Ismael Segura-Ulate, Navilla Apú, Bernal Cortés, Jordi Querol-Audi, Yamitzel Zaldívar, Carlos Alexander Ortega, Fernando Flores-Mora, Andrés Gatica-Arias, Germán Madrigal-Redondo
{"title":"交叉比较替代诊断方案，包括替代临床样本、RNA 提取方法和用于 COVID-19 诊断的核酸扩增技术。","authors":"Ismael Segura-Ulate, Navilla Apú, Bernal Cortés, Jordi Querol-Audi, Yamitzel Zaldívar, Carlos Alexander Ortega, Fernando Flores-Mora, Andrés Gatica-Arias, Germán Madrigal-Redondo","doi":"10.3389/fmolb.2024.1445142","DOIUrl":null,"url":null,"abstract":"<p><strong>Background: </strong>the gold-standard diagnostic protocol (GSDP) for COVID-19 consists of a nasopharyngeal swab (NPS) sample processed through traditional RNA extraction (TRE) and amplified with retrotranscription quantitative polymerase chain reaction (RT-qPCR). Multiple alternatives were developed to decrease time/cost of GSDP, including alternative clinical samples, RNA extraction methods and nucleic acid amplification. Thus, we carried out a cross comparison of various alternatives methods against GSDP and each other.</p><p><strong>Methods: </strong>we tested alternative diagnostic methods using saliva, heat-induced RNA release (HIRR) and a colorimetric retrotranscription loop-mediated isothermal amplification (RT-LAMP) as substitutions to the GSDP.</p><p><strong>Results: </strong>RT-LAMP using NPS processed by TRE showed high sensitivity (96%) and specificity (97%), closely matching GSDP. When saliva was processed by TRE and amplified with both RT-LAMP and RT-qPCR, RT-LAMP yielded high diagnostic parameters (88%-96% sensitivity and 95%-100% specificity) compared to RT-qPCR. Nonetheless, when saliva processed by TRE and detected by RT-LAMP was compared against the GSDP, the resulting diagnostic values for sensitivity (78%) and specificity (87%) were somewhat high but still short of those of the GSDP. Finally, saliva processed with HIRR and detected via RT-LAMP was the simplest and fastest method, but its sensitivity against GSDP was too low (56%) for any clinical application. Also, in this last method, the acidity of a large percentage of saliva samples (9%-22%) affected the pH-sensitive colorimetric indicator used in the test, requiring the exclusion of these acidic samples or an extra step for pH correction.</p><p><strong>Discussion: </strong>our comparison shows that RT-LAMP technology has diagnostic performance on par with RT-qPCR; likewise, saliva offers the same diagnostic functionality as NPS when subjected to a TRE method. Nonetheless, use of direct saliva after a HIRR and detected with RT-LAMP does not produce an acceptable diagnostic performance.</p>","PeriodicalId":12465,"journal":{"name":"Frontiers in Molecular Biosciences","volume":"11 ","pages":"1445142"},"PeriodicalIF":3.9000,"publicationDate":"2024-08-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11377848/pdf/","citationCount":"0","resultStr":"{\"title\":\"Cross comparison of alternative diagnostic protocols including substitution to the clinical sample, RNA extraction method and nucleic acid amplification technology for COVID-19 diagnosis.\",\"authors\":\"Ismael Segura-Ulate, Navilla Apú, Bernal Cortés, Jordi Querol-Audi, Yamitzel Zaldívar, Carlos Alexander Ortega, Fernando Flores-Mora, Andrés Gatica-Arias, Germán Madrigal-Redondo\",\"doi\":\"10.3389/fmolb.2024.1445142\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Background: </strong>the gold-standard diagnostic protocol (GSDP) for COVID-19 consists of a nasopharyngeal swab (NPS) sample processed through traditional RNA extraction (TRE) and amplified with retrotranscription quantitative polymerase chain reaction (RT-qPCR). Multiple alternatives were developed to decrease time/cost of GSDP, including alternative clinical samples, RNA extraction methods and nucleic acid amplification. Thus, we carried out a cross comparison of various alternatives methods against GSDP and each other.</p><p><strong>Methods: </strong>we tested alternative diagnostic methods using saliva, heat-induced RNA release (HIRR) and a colorimetric retrotranscription loop-mediated isothermal amplification (RT-LAMP) as substitutions to the GSDP.</p><p><strong>Results: </strong>RT-LAMP using NPS processed by TRE showed high sensitivity (96%) and specificity (97%), closely matching GSDP. When saliva was processed by TRE and amplified with both RT-LAMP and RT-qPCR, RT-LAMP yielded high diagnostic parameters (88%-96% sensitivity and 95%-100% specificity) compared to RT-qPCR. Nonetheless, when saliva processed by TRE and detected by RT-LAMP was compared against the GSDP, the resulting diagnostic values for sensitivity (78%) and specificity (87%) were somewhat high but still short of those of the GSDP. Finally, saliva processed with HIRR and detected via RT-LAMP was the simplest and fastest method, but its sensitivity against GSDP was too low (56%) for any clinical application. Also, in this last method, the acidity of a large percentage of saliva samples (9%-22%) affected the pH-sensitive colorimetric indicator used in the test, requiring the exclusion of these acidic samples or an extra step for pH correction.</p><p><strong>Discussion: </strong>our comparison shows that RT-LAMP technology has diagnostic performance on par with RT-qPCR; likewise, saliva offers the same diagnostic functionality as NPS when subjected to a TRE method. 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Cross comparison of alternative diagnostic protocols including substitution to the clinical sample, RNA extraction method and nucleic acid amplification technology for COVID-19 diagnosis.
