TITLE: OnsiteGene 4 Protocol Saliva Extract
AUTHORS: yliu 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This OnsiteGene protocol is designed for testing the normal saliva samples with nucleic acid extraction. A normal saliva  collection tube is used for the collection. The protocol uses the Star Array® Hi-Sense</span><span style = "vertical-align:super;">TM</span><span> COVID-19 Molecular Testing Kit 1.0 in the one-step real-time RT-qPCR test assay to qualitatively detect RNA from SARS-CoV-2 virus in the human respiratory specimen. It combines the reverse transcription technology and real-time PCR method to provide accurate detection of the SARS-CoV-2 coronavirus. The Star Array® magnetic beads RNA extraction instrument and kit can automatically extract and purify the RNA for 32 samples in 6 minutes. It is ideal for the saliva samples that contain high amount of PCR inhibitors and can't be processed by the direct amplification.  The triplex fluorescence design of the kit simultaneously detects the N1 and Orf1ab genes of the virus and the human RNase P gene as an internal control to ensure the sample quality. This protocol uses the Star Array® XDive</span><span style = "vertical-align:super;">TM </span><span>Superfast Real-Time PCR instrument to perform 40-cycle PCR in 8 minutes, and can test up to 16 samples or controls in each run. The total protocol time from sample collection to data interpretation is less than 15 minutes.</span></div><div class = "text-block">The sample collection steps is adapted from the published protocol:</div><div class = "text-block"><a href="https://www.protocols.io/view/saliva-collection-and-rna-extraction-for-sars-cov-bh6mj9c6" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.protocols.io/view/saliva-collection-and-rna-extraction-for-sars-cov-bh6mj9c6</span></a><span style = ":;"> </span></div></div>

[STEPS]
?. [Sample Collection Procedure]
Saliva sample should be collected with the assistance of a healthcare worker or technician.
?. [Sample Collection Procedure]
Before collection, clean hands using alcohol-based sanitizer or soap and water (no fragrances) and wear appropriate PPE (at minimum, gloves and a mask).
?. [Sample Collection Procedure]
Back away as far as possible from the sample donor during the whole process. While preparing collection materials, direct the sample donor to begin pooling saliva in their mouth.
?. [Sample Collection Procedure]
This protocol is intended for the collection of the normal saliva that naturally pools into the mouth. No coughing or sniffing prior to sample collection is required. Ideally, water should be avoided 10 minutes prior to collection. Other drinks, food, and nasal sprays should be avoided half an hour before sample collection.
?. [Sample Collection Procedure]
Ensure all collection materials are labelled with the correct identifying information.
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Open the lid of the saliva collection tube, and direct the sample provider to gently expel saliva into the container until at least 500 μL is collected. Securely replace the lid of the collection tube.
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The total volume measured should exclude any bubbles.
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Add 500 μL of OnsiteGene storage media to each sample tube and secure the lid of each tube.
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Sterilize the tube surface with 70% ethanol or a disinfecting wipe, and place the sample in a secondary container or an appropriately labeled biohazard bag.
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Dispose of gloves, and register the sample collection information (including date and time).
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Transfer the sample at room temperature for sample processing. The virus RNA remains stable at room temperature for 3-5 days.
?. [Real-Time PCR Procedure]
Vortex each sample collection tube for 5 seconds at 3000-5000 RPM.
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This step can be substituted by tapping or shaking each sample collection tube to mix the sample.
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Spin down the tube for 15 seconds using centrifuge.
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The purpose is to: 1) Remove liquid from the cap to avoid the contamination when cap is removed; 2) Spin down solid particles to the bottom of the tube and allow supernatant to be acquired.
?. [Real-Time PCR Procedure]
Transfer 900 µL supernatant of the sample into the cartridge of magnetic beads RNA extraction instrument from OnsiteGene.
?. [Real-Time PCR Procedure]
Load the cartridge into the extraction instrument to extract and purify the viral RNA. The duration of the process is 6 minutes without any hand-on work. The sample elution volume is 50 µL after the extraction. The extraction instrument can process 32 samples in one run.
?. [Real-Time PCR Procedure]
Transfer 7.5 µL extracted RNA sample (this sample can be stored at -20°C for 14 days) from the cartridge and 7.5 µL OnsiteGene Hi-SenseTM COVID-19 detection kit reaction mix (with primers and probes) into a superfast RT-qPCR reaction tube. Secure the lid of the tube.
?. [Real-Time PCR Procedure]
Spin down the tube for 15 seconds using centrifuge or spinner.
?. [Real-Time PCR Procedure]
This step can be substituted by hand-shaking the tube to get the samples to the bottom.
?. [Real-Time PCR Procedure]
Load up to 16 superfast RT-qPCR reaction tubes onto the OnsiteGene XDive Superfast Real-Time PCT instrument and run the following conditions:  ABC1 Step  Temperature  Time 2 1  50°C  30 sec 3 2  95°C  0 sec 4 3  60°C  2 sec 5 4  Read Fluorescence intensity and overhead  (10 sec) 6 5  Repeat steps 2-4 for 40 cycles.
ABC1 Step  Temperature  Time 2 1  50°C  30 sec 3 2  95°C  0 sec 4 3  60°C  2 sec 5 4  Read Fluorescence intensity and overhead  (10 sec) 6 5  Repeat steps 2-4 for 40 cycles.
?. [Real-Time PCR Procedure]
15.Report results per the following interpretation criteria:  ABCDE1ResultsIC CTSARS-Cov-2 TargetsInterpretation2 ORF1ab CT  N1 CT 3Negative ≥ 40≥ 40 Indicate the absence of SARS-Cov-2 RNA 4 Positive  any value  any value *  Indicate the presence of SARS-Cov-2 RNA 5 any value *  ≤ 40 6 Invalid ≥ 35≥ 40≥ 40 Inability to conclusively determine presence or absence of SARS-Cov-2 RNA. This may be due to 1) Internal Control failure; or 2) failure to detect sufficient specimen volume. The sample needs to be retested.
ABCDE1ResultsIC CTSARS-Cov-2 TargetsInterpretation2 ORF1ab CT  N1 CT 3Negative ≥ 40≥ 40 Indicate the absence of SARS-Cov-2 RNA 4 Positive  any value  any value *  Indicate the presence of SARS-Cov-2 RNA 5 any value *  ≤ 40 6 Invalid ≥ 35≥ 40≥ 40 Inability to conclusively determine presence or absence of SARS-Cov-2 RNA. This may be due to 1) Internal Control failure; or 2) failure to detect sufficient specimen volume. The sample needs to be retested.