Development and validation of a multiplex PCR assay with melt curve analysis for detecting simian Plasmodium in wild Macaca fascicularis
Abstract Accurate and sensitive detection of simian malaria parasites is essential for surveillance and risk assessment of zoonotic malaria. We developed and validated a SYBR Green-based real-time PCR assay targeting the msp1 gene to detect and differentiate P. knowlesi, P. cynomolgi, and P. inui. S...
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Nature Portfolio
2025-07-01
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| Series: | Scientific Reports |
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| Online Access: | https://doi.org/10.1038/s41598-025-07337-3 |
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| author | Thitiluck Swangsri Rucksak Rucksaken Wanat Sricharern Wang Nguitragool Naowarat Saralamba |
| author_facet | Thitiluck Swangsri Rucksak Rucksaken Wanat Sricharern Wang Nguitragool Naowarat Saralamba |
| author_sort | Thitiluck Swangsri |
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| description | Abstract Accurate and sensitive detection of simian malaria parasites is essential for surveillance and risk assessment of zoonotic malaria. We developed and validated a SYBR Green-based real-time PCR assay targeting the msp1 gene to detect and differentiate P. knowlesi, P. cynomolgi, and P. inui. Species-specific amplification was confirmed through distinct melting temperature (Tm) profiles. The assay demonstrated high analytical sensitivity, with a limit of detection of 10 copies/µL, excellent specificity with no cross-reactivity, and strong reproducibility, with low coefficients of variation for both cycle threshold (Ct) and Tm values. Amplification efficiency was within acceptable ranges, with R2 > 0.90 across standard curves. The assay was evaluated using 191 archived blood samples from wild M. fascicularis collected across three provinces in Thailand, with P. knowlesi detected in two samples. Both positive detections were confirmed by nested PCR and sequencing. This assay offers a rapid, cost-effective, and reliable tool for detecting simian malaria parasites in laboratory analyses and has potential for further application in field surveillance. |
| format | Article |
| id | doaj-art-c7fcb41c4f28438290c685afbd1f8428 |
| institution | Kabale University |
| issn | 2045-2322 |
| language | English |
| publishDate | 2025-07-01 |
| publisher | Nature Portfolio |
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| series | Scientific Reports |
| spelling | doaj-art-c7fcb41c4f28438290c685afbd1f84282025-08-20T04:01:35ZengNature PortfolioScientific Reports2045-23222025-07-011511910.1038/s41598-025-07337-3Development and validation of a multiplex PCR assay with melt curve analysis for detecting simian Plasmodium in wild Macaca fascicularisThitiluck Swangsri0Rucksak Rucksaken1Wanat Sricharern2Wang Nguitragool3Naowarat Saralamba4Department of Molecular Tropical Medicine and Genetics, Faculty of Tropical Medicine, Mahidol UniversityDepartment of Veterinary Nursing, Faculty of Veterinary Technology, Kasetsart UniversityDepartment of Veterinary Nursing, Faculty of Veterinary Technology, Kasetsart UniversityDepartment of Molecular Tropical Medicine and Genetics, Faculty of Tropical Medicine, Mahidol UniversityDepartment of Molecular Tropical Medicine and Genetics, Faculty of Tropical Medicine, Mahidol UniversityAbstract Accurate and sensitive detection of simian malaria parasites is essential for surveillance and risk assessment of zoonotic malaria. We developed and validated a SYBR Green-based real-time PCR assay targeting the msp1 gene to detect and differentiate P. knowlesi, P. cynomolgi, and P. inui. Species-specific amplification was confirmed through distinct melting temperature (Tm) profiles. The assay demonstrated high analytical sensitivity, with a limit of detection of 10 copies/µL, excellent specificity with no cross-reactivity, and strong reproducibility, with low coefficients of variation for both cycle threshold (Ct) and Tm values. Amplification efficiency was within acceptable ranges, with R2 > 0.90 across standard curves. The assay was evaluated using 191 archived blood samples from wild M. fascicularis collected across three provinces in Thailand, with P. knowlesi detected in two samples. Both positive detections were confirmed by nested PCR and sequencing. This assay offers a rapid, cost-effective, and reliable tool for detecting simian malaria parasites in laboratory analyses and has potential for further application in field surveillance.https://doi.org/10.1038/s41598-025-07337-3Multiplex PCRReal-time PCRPlasmodiumPlasmodium knowlesiPlasmodium cynomolgiMacaca fascicularis |
| spellingShingle | Thitiluck Swangsri Rucksak Rucksaken Wanat Sricharern Wang Nguitragool Naowarat Saralamba Development and validation of a multiplex PCR assay with melt curve analysis for detecting simian Plasmodium in wild Macaca fascicularis Scientific Reports Multiplex PCR Real-time PCR Plasmodium Plasmodium knowlesi Plasmodium cynomolgi Macaca fascicularis |
| title | Development and validation of a multiplex PCR assay with melt curve analysis for detecting simian Plasmodium in wild Macaca fascicularis |
| title_full | Development and validation of a multiplex PCR assay with melt curve analysis for detecting simian Plasmodium in wild Macaca fascicularis |
| title_fullStr | Development and validation of a multiplex PCR assay with melt curve analysis for detecting simian Plasmodium in wild Macaca fascicularis |
| title_full_unstemmed | Development and validation of a multiplex PCR assay with melt curve analysis for detecting simian Plasmodium in wild Macaca fascicularis |
| title_short | Development and validation of a multiplex PCR assay with melt curve analysis for detecting simian Plasmodium in wild Macaca fascicularis |
| title_sort | development and validation of a multiplex pcr assay with melt curve analysis for detecting simian plasmodium in wild macaca fascicularis |
| topic | Multiplex PCR Real-time PCR Plasmodium Plasmodium knowlesi Plasmodium cynomolgi Macaca fascicularis |
| url | https://doi.org/10.1038/s41598-025-07337-3 |
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