COVID-19 Nucleic Acid Amplification Test (NAAT) with qPCR: Sample Preparation Tools

COVID-19 Nucleic Acid RT-PCR Testing: Sample Preparation Tools

SARS-Cov-2 Nucleic Acid Amplification Test (NAAT) is a real-time test intended for the qualitative detection of RNA from the SARS-CoV-2 virus in upper respiratory specimens (such as nasal, nasopharyngeal, oropharyngeal swab specimens). The following article provides an outline to the NAAT sample preparation and the tools and equipment required in each stages.

Phases of sample preparation for the RT-PCR test for COVID-19: Proper sample collection, handling and preparation strongly influences the accuracy of RT-PCR test results.

Specimen Inactivation

 The specimens can be inactivated at 56°C using a dry bath incubator. Four E’s dry bath incubator with its removable blocks suiting different tubes is ideal for inactivation. A lot of companies are providing VTM buffers that can inactivate nucleic acids. For more information regarding such inactivation kits, please contact us.

RNA Extraction

Nucleic Acids needs to be isolated from the inactivated specimen samples. Nucleic acids are extracted through three key processes: lysis, washing/purification (elution if it is a solid phase extraction method like magnetic bead extraction is used). In cell lysis, lysis reagents are used to promote cell rupture and release nucleic acids. Wash buffers are then used to remove contaminants such as proteins, polysaccharides and lipids, thereby purifying the RNA. Elution buffers are used in case solid phase carriers like magnetic beads are used to bind the nucleic acids. Thorough mixing of the cell suspension with lysis reagent is achieved using a vortex mixer. In liquid phase extraction, once the samples are subjected to centrifugation RNA can be pipetted from the supernatant. Four E’s High Speed Micro Centrifuge with a maximum rpm of 15,000 is ideal for this process.

As in all molecular biology applications, RNAse/ DNAse-free pipette tips are required for pipetting, since RNA is destroyed very quickly by enzymes, such as RNAse. The Precipette^TM series Pipettes from Four E’s Scientific is ergonomically designed for fatigue-free pipetting, and has been designed to deliver the highest level of precision and accuracy.

For high throughput nucleic acid extraction, automated extractors based on magnetic bead extraction techniques can be used. Four E’s provide two automated Extractors MultiEx 032 and MultiEx 096 which provides maximum extraction throughput of 32 and 96 samples respectively. The automated system integrates all the purification phases including binding, mixing, washing, and elution, and delivers high-efficiency, high-quality purification. The system works with any prefilled magnetic bead-based reagent kit.

qPCR set up

The extracted RNA now needs to be added to a PCR tube with the qPCR reagent mix. The PCR master mix will include Reverse Transcription mix, PCR Enzyme, Primers and Probes. PCR tubes and Pipettes are used in preparing the master mix. Take specimen number N+2 tubes of RT-PCR mix, and then add extracted test sample RNA, extracted positive control RNA, and Negative control. Close caps, mix thoroughly, and spin briefly with the Mini Centrifuge. Place the reaction tubes into the  qPCR instrument and operate as per the recommended qPCR protocol from the qPCR reagent supplier. Four E's offer a range of qPCR instruments with maximum throughput of 16, 48 & 96 samples per run.

Four more detailed information regarding the tools required for COVID-19 testing or any Molecular Biology application, please contact us.

Please Note: COVID-19 Nucleic Acid Amplification Tests using RT-PCR Test Kits are intended for use by qualified clinical laboratory personnel instructed and trained in the techniques of real-time PCR and in vitro diagnostic procedures.