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Efficient SARS-CoV-2 Quantitative Reverse Transcriptase PCR Saliva Diagnostic Strategy utilizing Open-Source Pipetting Robots
Published on: February 11, 2022
Comparison of salivary collection and processing methods for quantitative HHV-8 detection
1School of Dentistry and Oral Health, Griffith University, Gold Coast, Qld, Australia; Molecular Basis of Disease Research Program, Griffith Health Institute, Griffith University, Gold Coast, Qld, Australia; Population & Social Health Research Program (Population Oral Health), Griffith Health Institute, Griffith University, Gold Coast, Qld, Australia.
The DNA Genotek P-021 kit with spin column extraction accurately quantifies viral loads in saliva, even after long-term room temperature storage. This method is ideal for studying salivary DNA viruses in resource-limited settings.
Area of Science:
- Molecular Biology
- Virology
- Diagnostic Technologies
Background:
- Saliva is a valuable diagnostic fluid for detecting infectious agents.
- Accurate quantification of viral load in saliva remains a challenge.
- Stabilizing solutions can prevent nucleic acid degradation, outperforming ice/freezing methods.
Purpose of the Study:
- To evaluate the quantitative accuracy of 10 different saliva collection and processing methods for viral load determination.
- To assess the performance of the DNA Genotek P-021 prototype kit for quantitative analysis of salivary DNA viruses.
- To determine the suitability of the P-021 kit for long-term storage and use in resource-poor settings.
Main Methods:
- Unstimulated whole mouth saliva was spiked with HHV-8 constructs and serially diluted.
- Samples were processed using 10 different collection/processing methods.
- Quantitative PCR (qPCR) was used to determine viral loads, with calibration curves analyzed by linear regression.
Main Results:
- Methods using commercial spin columns yielded linear calibration curves, wide dynamic ranges, and accurate viral loads.
- Ethanol precipitation with the P-021 kit resulted in non-linear curves and sample loss.
- DNA extraction from the P-021 kit using spin columns demonstrated excellent linearity, dynamic range, and limit of detection.
Conclusions:
- The P-021 kit, when combined with spin column extraction, allows for accurate viral load quantification down to 23 copies/μl.
- This system's quantitative capabilities and long-term storage stability make it highly suitable for studying salivary DNA viruses in resource-limited environments.
