ABSTRACT
Background. COVID-19 pandemic highlighted the importance of sensitive and specific tests that would be cost-efficient, fast and scalable. There are more than 200 COVID-19 detection tests available worldwide, with every country developing its own assays. Sample collection, preparing for a test, the test itself and interpretation of results have a strong impact on the clinical value of testing. The diversity of tests and workflows requires the analysis of their performance in clinics. Methods. Literature review, analysis of clinical reports, online resources, public and commercial reports were used to collect information about tests. The collected information was processed to obtain information relevant to this review. Results. COVID-19 tests based on the amplification of nucleic acids are reviewed. Tests employ polymerase chain reaction (PCR) or loop-mediated isothermal amplification (LAMP). The clinical value of these tests depends on the technologies used, as they differ for LAMP, real-time and standard PCR methods. The diversity of sample preparation protocols, different designs of the tests, used chemicals and protocols have a significant impact on tests. Tailoring a testing workflow to available infrastructure and selecting the most efficient combination of tests and protocols for each step in a testing workflow is crucial for the success. Conclusion. Strengths and weaknesses of different test systems and protocols that were reviewed herein can be helpful in selecting a testing workflow to achieve maximum clinical utility. © Serhiy Souchelnytskyi, Nazariy Souchelnytskyi, 2020
ABSTRACT
Introduction. Polymerase chain reaction (PCR)-based diagnostic tests use purifi ed nucleic acids (NAs) from clinical samples. The NAs purifi cation step adds time, cost, and aff ects the quality of testing. The objective of this study was to develop a protocol for direct use of saliva in tests for genetic markers, without purifi cation of nucleic acids. Methods. PCR, real-time RT-PCR and isothermal amplifi cation tests were used for direct detection of genetic markers, without purifi cation of nucleic acids. Results. We report a protocol for the direct detection of genetic markers in saliva. The protocol is based on a collection of saliva in a solution containing a detergent and ethanol and is compatible with isothermal amplifi cation (LAMP), real-time RT-PCR and RT-PCR. SARS-CoV-2 and GAPDH markers were used as reference markers. We observed that mild detergents allow effi cient detection of external reference and intracellular endogenous markers, while strong detergent, e.g. sodium dodecyl sulfate, inhibited the PCR reaction. Under these conditions, saliva samples can be stored for 24 h at +4°C or -18°C with the preservation of markers. Storage at room temperature led to the deterioration of marker detection. Snap heating of saliva samples at the time of collection, followed by storage at room temperature, provided partial protection. Conclusion. The protocol presented in this report describes the collection and storage of saliva for direct detection of genetic markers and is compatible with PCR and LAMP tests. © 2021 Croatian Veterinary Institute. All rights reserved.