Department of Medical Technology, Faculty of Allied Health Sciences, Naresuan University, Phitsanulok, Thailand.
Am J Trop Med Hyg. 2023 Sep 11;109(4):770-777. doi: 10.4269/ajtmh.23-0130. Print 2023 Oct 4.
There are many techniques for malaria diagnosis. Currently, the nested polymerase chain reaction (PCR) method based on a small subunit ribosomal RNA gene (18S rRNA) has been used as a confirmatory method. However, this method is time-consuming, laborious, and costly. Therefore, the objective of this study was to develop nested multiplex PCR for Plasmodium species identification using the dihydropterin pyrophosphokinase-dihydropteroate synthase (hppk-dhps) gene. Genus- and species-specific primers for the hppk-dhps gene were designed. The performance of the novel nested multiplex PCR was compared with 18S rRNA nested PCR. A total of 115 blood samples were used in this study, including 84 infected samples and 31 uninfected samples. Analysis of the blood samples by nested multiplex PCR targeting the hppk-dhps gene identified 81 infected cases. The level of agreement between this novel method and 18S rRNA nested PCR was 97.4%. Further, the novel method successfully detected all human malaria parasites except Plasmodium ovale and detected mixed Plasmodium falciparum/Plasmodium vivax infections. The sensitivity and specificity obtained from this novel method were 96.4% and 100%, respectively. The limit of detection of the hppk-dhps nested multiplex PCR for P. falciparum and P. vivax was 500 parasites/µL and 4 parasites/µL, respectively. The lowest parasite gDNA detected by this method was 0.5 ng/µL for P. falciparum and 0.1 ng/µL for P. vivax. These results corroborate that the hppk-dhps gene is a novel amplification target for the detection of human malaria. This novel target PCR-based method is a beneficial approach for malaria diagnosis, as well as species identification and differentiation.
疟疾的诊断方法有很多。目前，基于小亚基核糖体 RNA 基因（18S rRNA）的巢式聚合酶链反应（PCR）方法已被用作确认方法。然而，这种方法既费时、费力又昂贵。因此，本研究的目的是开发一种基于二氢喋啶磷酸核糖基转移酶-二氢叶酸合成酶（hppk-dhps）基因的疟原虫种属鉴定巢式多重 PCR 方法。设计了用于 hppk-dhps 基因的种属和种特异性引物。比较了新型巢式多重 PCR 与 18S rRNA 巢式 PCR 的性能。本研究共使用了 115 份血样，包括 84 份感染样本和 31 份未感染样本。用针对 hppk-dhps 基因的巢式多重 PCR 分析血样，鉴定出 81 例感染病例。该新方法与 18S rRNA 巢式 PCR 的一致性为 97.4%。此外，该新方法成功检测到除卵形疟原虫和间日疟原虫/恶性疟原虫混合感染外的所有人类疟原虫。该新方法获得的灵敏度和特异性分别为 96.4%和 100%。hppk-dhps 巢式多重 PCR 检测恶性疟原虫和间日疟原虫的检测限分别为 500 个/µL 和 4 个/µL。该方法检测到的最低疟原虫 gDNA 量为恶性疟原虫 0.5 ng/µL，间日疟原虫 0.1 ng/µL。这些结果证实 hppk-dhps 基因是一种新型的人类疟疾检测扩增靶标。这种基于新型靶标 PCR 的方法对疟疾诊断以及种属鉴定和区分具有重要意义。
