Zhu Huiyin, Zhu Daiqian, Li Yuting, Li Yun, Song Xiaonan, Mo Jinyu, Liu Long, Liu Zhixin, Wang Siqi, Yao Yi, Yan He, Wu Kai, Wang Wei, Yin Jianhai, Lin Min, Li Jian
National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention (Chinese Center for Tropical Diseases Research), NHC Key Laboratory of Parasite and Vector Biology, WHO Collaborating Centre for Tropical Diseases, National Center for International Research on Tropical Diseases, Ministry of Science and Technology, Shanghai, China; School of Basic Medical Sciences, Hubei University of Medicine, Shiyan, China; Department of Pediatrics, Taihe Hospital, Hubei University of Medicine, Shiyan, China.
School of Basic Medical Sciences, Hubei University of Medicine, Shiyan, China.
Int J Parasitol Drugs Drug Resist. 2024 Dec;26:100568. doi: 10.1016/j.ijpddr.2024.100568. Epub 2024 Oct 28.
Malaria remains a major public health concern. The rapid spread of resistance to antimalarial drugs is a major challenge for malaria eradication. Timely and accurate molecular monitoring based on practical detection methods is a critical step toward malaria control and elimination. In this study, two rapid detection techniques, allele-specific PCR (AS‒PCR) and recombinase-aided amplification (RAA) combined with CRISPR/Cas12a, were established, optimized and assessed to detect single nucleotide polymorphisms in the Plasmodium falciparum exonuclease (Pfexo) gene related to suspected piperaquine resistance. Moreover, phosphorothioate and artificial mismatches were introduced into the allele-specific primers for AS‒PCR, and crRNA-mismatched bases were introduced into the RAA‒CRISPR/Cas12a assay because crRNAs designed according to conventional rules fail to discriminate genotypes. As a result, the detection limits of the AS‒PCR and RAA‒CRISPR/Cas12a assays were 10 copies/μL and 10 copies/μL, respectively. The detection threshold for dried blood spots was 100‒150 parasites/μL, with no cross-reactivity against other genotypes. The average cost of AS‒PCR is approximately $1 per test and takes 2-3 h, whereas that of the RAA‒CRISPR/Cas12a system is approximately $7 per test and takes 1 h or less. Therefore, we provide more options for testing single nucleotide polymorphisms in the Pfexo gene, considering economic conditions and the availability of instruments, equipment, and reagents, which can contribute to the molecular monitoring of antimalarial resistance.
疟疾仍然是一个重大的公共卫生问题。抗疟药物耐药性的迅速传播是根除疟疾的一项重大挑战。基于实用检测方法的及时、准确的分子监测是疟疾控制和消除的关键一步。在本研究中，建立、优化并评估了两种快速检测技术，即等位基因特异性PCR（AS-PCR）和重组酶辅助扩增（RAA）结合CRISPR/Cas12a，以检测恶性疟原虫核酸外切酶（Pfexo）基因中与疑似哌喹耐药相关的单核苷酸多态性。此外，在AS-PCR的等位基因特异性引物中引入了硫代磷酸酯和人工错配，并在RAA-CRISPR/Cas12a检测中引入了crRNA错配碱基，因为按照常规规则设计的crRNA无法区分基因型。结果，AS-PCR和RAA-CRISPR/Cas12a检测的检测限分别为10拷贝/μL和10拷贝/μL。干血斑的检测阈值为100-150个寄生虫/μL，对其他基因型无交叉反应。AS-PCR的平均成本约为每次检测1美元，耗时2-3小时，而RAA-CRISPR/Cas12a系统的平均成本约为每次检测7美元，耗时1小时或更短。因此，考虑到经济条件以及仪器、设备和试剂的可用性，我们为检测Pfexo基因中的单核苷酸多态性提供了更多选择，这有助于抗疟耐药性的分子监测。
