دورية أكاديمية
أعرض في EDS

Improved protocol for Bst polymerase and reverse transcriptase production and application to a point-of-care diagnostics system.

التفاصيل البيبلوغرافية
العنوان: Improved protocol for Bst polymerase and reverse transcriptase production and application to a point-of-care diagnostics system.
المؤلفون: de Souza LR; Center for Molecular Biology and Genetic Engineering (CBMEG), Universidade Estadual de Campinas (UNICAMP), Campinas/SP 13083-875, Brazil.; Center for Medicinal Chemistry (CQMED), Universidade Estadual de Campinas (UNICAMP), Campinas/SP 13083-886, Brazil., Silva IEPD; Center for Molecular Biology and Genetic Engineering (CBMEG), Universidade Estadual de Campinas (UNICAMP), Campinas/SP 13083-875, Brazil.; Center for Medicinal Chemistry (CQMED), Universidade Estadual de Campinas (UNICAMP), Campinas/SP 13083-886, Brazil.; Department of Biochemistry and Tissue Biology, Institute of Biology, Universidade Estadual de Campinas (UNICAMP), Campinas/SP 13083-862, Brazil., Celis-Silva G; Center for Molecular Biology and Genetic Engineering (CBMEG), Universidade Estadual de Campinas (UNICAMP), Campinas/SP 13083-875, Brazil.; Center for Medicinal Chemistry (CQMED), Universidade Estadual de Campinas (UNICAMP), Campinas/SP 13083-886, Brazil., Raddatz BW; Hilab, Curitiba/PR 81270-185, Brazil., Imamura LM; Hilab, Curitiba/PR 81270-185, Brazil., Kim EYS; Hilab, Curitiba/PR 81270-185, Brazil., Valderrama GV; Center for Molecular Biology and Genetic Engineering (CBMEG), Universidade Estadual de Campinas (UNICAMP), Campinas/SP 13083-875, Brazil.; Center for Medicinal Chemistry (CQMED), Universidade Estadual de Campinas (UNICAMP), Campinas/SP 13083-886, Brazil., Riedi HP; Hilab, Curitiba/PR 81270-185, Brazil., Rogal SR Jr; Hilab, Curitiba/PR 81270-185, Brazil., Almeida BMM; Hilab, Curitiba/PR 81270-185, Brazil., Figueredo MVM; Hilab, Curitiba/PR 81270-185, Brazil., Bengtson MH; Center for Medicinal Chemistry (CQMED), Universidade Estadual de Campinas (UNICAMP), Campinas/SP 13083-886, Brazil.; Department of Biochemistry and Tissue Biology, Institute of Biology, Universidade Estadual de Campinas (UNICAMP), Campinas/SP 13083-862, Brazil., Massirer KB; Center for Molecular Biology and Genetic Engineering (CBMEG), Universidade Estadual de Campinas (UNICAMP), Campinas/SP 13083-875, Brazil.; Center for Medicinal Chemistry (CQMED), Universidade Estadual de Campinas (UNICAMP), Campinas/SP 13083-886, Brazil.
المصدر: Experimental biology and medicine (Maywood, N.J.) [Exp Biol Med (Maywood)] 2023 Oct; Vol. 248 (19), pp. 1671-1683. Date of Electronic Publication: 2023 Dec 13.
نوع المنشور: Journal Article; Research Support, Non-U.S. Gov't
اللغة: English
بيانات الدورية: Publisher: Frontiers Media S.A Country of Publication: Switzerland NLM ID: 100973463 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1535-3699 (Electronic) Linking ISSN: 15353699 NLM ISO Abbreviation: Exp Biol Med (Maywood) Subsets: MEDLINE
أسماء مطبوعة: Publication: 2024- : Lausanne, Switzerland : Frontiers Media S.A.
Original Publication: Maywood, NJ : The Society, c2001-
مواضيع طبية MeSH: COVID-19*/diagnosis, Humans ; RNA-Directed DNA Polymerase ; Sensitivity and Specificity ; SARS-CoV-2/genetics ; Molecular Diagnostic Techniques/methods ; Point-of-Care Testing ; Reverse Transcriptase Polymerase Chain Reaction ; COVID-19 Testing
مستخلص: The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has raised awareness in the scientific community about the importance of being prepared for sanitary emergencies. Many measures implemented during the COVID pandemic are now being expanded to other applications. In the field of molecular and immunological diagnostics, the need to massively test the population worldwide resulted in the application of a variety of methods to detect viral infection. Besides gold standard reverse transcription quantitative polymerase chain reaction (RT-qPCR), the use of reverse transcription loop-mediated isothermal amplification (RT-LAMP) arose as an alternative and sensitive method to amplify and detect viral genetic material. We have used openly available protocols and have improved the protein production of RT-LAMP enzymes Bst polymerase and HIV -reverse transcriptase. To optimize enzyme production, we tested different protein tags, and we shortened the protein purification protocol, resulting in reduced processing time and handling of the enzymes and, thus, preserved the protein activity with high purity. The enzymes showed significant stability at 4 °C and 25 °C, over 60 days, and were highly reliable when used as a one-step RT-LAMP reaction in a portable point-of-care device with clinical samples. The enzymes and the reaction setup can be further expanded to detect other infectious diseases agents.
Competing Interests: Declaration of Conflicting InterestsMVMF is the CEO at Hi Technologies LTDA; SRR is the CTO at Hi Technologies LTDA; BMMA is the CMO at Hi Technologies LTDA. BWR, LMI, EYSK, HPR work or worked at Hi Technologies LTDA and do not declare any competing interest.
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فهرسة مساهمة: Keywords: Bst polymerase; RNA virus detection; RT-LAMP; SARS-CoV-2; point-of-care; reverse transcriptase
المشرفين على المادة: EC 2.7.7.49 (RNA-Directed DNA Polymerase)
تواريخ الأحداث: Date Created: 20231213 Date Completed: 20231216 Latest Revision: 20240614
رمز التحديث: 20240614
مُعرف محوري في PubMed: PMC10723028
DOI: 10.1177/15353702231215815
PMID: 38088106
قاعدة البيانات: MEDLINE
الوصف
تدمد:1535-3699
DOI:10.1177/15353702231215815