Sang-Soo Lee , Ah Leum Kim , Jae-Hyung Park , Da-Hye Lee , Young-Kyung Bae
{"title":"优化测量 SARS-CoV-2 RNA 的双工数字 PCR。","authors":"Sang-Soo Lee , Ah Leum Kim , Jae-Hyung Park , Da-Hye Lee , Young-Kyung Bae","doi":"10.1016/j.jviromet.2024.114911","DOIUrl":null,"url":null,"abstract":"<div><p>Quantitative PCR (qPCR) is the gold standard for detecting nucleic acid sequences specific to the target pathogen. For COVID-19 diagnosis, several molecular assays have been developed. In this study, we present an optimization strategy for the measurement of SARS-CoV-2 RNA via multiplex qPCR and digital PCR (dPCR). Compared to qPCR, both droplet and chip-based dPCR, which are known to be more sensitive and accurate, showed a better resilience to suboptimal assay compositions and cycling conditions following the proposed optimizations. In particular, the formation of heterodimers among assays greatly interfered with qPCR results, but only minimally with dPCR results. In dPCR, existing heterodimers lowered the PCR efficiency, producing a dampened fluorescent signal in positive partitions. This can be corrected by adjusting the PCR cycling conditions, after which dPCR shows the capability of measuring the expected copy number. In addition, we present a process to improve the existing <em>RdRp</em> assay by correcting the primer sequences and matching the melting temperature, ultimately producing highly sensitive and robust assays. The results of this study can reduce the cost and time of SARS-CoV-2 diagnosis while increasing accuracy. Furthermore, our results suggest that dPCR is a reliable method for the accurate measurement of nucleic acid targets.</p></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":null,"pages":null},"PeriodicalIF":2.2000,"publicationDate":"2024-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Optimization of duplex digital PCR for the measurement of SARS-CoV-2 RNA\",\"authors\":\"Sang-Soo Lee , Ah Leum Kim , Jae-Hyung Park , Da-Hye Lee , Young-Kyung Bae\",\"doi\":\"10.1016/j.jviromet.2024.114911\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>Quantitative PCR (qPCR) is the gold standard for detecting nucleic acid sequences specific to the target pathogen. For COVID-19 diagnosis, several molecular assays have been developed. In this study, we present an optimization strategy for the measurement of SARS-CoV-2 RNA via multiplex qPCR and digital PCR (dPCR). Compared to qPCR, both droplet and chip-based dPCR, which are known to be more sensitive and accurate, showed a better resilience to suboptimal assay compositions and cycling conditions following the proposed optimizations. In particular, the formation of heterodimers among assays greatly interfered with qPCR results, but only minimally with dPCR results. In dPCR, existing heterodimers lowered the PCR efficiency, producing a dampened fluorescent signal in positive partitions. This can be corrected by adjusting the PCR cycling conditions, after which dPCR shows the capability of measuring the expected copy number. In addition, we present a process to improve the existing <em>RdRp</em> assay by correcting the primer sequences and matching the melting temperature, ultimately producing highly sensitive and robust assays. The results of this study can reduce the cost and time of SARS-CoV-2 diagnosis while increasing accuracy. Furthermore, our results suggest that dPCR is a reliable method for the accurate measurement of nucleic acid targets.</p></div>\",\"PeriodicalId\":17663,\"journal\":{\"name\":\"Journal of virological methods\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":2.2000,\"publicationDate\":\"2024-03-04\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Journal of virological methods\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S0166093424000351\",\"RegionNum\":4,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"BIOCHEMICAL RESEARCH METHODS\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of virological methods","FirstCategoryId":"3","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0166093424000351","RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
Optimization of duplex digital PCR for the measurement of SARS-CoV-2 RNA
Quantitative PCR (qPCR) is the gold standard for detecting nucleic acid sequences specific to the target pathogen. For COVID-19 diagnosis, several molecular assays have been developed. In this study, we present an optimization strategy for the measurement of SARS-CoV-2 RNA via multiplex qPCR and digital PCR (dPCR). Compared to qPCR, both droplet and chip-based dPCR, which are known to be more sensitive and accurate, showed a better resilience to suboptimal assay compositions and cycling conditions following the proposed optimizations. In particular, the formation of heterodimers among assays greatly interfered with qPCR results, but only minimally with dPCR results. In dPCR, existing heterodimers lowered the PCR efficiency, producing a dampened fluorescent signal in positive partitions. This can be corrected by adjusting the PCR cycling conditions, after which dPCR shows the capability of measuring the expected copy number. In addition, we present a process to improve the existing RdRp assay by correcting the primer sequences and matching the melting temperature, ultimately producing highly sensitive and robust assays. The results of this study can reduce the cost and time of SARS-CoV-2 diagnosis while increasing accuracy. Furthermore, our results suggest that dPCR is a reliable method for the accurate measurement of nucleic acid targets.
期刊介绍:
The Journal of Virological Methods focuses on original, high quality research papers that describe novel and comprehensively tested methods which enhance human, animal, plant, bacterial or environmental virology and prions research and discovery.
The methods may include, but not limited to, the study of:
Viral components and morphology-
Virus isolation, propagation and development of viral vectors-
Viral pathogenesis, oncogenesis, vaccines and antivirals-
Virus replication, host-pathogen interactions and responses-
Virus transmission, prevention, control and treatment-
Viral metagenomics and virome-
Virus ecology, adaption and evolution-
Applied virology such as nanotechnology-
Viral diagnosis with novelty and comprehensive evaluation.
We seek articles, systematic reviews, meta-analyses and laboratory protocols that include comprehensive technical details with statistical confirmations that provide validations against current best practice, international standards or quality assurance programs and which advance knowledge in virology leading to improved medical, veterinary or agricultural practices and management.