Rapid and supersensitive allele detection of Plasmodium falciparum chloroquine resistance via a Pyrococcus furiosus argonaute-triggered dual-signal biosensing platform
Abstract Background Malaria remains a serious public health problem worldwide, particularly in Africa. Resistance to antimalarial drugs is an essential issue for malaria control and elimination. Currently, polymerase chain reaction (PCR) combined with Sanger sequencing is regarded as the gold standa...
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2024-11-01
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| Online Access: | https://doi.org/10.1186/s13071-024-06575-0 |
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| author | Liying Chen Wencheng Chen Huagui Wei Wenai Lin Cheng Zhang Hongfei Hu Chunfang Wang Jiangtao Chen Xueyan Liang Daiqian Zhu Junli Wang Zongyun Lin Yuxia Wei Jian Li Min Lin |
| author_facet | Liying Chen Wencheng Chen Huagui Wei Wenai Lin Cheng Zhang Hongfei Hu Chunfang Wang Jiangtao Chen Xueyan Liang Daiqian Zhu Junli Wang Zongyun Lin Yuxia Wei Jian Li Min Lin |
| author_sort | Liying Chen |
| collection | DOAJ |
| description | Abstract Background Malaria remains a serious public health problem worldwide, particularly in Africa. Resistance to antimalarial drugs is an essential issue for malaria control and elimination. Currently, polymerase chain reaction (PCR) combined with Sanger sequencing is regarded as the gold standard for mutation detection. However, this method fails to meet the requirements of point-of-care testing (POCT) because of its time-consuming, expensive instruments and professional dependence. To support this strategy, we developed a novel diagnostic platform that combines recombinase polymerase amplification (RPA) with the Pyrococcus furiosus argonaute (PfAgo) protein and was designed to detect gene mutations related to antimalarial drug resistance. The Pfcrt haplotypes CVMNK and CVIET of chloroquine resistance (CQR) were used as examples and were assessed in this study. Methods By meticulously designing strategies, RPA primers, guide DNAs, and probes were screened, the reaction was optimized, and the resulting parameters were employed to ascertain the genotype of Pfcrt. The recombinant plasmids pUC57/Pfcrt-CVIET and pUC57/Pfcrt-CVMNK were constructed and diluted for sensitivity detection. The pUC57/Pfcrt-CVIET plasmid mixture was added to the pUC57/Pfcrt-CVMNK plasmid mixture in different additions to configure several specific proportions of mixed plasmid mixtures. The RPA-PfAgo platform was used, and the mixed plasmid was detected simultaneously via nest-PCR (nPCR) and Sanger sequencing. The platform was then evaluated on 85 clinical samples and compared with Sanger sequencing. Results The entire process achieves the key mutation Pfcrt-CVMNK/CVIET genotype identification of CQR within 90 min. The platform achieved 1.8 × 104 copies/μL sensitivity and could detect as little as 3% CVIET in mixed plasmids, which is a higher sensitivity than that of Sanger sequencing (5%). Notably, the platform shows 100% concordance with the gold standard method when 85 clinical samples are tested. The sensitivity and specificity were 100% for the 85 clinical samples. Conclusions This study established an RPA-PfAgo platform for genotyping the key mutation Pfcrt-CVMNK/CVIET of CQR. This method can rapidly produce reliable results and avoid the disadvantages of nPCR with sequencing. This approach has the characteristics of a short operation time, low device dependence, and a good match to the POCT strategy, suggesting that the platform can be easily applied locally or on site. Graphical abstract |
| format | Article |
| id | doaj-art-d8438f6b741a48b9bbcee63b2827b4dc |
| institution | Kabale University |
| issn | 1756-3305 |
| language | English |
| publishDate | 2024-11-01 |
| publisher | BMC |
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| series | Parasites & Vectors |
| spelling | doaj-art-d8438f6b741a48b9bbcee63b2827b4dc2024-12-01T12:12:21ZengBMCParasites & Vectors1756-33052024-11-0117111310.1186/s13071-024-06575-0Rapid and supersensitive allele detection of Plasmodium falciparum chloroquine resistance via a Pyrococcus furiosus argonaute-triggered dual-signal biosensing platformLiying Chen0Wencheng Chen1Huagui Wei2Wenai Lin3Cheng Zhang4Hongfei Hu5Chunfang Wang6Jiangtao Chen7Xueyan Liang8Daiqian Zhu9Junli Wang10Zongyun Lin11Yuxia Wei12Jian Li13Min Lin14Department of Reproductive Medicine, Affiliated Hospital of Youjiang Medical University for NationalitiesLaboratory Medical Center, Affiliated Hospital of Youjiang Medical University for NationalitiesDepartment of Reproductive Medicine, Affiliated Hospital of Youjiang Medical University for NationalitiesDepartment of Reproductive Medicine, Affiliated Hospital of Youjiang Medical University for NationalitiesDepartment of Reproductive Medicine, Affiliated Hospital of Youjiang Medical University for NationalitiesLaboratory Medical Center, Affiliated Hospital of Youjiang Medical University for NationalitiesLaboratory Medical Center, Affiliated Hospital of Youjiang Medical University for NationalitiesLaboratory Medical Center, Huizhou Municipal Central HospitalLaboratory Medical Center, Huizhou Municipal Central HospitalSchool of Basic Medicine