TITLE: One-step RT-PCR Ins214EPE assay for Omicron (B.1.1.529) variant detection 
AUTHORS: Nikita Yolshin  , Kirill  Varchenko, Kseniya  Komissarova, Daria  Danilenko, Andrey  Komissarov, Dmitry   Lioznov

[DESCRIPTION]
On 26 November 2021 WHO designated a new variant of concern B.1.1.529 named Omicron. This variant has a large number of mutations, some of which are concerning. Preliminary evidence suggests an increased risk of reinfection with this variant and reduced neutralization by convalescent and vaccinated sera, as compared to other VOCs. Implementation of the high-throughput rRT-PCR screening for Omicron is of great importance for monitoring the spread of this VOC in the population, especially in resource-limited countries lacking sufficient sequencing capacity.   

Omicron lineage B.1.1.529 (BA.1) has some indels that turned out to be a good target for its detection. In the current protocol, we use ins214EPE for this purpose. Here we describe the 1-step quantitative multiplex RT-qPCR assay consisting of the newly developed Ins214EPE detection set and widely used Hong Kong University N gene assay for SARS-CoV-2 detection (Chu et al., 2020).

  
The assay was validated on the Omicron variant RNA kindly provided by the Pathogenic Microorganisms Variability Laboratory (Dr. Vladimir Guschin, Gamaleya Institute, Moscow, Russia) and RNA from the collection of Smorodintsev Research Institute of Influenza. 


 

Omicron RNA specimens were positive in the assay as expected. Negative controls were found negative. 10-fold serial dilutions of Omicron RNA were used to assess ins214EPE assay amplification efficiency. The amplification efficiency was 98,9% (R2 = 0,99).

   
  

The developed rRT-PCR assay demonstrates high specificity. It was tested on 26 clinical samples (RNA extracted from oropharyngeal swabs) with previously characterized viruses belonging to 8 different SARS-CoV-2 lineages (including Delta B.1.617.2+AY.*) Specific signal was detected only in samples with SARS-CoV-2 Omicron lineage RNA (confirmed by whole-genome sequencing). Specificity was additionally tested on clinical samples positive for other respiratory viruses from the collection of Smorodintsev Research Institute of Influenza - influenza, parainfluenza, human seasonal coronaviruses (OC43, NL63, 229E, HKU1), hRSV, rhinoviruses, bocaviruses, metapneumovirus (33 in total) - with no false-positive results.

 
              Ins214EPE Cq    6x  B.1.1.7       2x  B.1.351       5x  AT.1       6x  B.1.617.2       4x  AY.122          P.1          B.1.1.529  28,72       B.1.1.529  26,29  

 
 
     virus      RP   Cq      SARS   Cq      Ins214   Cq        RSV   A      28,76                      RSV   A      30,56                      RSV   A      27,70                      RSV   B      31,49                      RSV   B      30,98                      RSV   B      32,33                      NL63      32,20                      NL63      30,42                      NL63      24,95                      Oc43      30,34                      Oc43      30,69                      Oc43      28,64                      HKU1      30,06                      HKU1      28,30                      HKU1      30,73                      229E      29,11                      229E      32,52                      229E      29,37                      BoV      32,26                      BoV      30,75                      BoV      27,25                      Rv      32,85                      Rv      33,76                      Rv      27,75                      Piv1      28,63                      Piv2      24,72                      Piv3      27,01                      Piv4      23,90                      Adv      29,47                      MPV      30,12                      HIV   A      29,13                      HIV   A      28,45                      HIV   A      28,16      39,06               c+      34,15      26,61      28,44   

 
 

Analytical sensitivity determination is underway.

We consider developed assay to be useful in wide populational RT-PCR screening to assess the spread of Omicron variant.



[STEPS]
1. Order oligonucleotides with following sequences 5'->3':
 
     Ins214EPE F      ATATTCTAAGCACACGCCTATT        Ins214EPE P1      ROX-AGCCAGAAGATCTCCCTCAGGGTT-BHQ        Ins214EPE P2      ROX-TGCGTGAGCCAGAAGATCTCCCT-BHQ        Ins214EPE R      GGCAAATCTACCAATGGTTCTA   
 
2. Briefly vortex and centrifuge reagents before use.
Prepare the PCR reaction mixture following the specifications below:

 
          2X RT-PCR Buffer      12.5   μl              25X RT-PCR Enzyme Mix      1 μl              Ins214EPE F      400 nM        Ins214EPE P    400 nM        Ins214EPE R      400 nM             RNA template      5 μl              Nuclease-free water      to 25 μl       
 

 

3. Perform the amplification in a qPCR thermocycler with appropriate temperature profile:


  

4. How to interpret the results: detection of fluorescence of FAM probe (HKU-NP) means thepresence of SARS-CoV-2 RNA in the sample, detection of fluorescence in ROX channel (Ins214EPE probe) means the presence of B.1.1.529 (BA.1)(omicron) RNA.