Development of a novel strategy to reduce diagnostic errors in real-time polymerase chain reaction using probe-based techniques
Abstract Real-time PCR assays are valuable tools for the rapid and accurate diagnosis of infectious diseases by identifying the nucleic acid sequences of pathogens. Here, we aimed to develop a recombinant plasmid-based standard for validating the sensitivity of different molecular diagnostic methods...
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Nature Portfolio
2024-11-01
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| Series: | Scientific Reports |
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| Online Access: | https://doi.org/10.1038/s41598-024-78654-2 |
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| author | Hyoung Jun Kim Morten Schiøtt Niels Jørgen Olesen Euna Choi Bok Kyung Ku Kyoung Ki Lee Hye Young Jeong Ilseob Lee Seong Mok Kim Miyoung Cho Young Chul Kim |
| author_facet | Hyoung Jun Kim Morten Schiøtt Niels Jørgen Olesen Euna Choi Bok Kyung Ku Kyoung Ki Lee Hye Young Jeong Ilseob Lee Seong Mok Kim Miyoung Cho Young Chul Kim |
| author_sort | Hyoung Jun Kim |
| collection | DOAJ |
| description | Abstract Real-time PCR assays are valuable tools for the rapid and accurate diagnosis of infectious diseases by identifying the nucleic acid sequences of pathogens. Here, we aimed to develop a recombinant plasmid-based standard for validating the sensitivity of different molecular diagnostic methods adopting the Jonstrup assay (J assay). Chimeric plasmid DNA (cpDNA) harboring various pathogen genes and the target site of the J assay was constructed. Our findings revealed that the J assay could detect a single copy of the target gene similar to digital droplet PCR. The detection sensitivity of each established real-time PCR method for various disease diagnoses was evaluated using the cpDNA. Although most methods showed high sensitivity, similar to that of the J assay, the VHS Garver and SARS-CoV-2 diagnostic methods exhibited detection sensitivities tenfold lower than that of the J assay. To ensure accuracy of results and avoid genetic contamination from positive controls, we introduced an additional probe attachment site emitting distinct fluorescent signals within the cpDNA. Target genes and exogenous sequences within the plasmid DNA were simultaneously detected in a single assay using this unique method. Our approach will help improve the sensitivity of diagnostic methods and develop novel diagnostic methods based on molecular techniques. |
| format | Article |
| id | doaj-art-d4fb0bcde0ca460cba29bf005e411419 |
| institution | OA Journals |
| issn | 2045-2322 |
| language | English |
| publishDate | 2024-11-01 |
| publisher | Nature Portfolio |
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| series | Scientific Reports |
| spelling | doaj-art-d4fb0bcde0ca460cba29bf005e4114192025-08-20T02:13:39ZengNature PortfolioScientific Reports2045-23222024-11-0114111110.1038/s41598-024-78654-2Development of a novel strategy to reduce diagnostic errors in real-time polymerase chain reaction using probe-based techniquesHyoung Jun Kim0Morten Schiøtt1Niels Jørgen Olesen2Euna Choi3Bok Kyung Ku4Kyoung Ki Lee5Hye Young Jeong6Ilseob Lee7Seong Mok Kim8Miyoung Cho9Young Chul Kim10WOAH Reference Laboratory for VHS, National Institute of Fisheries ScienceEU Reference Laboratory for Crustacean Diseases, National Institute of Aquatic Resources, Technical University of DenmarkEU Reference Laboratory for Crustacean Diseases, National Institute of Aquatic Resources, Technical University of DenmarkWOAH Reference Laboratory for VHS, National Institute of Fisheries ScienceAnimal Disease Diagnostic Division, Animal and Plant Quarantine Agency 177Animal Disease Diagnostic Division, Animal and Plant Quarantine Agency 177Animal Disease Diagnostic Division, Animal and Plant Quarantine Agency 177Animal Disease Diagnostic Division, Animal and Plant Quarantine Agency 177WOAH Reference Laboratory for VHS, National Institute of Fisheries ScienceWOAH Reference Laboratory for VHS, National Institute of Fisheries ScienceWOAH Reference Laboratory for VHS, National Institute of Fisheries ScienceAbstract Real-time PCR assays are valuable tools for the rapid and accurate diagnosis of infectious diseases by identifying the nucleic acid sequences of pathogens. Here, we aimed to develop a recombinant plasmid-based standard for validating the sensitivity of different molecular diagnostic methods adopting the Jonstrup assay (J assay). Chimeric plasmid DNA (cpDNA) harboring various pathogen genes and the target site of the J assay was constructed. Our findings revealed that the J assay could detect a single copy of the target gene similar to digital droplet PCR. The detection sensitivity of each established real-time PCR method for various disease diagnoses was evaluated using the cpDNA. Although most methods showed high sensitivity, similar to that of the J assay, the VHS Garver and SARS-CoV-2 diagnostic methods exhibited detection sensitivities tenfold lower than that of the J assay. To ensure accuracy of results and avoid genetic contamination from positive controls, we introduced an additional probe attachment site emitting distinct fluorescent signals within the cpDNA. Target genes and exogenous sequences within the plasmid DNA were simultaneously detected in a single assay using this unique method. Our approach will help improve the sensitivity of diagnostic methods and develop novel diagnostic methods based on molecular techniques.https://doi.org/10.1038/s41598-024-78654-2Viral hemorrhagic septicemiaStandardizationReal-time PCRFalse-positiveFalse-negativePositive control |
| spellingShingle | Hyoung Jun Kim Morten Schiøtt Niels Jørgen Olesen Euna Choi Bok Kyung Ku Kyoung Ki Lee Hye Young Jeong Ilseob Lee Seong Mok Kim Miyoung Cho Young Chul Kim Development of a novel strategy to reduce diagnostic errors in real-time polymerase chain reaction using probe-based techniques Scientific Reports Viral hemorrhagic septicemia Standardization Real-time PCR False-positive False-negative Positive control |
| title | Development of a novel strategy to reduce diagnostic errors in real-time polymerase chain reaction using probe-based techniques |
| title_full | Development of a novel strategy to reduce diagnostic errors in real-time polymerase chain reaction using probe-based techniques |
| title_fullStr | Development of a novel strategy to reduce diagnostic errors in real-time polymerase chain reaction using probe-based techniques |
| title_full_unstemmed | Development of a novel strategy to reduce diagnostic errors in real-time polymerase chain reaction using probe-based techniques |
| title_short | Development of a novel strategy to reduce diagnostic errors in real-time polymerase chain reaction using probe-based techniques |
| title_sort | development of a novel strategy to reduce diagnostic errors in real time polymerase chain reaction using probe based techniques |
| topic | Viral hemorrhagic septicemia Standardization Real-time PCR False-positive False-negative Positive control |
| url | https://doi.org/10.1038/s41598-024-78654-2 |
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