodium vivax.
METHODS
A multiplex-nested RT-PCR targeting Pfs25 and Pvs25 mRNA specific to mature gametocytes of P. falciparum and P. vivax, respectively, was developed. The assay was evaluated using blood samples collected in rainy and dry seasons from febrile patients,in a malaria-endemic area in Thailand. Malaria diagnosis was performed by Giemsa-stained blood smears and 18S rRNA PCR.
RESULTS
The multiplex-nested RT-PCR detected Pfs25 mRNA in 75 of 86 (87.2%) P. falciparum-infected individuals and Pvs25 mRNA in 82 of 90 (91.1%) P. vivax malaria patients diagnosed by 18S rRNA PCR. Gametocytes were detected in 38 (eight P. falciparum and 30 P. vivax) of 157 microscopy positive samples, implying that a large number of patients harbour sub-microscopic gametocytaemia. No seasonal differences in gametocyte carriage were observed for both malaria species diagnosed by multiplex-nested RT-PCR. With single-nested RT-PCR targeting Pfs25 or Pvs25 mRNA as standard, the multiplex-nested RT-PCR offered sensitivities of 97.4% and 98.9% and specificities of 100% and 98.8% for diagnosing mature gametocytes of P. falciparum and P. vivax, respectively. The minimum detection limit of the multiplex-nested PCR was 10 copies of templates.
CONCLUSIONS
The multiplex-nested RT-PCR developed herein is useful for simultaneous assessment of both P. falciparum and P. vivax gametocyte carriage that is prevalent and generally sympatric in several malaria-endemic areas outside Africa.
背景
配子体携带对于疟疾传播和疾病流行至关重要；因此，它是疟疾控制策略的目标。受感染的个体可能携带有低于显微镜检测阈值的配子体，但可以通过针对配子体特异性 mRNA 的逆转录聚合酶链反应 (RT-PCR) 检测到。迄今为止，RT-PCR 主要应用于恶性疟原虫配子体的诊断，但很少用于间日疟原虫配子体的诊断。
方法
本研究开发了一种针对恶性疟原虫和间日疟原虫成熟配子体的 Pfs25 和 Pvs25 mRNA 的多重巢式 RT-PCR。该检测方法使用来自泰国疟疾流行地区发热患者在雨季和旱季采集的血液样本进行评估。疟疾诊断采用吉姆萨染色血涂片和 18S rRNA PCR 进行。
结果
多重巢式 RT-PCR 检测到 86 例恶性疟原虫感染患者中的 75 例（87.2%）存在 Pfs25 mRNA，90 例间日疟原虫感染患者中的 82 例（91.1%）存在 Pvs25 mRNA，这些患者均通过 18S rRNA PCR 诊断。在 38 例显微镜阳性样本（8 例恶性疟原虫和 30 例间日疟原虫）中检测到配子体，这表明大量患者存在亚微观配子体血症。通过多重巢式 RT-PCR 诊断的两种疟疾在雨季和旱季均未观察到配子体携带量的差异。以针对 Pfs25 或 Pvs25 mRNA 的单重巢式 RT-PCR 为标准，该多重巢式 RT-PCR 检测恶性疟原虫和间日疟原虫成熟配子体的灵敏度分别为 97.4%和 98.9%，特异性均为 100%。该多重巢式 PCR 的最低检测限为 10 个模板拷贝。
结论
本研究开发的多重巢式 RT-PCR 可用于同时评估非洲以外几个疟疾流行地区普遍共存的恶性疟原虫和间日疟原虫配子体携带情况。
Zotero 插件