Rapid detection of mutations in the suspected piperaquine resistance gene E415G-exo in Plasmodium falciparum exonuclease via AS‒PCR and RAA with CRISPR/Cas12a
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, tw...
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Elsevier
2024-12-01
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| Series: | International Journal for Parasitology: Drugs and Drug Resistance |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2211320724000496 |
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| author | Huiyin Zhu Daiqian Zhu Yuting Li Yun Li Xiaonan Song Jinyu Mo Long Liu Zhixin Liu Siqi Wang Yi Yao He Yan Kai Wu Wei Wang Jianhai Yin Min Lin Jian Li |
| author_facet | Huiyin Zhu Daiqian Zhu Yuting Li Yun Li Xiaonan Song Jinyu Mo Long Liu Zhixin Liu Siqi Wang Yi Yao He Yan Kai Wu Wei Wang Jianhai Yin Min Lin Jian Li |
| author_sort | Huiyin Zhu |
| collection | DOAJ |
| description | 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 104 copies/μL and 103 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. |
| format | Article |
| id | doaj-art-9c3b5899fbe04b039e9883e7d9107136 |
| institution | Kabale University |
| issn | 2211-3207 |
| language | English |
| publishDate | 2024-12-01 |
| publisher | Elsevier |
| record_format | Article |
| series | International Journal for Parasitology: Drugs and Drug Resistance |
| spelling | doaj-art-9c3b5899fbe04b039e9883e7d91071362024-12-12T05:21:14ZengElsevierInternational Journal for Parasitology: Drugs and Drug Resistance2211-32072024-12-0126100568Rapid detection of mutations in the suspected piperaquine resistance gene E415G-exo in Plasmodium falciparum exonuclease via AS‒PCR and RAA with CRISPR/Cas12aHuiyin Zhu0Daiqian Zhu1Yuting Li2Yun Li3Xiaonan Song4Jinyu Mo5Long Liu6Zhixin Liu7Siqi Wang8Yi Yao9He Yan10Kai Wu11Wei Wang12Jianhai Yin13Min Lin14Jian Li15National 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, ChinaSchool of Basic Medical Sciences, Hubei University of Medicine, Shiyan, ChinaSchool of Basic Medical Sciences, Hubei University of Medicine, Shiyan, ChinaSchool of Basic Medical Sciences, Hubei University of Medicine, Shiyan, ChinaSchool of Basic Medical Sciences, Hubei University of Medicine, Shiyan, ChinaSchool of Basic Medical Sciences, Hubei University of Medicine, Shiyan, ChinaSchool of Basic Medical Sciences, Hubei University of Medicine, Shiyan, ChinaSchool of Basic Medical Sciences, Hubei University of Medicine, Shiyan, ChinaNational 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, ChinaDepartment of Pediatrics, Taihe Hospital, Hubei University of Medicine, Shiyan, ChinaNational 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, ChinaWuhan Center for Disease Control and Prevention, Wuhan, ChinaKey Laboratory of National Health Commission on Technology for Parasitic Diseases Prevention and Control, Jiangsu Provincial Key Laboratory on Parasites and Vector Control Technology, Jiangsu Institute of Parasitic Diseases, Wuxi, ChinaNational 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; Corresponding author.School of Food Engineering and Biotechnology, Hanshan Normal University, Chaozhou, China; Corresponding author.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; Corresponding author. School of Basic Medical Sciences, Hubei University of Medicine, Shiyan, China.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 104 copies/μL and 103 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.http://www.sciencedirect.com/science/article/pii/S2211320724000496Plasmodium falciparumSingle nucleotide polymorphismPfexo geneAllele-specific PCRRecombinase-aided amplificationCRISPR/Cas12a |
| spellingShingle | Huiyin Zhu Daiqian Zhu Yuting Li Yun Li Xiaonan Song Jinyu Mo Long Liu Zhixin Liu Siqi Wang Yi Yao He Yan Kai Wu Wei Wang Jianhai Yin Min Lin Jian Li Rapid detection of mutations in the suspected piperaquine resistance gene E415G-exo in Plasmodium falciparum exonuclease via AS‒PCR and RAA with CRISPR/Cas12a International Journal for Parasitology: Drugs and Drug Resistance Plasmodium falciparum Single nucleotide polymorphism Pfexo gene Allele-specific PCR Recombinase-aided amplification CRISPR/Cas12a |
| title | Rapid detection of mutations in the suspected piperaquine resistance gene E415G-exo in Plasmodium falciparum exonuclease via AS‒PCR and RAA with CRISPR/Cas12a |
| title_full | Rapid detection of mutations in the suspected piperaquine resistance gene E415G-exo in Plasmodium falciparum exonuclease via AS‒PCR and RAA with CRISPR/Cas12a |
| title_fullStr | Rapid detection of mutations in the suspected piperaquine resistance gene E415G-exo in Plasmodium falciparum exonuclease via AS‒PCR and RAA with CRISPR/Cas12a |
| title_full_unstemmed | Rapid detection of mutations in the suspected piperaquine resistance gene E415G-exo in Plasmodium falciparum exonuclease via AS‒PCR and RAA with CRISPR/Cas12a |
| title_short | Rapid detection of mutations in the suspected piperaquine resistance gene E415G-exo in Plasmodium falciparum exonuclease via AS‒PCR and RAA with CRISPR/Cas12a |
| title_sort | rapid detection of mutations in the suspected piperaquine resistance gene e415g exo in plasmodium falciparum exonuclease via as pcr and raa with crispr cas12a |
| topic | Plasmodium falciparum Single nucleotide polymorphism Pfexo gene Allele-specific PCR Recombinase-aided amplification CRISPR/Cas12a |
| url | http://www.sciencedirect.com/science/article/pii/S2211320724000496 |
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