RT–PCR protocol for the detection ORF1 11288–11296 deletion (NSP6 106-108del) in SARS-CoV-2 genome
Nikita Yolshin, Artem Fadeev, Andrey Komissarov
Abstract
ORF1 deletion is observed in some new SARS-CoV-2 lineages and is lineage defining for B.1.1.7 and P.1. Biological effect of this mutation is still unknown. Developed RT-PCR was tested on numerous samples of lineages B.1.1.7, B.1.351, P.1 (validated by sequencing) and samples without this mutation with no false-postive results.
Steps
Order oligonucleotides with following sequences:
| A | B |
|---|---|
| Name | Sequence (5’ –> 3’) |
| ORF1-del-F | GGTTGGATATGGTTGATACTAGTTTGAAG |
| ORF1-del-R | TGTCAAGACATTCATAAGTGTCCACA |
| ORF1-del-P | Cy5.5-ACTGTGTTATGTATGCATCAGCTGTAGTGTTACTAATCC-BHQ3 |
Briefly vortex and centrifuge reagents before use.Prepare the PCR reaction mixture following the specifications below: | A | B | | --- | --- | | 2X RT-PCR Buffer | 12.5 μl | | 25X RT-PCR Enzyme Mix | 1 μl | | ORF1-del primers | 600 nM each | | ORF1-del probe | 400 nM | | RNA template | 5 μl | | Nuclease-free water | to 25 μl |
AgPath-ID™ One-Step RT-PCR Reagents was used in our case Use SARS-CoV-2 positive samples as the template. In the case of screening of unknown samples use multiplex RT-PCR with some oligonucleotides for detection SARS-CoV-2 as a control reaction. This primer set for SARS-CoV-2 detection can be added to the test without decrease of sensitivity of the ORF1 deletion test:
| A | B |
|---|---|
| Name | Sequence (5’ – 3’) |
| 2-SARS-CoV-2-ORF-1-F | AGAGCTATGAATTGCAGAC |
| 2-SARS-CoV-2-ORF-1-R | GGGAAATACAAAATTTGGACA |
| 2-SARS-CoV-2-ORF-1-P | FAM-AATTGGCAAAGAAATTTGACACCTTCA-BHQ-1 |
Perform the amplification in a general thermocycler with appropriate temperature profile:
Interprete the results: detection of fluorescence probe “ORF1-del-P” (Cy5 channel in our case) means presence of ORF deletion 11288–11296