Science, Hubei University of MedicineDepartment of Reproductive Medicine, Affiliated Hospital of Youjiang Medical University for NationalitiesDepartment of Reproductive Medicine, Affiliated Hospital of Youjiang Medical University for NationalitiesDepartment of Reproductive Medicine, Affiliated Hospital of Youjiang Medical University for NationalitiesSchool of Basic Medicine Science, Hubei University of MedicineDepartment of Reproductive Medicine, Affiliated Hospital of Youjiang Medical University for NationalitiesAbstract Background Malaria remains a serious public health problem worldwide, particularly in Africa. Resistance to antimalarial drugs is an essential issue for malaria control and elimination. Currently, polymerase chain reaction (PCR) combined with Sanger sequencing is regarded as the gold standard for mutation detection. However, this method fails to meet the requirements of point-of-care testing (POCT) because of its time-consuming, expensive instruments and professional dependence. To support this strategy, we developed a novel diagnostic platform that combines recombinase polymerase amplification (RPA) with the Pyrococcus furiosus argonaute (PfAgo) protein and was designed to detect gene mutations related to antimalarial drug resistance. The Pfcrt haplotypes CVMNK and CVIET of chloroquine resistance (CQR) were used as examples and were assessed in this study. Methods By meticulously designing strategies, RPA primers, guide DNAs, and probes were screened, the reaction was optimized, and the resulting parameters were employed to ascertain the genotype of Pfcrt. The recombinant plasmids pUC57/Pfcrt-CVIET and pUC57/Pfcrt-CVMNK were constructed and diluted for sensitivity detection. The pUC57/Pfcrt-CVIET plasmid mixture was added to the pUC57/Pfcrt-CVMNK plasmid mixture in different additions to configure several specific proportions of mixed plasmid mixtures. The RPA-PfAgo platform was used, and the mixed plasmid was detected simultaneously via nest-PCR (nPCR) and Sanger sequencing. The platform was then evaluated on 85 clinical samples and compared with Sanger sequencing. Results The entire process achieves the key mutation Pfcrt-CVMNK/CVIET genotype identification of CQR within 90 min. The platform achieved 1.8 × 104 copies/μL sensitivity and could detect as little as 3% CVIET in mixed plasmids, which is a higher sensitivity than that of Sanger sequencing (5%). Notably, the platform shows 100% concordance with the gold standard method when 85 clinical samples are tested. The sensitivity and specificity were 100% for the 85 clinical samples. Conclusions This study established an RPA-PfAgo platform for genotyping the key mutation Pfcrt-CVMNK/CVIET of CQR. This method can rapidly produce reliable results and avoid the disadvantages of nPCR with sequencing. This approach has the characteristics of a short operation time, low device dependence, and a good match to the POCT strategy, suggesting that the platform can be easily applied locally or on site. Graphical abstracthttps://doi.org/10.1186/s13071-024-06575-0Pyrococcus furiosus argonaute (PfAgo)Recombinase polymerase amplification (RPA)Plasmodium falciparum chloroquine resistance transporter (Pfcrt)GenotypingHaplotype detectionPoint-of-care testing (POCT) |
| spellingShingle | Liying Chen Wencheng Chen Huagui Wei Wenai Lin Cheng Zhang Hongfei Hu Chunfang Wang Jiangtao Chen Xueyan Liang Daiqian Zhu Junli Wang Zongyun Lin Yuxia Wei Jian Li Min Lin Rapid and supersensitive allele detection of Plasmodium falciparum chloroquine resistance via a Pyrococcus furiosus argonaute-triggered dual-signal biosensing platform Parasites & Vectors Pyrococcus furiosus argonaute (PfAgo) Recombinase polymerase amplification (RPA) Plasmodium falciparum chloroquine resistance transporter (Pfcrt) Genotyping Haplotype detection Point-of-care testing (POCT) |
| title | Rapid and supersensitive allele detection of Plasmodium falciparum chloroquine resistance via a Pyrococcus furiosus argonaute-triggered dual-signal biosensing platform |
| title_full | Rapid and supersensitive allele detection of Plasmodium falciparum chloroquine resistance via a Pyrococcus furiosus argonaute-triggered dual-signal biosensing platform |
| title_fullStr | Rapid and supersensitive allele detection of Plasmodium falciparum chloroquine resistance via a Pyrococcus furiosus argonaute-triggered dual-signal biosensing platform |
| title_full_unstemmed | Rapid and supersensitive allele detection of Plasmodium falciparum chloroquine resistance via a Pyrococcus furiosus argonaute-triggered dual-signal biosensing platform |
| title_short | Rapid and supersensitive allele detection of Plasmodium falciparum chloroquine resistance via a Pyrococcus furiosus argonaute-triggered dual-signal biosensing platform |
| title_sort | rapid and supersensitive allele detection of plasmodium falciparum chloroquine resistance via a pyrococcus furiosus argonaute triggered dual signal biosensing platform |
| topic | Pyrococcus furiosus argonaute (PfAgo) Recombinase polymerase amplification (RPA) Plasmodium falciparum chloroquine resistance transporter (Pfcrt) Genotyping Haplotype detection Point-of-care testing (POCT) |
| url | https://doi.org/10.1186/s13071-024-06575-0 |
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