TITLE: OnsiteGene 2 Protocol Saliva Direct
AUTHORS: yliu 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This OnsiteGene protocol is designed for testing the normal saliva samples without nucleic acid extraction. A convenient saliva swab is used to collect about 50 μL of saliva for the detection. 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. Along with the Star Array® SA</span><span style = "vertical-align:super;">TM </span><span>Direct Extract Buffer, the protocol supports direct amplification without the need of specific RNA extraction equipment and kit. It significantly reduces the loss of RNA from the sample extraction and purification process, saves time and workload from sample preparation, and minimized the burdens on supply chain. 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 11 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/salivadirect-rna-extraction-free-sars-cov-2-diagno-bkjgkujw" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.protocols.io/view/salivadirect-rna-extraction-free-sars-cov-2-diagno-bkjgkujw</span></a></div></div>

[STEPS]
?. [Sample Collection Procedure]
Saliva sample should be collected with the assistance of a healthcare worker or technician.
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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.
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Ensure all collection materials are labelled with the correct identifying information.
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Remove the lid of the collection tube containing 450 µL sample storage solution.
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Take the saliva swab out from the package and direct the sample donor to soak the swab pad in the mouth with saliva for 15 seconds.
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Place the soaked swab into the storage solution in the tube. Remove stick through the opening and leave swab pad in the tube. Discard stick and secure the lid.
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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.
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Transfer 20 µL supernatant of the sample storage solution to a tube containing 20 µL SATM direct RNA release buffer. Secure the lid of the tube.
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Heat the tube with the sample for 1 minute at 95°C on a heating block.
?. [Real-Time PCR Procedure]
Transfer 7.5 µL viral RNA sample solution (this sample can be stored at -20°C for 14 days) 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]
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.