Background: the gold-standard diagnostic protocol (GSDP) for COVID-19 consists of a nasopharyngeal swab (NPS) sample processed through traditional RNA extraction (TRE) and amplified with retrotranscription quantitative polymerase chain reaction (RT-qPCR). Multiple alternatives were developed to decrease time/cost of GSDP, including alternative clinical samples, RNA extraction methods and nucleic acid amplification. Thus, we carried out a cross comparison of various alternatives methods against GSDP and each other.
Methods: we tested alternative diagnostic methods using saliva, heat-induced RNA release (HIRR) and a colorimetric retrotranscription loop-mediated isothermal amplification (RT-LAMP) as substitutions to the GSDP.
Results: RT-LAMP using NPS processed by TRE showed high sensitivity (96%) and specificity (97%), closely matching GSDP. When saliva was processed by TRE and amplified with both RT-LAMP and RT-qPCR, RT-LAMP yielded high diagnostic parameters (88%-96% sensitivity and 95%-100% specificity) compared to RT-qPCR. Nonetheless, when saliva processed by TRE and detected by RT-LAMP was compared against the GSDP, the resulting diagnostic values for sensitivity (78%) and specificity (87%) were somewhat high but still short of those of the GSDP. Finally, saliva processed with HIRR and detected via RT-LAMP was the simplest and fastest method, but its sensitivity against GSDP was too low (56%) for any clinical application. Also, in this last method, the acidity of a large percentage of saliva samples (9%-22%) affected the pH-sensitive colorimetric indicator used in the test, requiring the exclusion of these acidic samples or an extra step for pH correction.
Discussion: our comparison shows that RT-LAMP technology has diagnostic performance on par with RT-qPCR; likewise, saliva offers the same diagnostic functionality as NPS when subjected to a TRE method. Nonetheless, use of direct saliva after a HIRR and detected with RT-LAMP does not produce an acceptable diagnostic performance.
期刊介绍：
Much of contemporary investigation in the life sciences is devoted to the molecular-scale understanding of the relationships between genes and the environment — in particular, dynamic alterations in the levels, modifications, and interactions of cellular effectors, including proteins. Frontiers in Molecular Biosciences offers an international publication platform for basic as well as applied research; we encourage contributions spanning both established and emerging areas of biology. To this end, the journal draws from empirical disciplines such as structural biology, enzymology, biochemistry, and biophysics, capitalizing as well on the technological advancements that have enabled metabolomics and proteomics measurements in massively parallel throughput, and the development of robust and innovative computational biology strategies. We also recognize influences from medicine and technology, welcoming studies in molecular genetics, molecular diagnostics and therapeutics, and nanotechnology.
Our ultimate objective is the comprehensive illustration of the molecular mechanisms regulating proteins, nucleic acids, carbohydrates, lipids, and small metabolites in organisms across all branches of life.
In addition to interesting new findings, techniques, and applications, Frontiers in Molecular Biosciences will consider new testable hypotheses to inspire different perspectives and stimulate scientific dialogue. The integration of in silico, in vitro, and in vivo approaches will benefit endeavors across all domains of the life sciences.
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