Nov 10, 2020
Version 2
OnsiteGene 4 Protocol Saliva Extract V.2
- yliu1
- 1OnsiteGene Inc.
- OnsiteGene 1
- XPRIZE Rapid Covid Testing
Protocol Citation: yliu 2020. OnsiteGene 4 Protocol Saliva Extract. protocols.io https://dx.doi.org/10.17504/protocols.io.bpjemkjeVersion created by yliu
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 10, 2020
Last Modified: November 10, 2020
Protocol Integer ID: 44358
Keywords: XDive, superfast PCR, superfast RT-qPCR, fast PCR,
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Abstract
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-SenseTM 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® XDiveTM 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.
The sample collection steps is adapted from the published protocol:
Materials
Instruments and Consumables
| Instrument and Consumable | Supplier | Catalog # | |
| Real-Time PCR | OnsiteGene | XDIVE-16 | |
| RNA extraction instrument | OnsiteGene | IS3000001 | |
| Mini centrifuge | Heathrow Scientific | HS120395 | |
| Vortex | Four E's Scientific | 4ESS-MI0101002D | |
| Reaction tube and cap | OnsiteGene | CM1010001 | |
| Pipette set | Microlit | RBOKit | |
| Pipette tips | Extragene | TS-10-RS |
Sample Colletion and Extraction Kit
| Reagent or Consumable | Supplier | Catalog # | |
| 1.5ml microcentrifuge tube | Biologix | 88-915X | |
| Purifier extraction kit | OnsiteGene | KT3000001 | |
| Purifier cartridge | OnsiteGene | CM3000001 | |
| Storage solution | OnsiteGene | RG0000001 |
PCR Reaction Reagents
| Reagent | Supplier | Catalog # | |
| Hi-Sense COVID detection kit | OnsiteGene | KT1010001 | |
| External positive control | ZeptoMetrix | NATSARS(COV2)-ERC |
Primer and Probe Sequences
| Target Number | Target Gene | Sequence/antibody | |
| 1_F | N1 | GACCCCAAAATCAGCGAAAT | |
| 1_R | N1 | TCTGGTTACTGCCAGTTGAATCTG | |
| 1_P | N1 | ACCCCGCATTACGTTTGGTGGACC | |
| 2_F | ORF1ab | TATGTGGAAAGGTTATGGCTGTAG | |
| 2_R | ORF1ab | GATTGTGCATCAGCTGACTGAAG | |
| 2_P | ORF1ab | TTGTGATCAACTCCGCGAACCCATG | |
| 3_F | Rnase P | AGATTTGGACCTGCGAGCG | |
| 3_R | Rnase P | GAGCGGCTGTCTCCACAAGT | |
| 3_P | Rnase P | TTCTGACCTGAAGGCTCTGCGCG |
Safety warnings
Most institutions will require samples potentially containing full-length SARS-CoV-2 RNA to be handled in a biosafety level 2 cabinet. Please seek guidance from your local biosafety office on specific recommendations for working with samples which could contain live SARS-CoV-2 virus.
Sample Collection Procedure
Sample Collection Procedure
1m
1m
Saliva sample should be collected with the assistance of a healthcare worker or technician.
Before collection, clean hands using alcohol-based sanitizer or soap and water (no fragrances) and wear appropriate PPE (at minimum, gloves and a mask).
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.
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.
Ensure all collection materials are labelled with the correct identifying information.
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.
1m
The total volume measured should exclude any bubbles.
Add 500 μL of OnsiteGene storage media to each sample tube and secure the lid of each tube.
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.
Dispose of gloves, and register the sample collection information (including date and time).
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
Real-Time PCR Procedure
9m 45s
9m 45s
Vortex each sample collection tube for 5 seconds at 3000-5000 RPM.
5s
This step can be substituted by tapping or shaking each sample collection tube to mix the sample.
Spin down the tube for 15 seconds using centrifuge.
15s
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.
Transfer 900 µL supernatant of the sample into the cartridge of magnetic beads RNA extraction instrument from OnsiteGene.
5s
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.
6m
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.
5s
Spin down the tube for 15 seconds using centrifuge or spinner.
15s
This step can be substituted by hand-shaking the tube to get the samples to the bottom.
Load up to 16 superfast RT-qPCR reaction tubes onto the OnsiteGene XDive Superfast Real-Time PCT instrument and run the following conditions:
| Step | Temperature | Time | |
| 1 | 50°C | 30 sec | |
| 2 | 95°C | 0 sec | |
| 3 | 60°C | 2 sec | |
| 4 | Read Fluorescence intensity and overhead | (10 sec) | |
| 5 | Repeat steps 2-4 for 40 cycles. |
a) XDive takes 8 minutes to complete 40 thermal cycles with fluorescence imaging.
b) Run external positive control and external negative control twice per day using 5 µL of positive control and no-template control (NTC - water). The control tests are recommended to be performed at the beginning and the end of the work/day.
8m
15.Report results per the following interpretation criteria:
| Results | IC CT | SARS-Cov-2 Targets | Interpretation | ||
| ORF1ab CT | N1 CT | ||||
| Negative | < 35 | ≥ 40 | ≥ 40 | Indicate the absence of SARS-Cov-2 RNA | |
| Positive | any value | < 40 | any value * | Indicate the presence of SARS-Cov-2 RNA | |
| any value * | ≤ 40 | ||||
| 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. | |
* In the case of one SARS-Cov-2 target positive (CT < 40) and one SARS-Cov-2 target negative (CT ≥ 40), the result is suggestive of: 1) a sample at concentrations near or below the limit of detection of the test, 2) a mutation in one of the target regions, or 3) other factors.