Almeida-de-Oliveira Natália Ketrin, Moreira Otacílio C, de Lavigne Aline Rosa, Mendonça-Lima Leila, Werneck Guilherme Loureiro, Daniel-Ribeiro Cláudio Tadeu, Ferreira-da-Cruz Maria de Fátima
Fundação Oswaldo Cruz-Fiocruz, Instituto Oswaldo Cruz, Laboratório de Pesquisa em Malária, Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz-Fiocruz, Instituto Oswaldo Cruz, Laboratório de Biologia Molecular e Doenças Endêmicas, Rio de Janeiro, RJ, Brasil.
Mem Inst Oswaldo Cruz. 2019 Jan 31;114:e180350. doi: 10.1590/0074-02760180350.
The prompt diagnosis of plasmodial species for effective treatment prevents worsening of individual health and avoids transmission maintenance or even malaria reintroduction in areas where Plasmodium does not exist. Polymerase chain reaction (PCR) allows for the detection of parasites below the threshold of microscopic examination.
Our aim was to develop a real-time PCR test to reduce diagnostic errors and increase efficacy.
The lower limit of quantification and the linearity/analytical sensitivity to measure sensitivity or limit of detection (LoD) were determined. Intra-assay variations (repeatability) and alterations between assays, operators, and instruments (reproducibility) were also assessed to set precision.
The linearity in SYBR™ Green and TaqMan™ systems was 106 and 102 copies and analytical sensitivity 1.13 and 1.17 copies/μL, respectively. Real-time PCR was more sensitive than conventional PCR, showing a LoD of 0.01 parasite (p)/μL. Reproducibility and repeatability (precision) were 100% for up to 0.1 p/μL in SYBR™ Green and 1 p/μL in TaqMan™ and conventional PCR.
Real-time PCR may replace conventional PCR in reference laboratories for P. vivax detection due to its rapidity. The TaqMan™ system is the most indicated when quantification assays are required. Performing tests in triplicate when diagnosing Plasmodium-infected-asymptomatic individuals is recommended to minimise diagnostic errors.
对疟原虫种类进行快速诊断以实现有效治疗，可防止个体健康状况恶化，并避免在不存在疟原虫的地区维持传播或甚至重新引入疟疾。聚合酶链反应（PCR）能够检测低于显微镜检查阈值的寄生虫。
我们的目标是开发一种实时PCR检测方法，以减少诊断错误并提高效率。
确定定量下限以及用于测量灵敏度或检测限（LoD）的线性/分析灵敏度。还评估了实验内变异（重复性）以及不同实验、操作人员和仪器之间的变异（再现性），以确定精密度。
SYBR™ Green和TaqMan™系统的线性分别为106和102拷贝，分析灵敏度分别为1.13和1.17拷贝/μL。实时PCR比传统PCR更灵敏，检测限为0.01个寄生虫（p）/μL。对于SYBR™ Green中高达0.1 p/μL以及TaqMan™和传统PCR中高达1 p/μL的情况，再现性和重复性（精密度）均为100%。
由于实时PCR速度快，在参考实验室中检测间日疟原虫时，它可能会取代传统PCR。当需要定量检测时，TaqMan™系统是最适用的。建议在诊断疟原虫感染的无症状个体时进行一式三份检测，以尽量减少诊断错误。
