دورية أكاديمية
Development of a Nanoplate-Based Digital PCR Test Method for Quantitative Detection of Human Adenovirus DNA.
| العنوان: | Development of a Nanoplate-Based Digital PCR Test Method for Quantitative Detection of Human Adenovirus DNA. |
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| المؤلفون: | Doğantürk YE; Department of Medical Microbiology, İstanbul University-Cerrahpaşa School of Medicine, İstanbul, Türkiye.; Department of Medical Microbiology, İstanbul Aydın University School of Medicine, İstanbul, Türkiye., Dağ-Güzel A; Department of Medical Microbiology, İstanbul University-Cerrahpaşa School of Medicine, İstanbul, Türkiye.; Medical Laboratory Techniques, Arel University Junior Technical Collage, İstanbul, Türkiye., Kuşkucu MA; Department of Medical Microbiology, İstanbul University-Cerrahpaşa School of Medicine, İstanbul, Türkiye.; Department of Medical Microbiology, Koç University School of Medicine, İstanbul, Türkiye. |
| المصدر: | Infectious diseases & clinical microbiology [Infect Dis Clin Microbiol] 2023 Dec 29; Vol. 5 (4), pp. 353-366. Date of Electronic Publication: 2023 Dec 29 (Print Publication: 2023). |
| نوع المنشور: | Journal Article |
| اللغة: | English |
| بيانات الدورية: | Publisher: TÜRK KLİNİK MİKROBİYOLOJİ VE İNFEKSİYON HAS DR İKT Country of Publication: Turkey NLM ID: 9918680988406676 Publication Model: eCollection Cited Medium: Internet ISSN: 2667-646X (Electronic) Linking ISSN: 2667646X NLM ISO Abbreviation: Infect Dis Clin Microbiol Subsets: PubMed not MEDLINE |
| أسماء مطبوعة: | Original Publication: İstanbul: TÜRK KLİNİK MİKROBİYOLOJİ VE İNFEKSİYON HAS DR İKT İ |
| مستخلص: | Objective: Digital polymerase chain reaction (dPCR) assay is an advanced PCR technique that allows for the simultaneous detection and absolute quantification of diverse pathogens.Commercially validated kits available for detecting all subtypes of human adenovirus (HAdV) are limited. This study aimed to demonstrate the development of an in-house nanoplate-based dPCR assay with high sensitivity, even at low copy numbers. Materials and Methods: In this methodological study, the standardized HAdV DNA was prepared by amplifying the specific hexon gene region with real-time PCR and purifying the HAdV DNA using magnetic beads from HAdV-positive extractions. Dilutions were tested in triplicate during three independent runs to determine the dynamic range, the limit of detection (LoD), the limit of quantification (LoQ), precision, and reproducibility. The primer and probe sequences used in the study were selected based on a literature review to ensure the detection of all HAdV serotypes in a single run. The selected primers were verified using the US National Center for Biotechnology Information (NBCI) nBLAST tools, and the target sequence was determined using the BioEdit software. The DNA concentration of the stock solution was measured using a Qubit fluorometer. The estimated copy number of the stock solution per milliliter was calculated based on the length of the amplified base sequence and fluorometer measurement. Results: The dynamic range of the test was determined to be from 770.4 to 0.9476 cp/μl, with the LoD and LoQ values both being 0.9476 cp/μl. The coefficient of determination (r 2 ) value of the test was 0.9986. Conclusion: The results demonstrated that the dPCR method could be an ideal tool for the diagnosis and absolute quantification of human adenoviruses, especially in low copy numbers. In order to determine the reproducibility of the test and validate the method for field use, it needs to be developed and adapted in various laboratories and supported by clinical studies. Competing Interests: The authors declare no conflict of interest. (Copyright © 2024 Infectious Diseases and Clinical Microbiology.) |
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| فهرسة مساهمة: | Keywords: HAdV; PCR; adenovirus; dPCR; validation |
| تواريخ الأحداث: | Date Created: 20240418 Latest Revision: 20240419 |
| رمز التحديث: | 20240419 |
| مُعرف محوري في PubMed: | PMC10986707 |
| DOI: | 10.36519/idcm.2023.255 |
| PMID: | 38633848 |
| قاعدة البيانات: | MEDLINE |
| تدمد: | 2667-646X |
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| DOI: | 10.36519/idcm.2023.255 |