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.
Highlights
Polymerase chain reaction (PCR)-based diagnostic tests use purified nucleic acids (NAs) from clinical samples
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 amplification (LAMP), real-time
Summary
Polymerase chain reaction (PCR)-based diagnostic tests use purified nucleic acids (NAs) from clinical samples. The NAs purification step adds time, cost, and affects the quality of testing. Polymerase chain reaction is a frequently used highly sensitive diagnostic method for detecting nucleic acids. Accessibility of NAs for amplification is crucial for the performance and sensitivity of a PCR test. The considerations of DNA accessibility and the complex nature of clinical samples prompted the introduction of a nucleic acid purification step in PCR diagnostic tests. This purification step increases the time and cost of each assay and requires dedicated laboratory instrumentation [3; 4]. Failure in the purification procedure may jeopardize an assay [5]
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