qPCR tests for SARS-CoV-2 RNA

 

During the acute phase of the Covid-19 disease, the existence of virus-genome can be proven by PCR, one of the most sensitive methods for the detection of nucleic acids.

Our assays are based on published and thoroughly tested PCR primers and probes, both by the developing institutes and by us.  The assays can be performed on any standard qCPR machine with channels for FAM, JOE and ROX. We could show that the broadly used e-gene assay can also detect nucleic acids that are not originating from viruses and have therefore replaced this assay with a new one, that was developed by the University of Frankfurt (Toptan, Widera et al.2020).

All assays have been tested with standard Viral Transfer Media (PBS). However, for highest yield and more safety and sensitivity we strongly recommend to use the GenXPro ReLy buffer for direct lysis of the swab-content

 

Triplex Assay:

The Triplex assay targets two highly specific regions on the SARS-CoV-2 genome and a human RNA specific target.

The human RNA target proofs if all steps of the process have worked, from swab-RNA extraction via reverse transcription to the qPCR.

The test works especially well with GenXPros ReLy buffer for direct lysis of the buccal swab-content.

Requirements: qPCR machine with FAM, JOE and ROX channel.

The CoV-2 specific assay was designed by the virology department of the J.W.Goethe University of Frankfurt am Main (Toptan, Widera et al. 2020).

 

Duplex Assay:

The duplex assay targets one highly specific region on the SARS-CoV-2 genome and a human RNA specific target in a single assay.

The human RNA target proofs if all steps of the process have worked, from swab-RNA extraction via reverse transcription to the qPCR.

The test works especially well with GenXPros ReLy buffer for direct lysis of the buccal swab-content.

Requirements: qPCR machine with FAM, and JOE channel.

The CoV-2 specific assay was designed by the virology department of the J.W.Goethe University of Frankfurt am Main (Toptan, Widera et al. 2020).

 

Single Plex Assay

 

The test is performed optionally in two steps using the highly sensitive M-gene assay followed optionally by second gene assay. Each is performed in a separate qPCR tube.

In addition we offer a human RNA target assay to proof if all steps of the process have worked, from swab-RNA extraction via reverse transcription to the qPCR.

The test works especially well with GenXPros ReLy buffer for direct lysis of the buccal swab-content.

Requirements: qPCR machine with FAM channel

 

 

Single Plex WHO Assay

 

This single-plex assay corresponds to WHO assay as designed by the Charité / Drosten.

The test is performed in two steps using a highly sensitive E-gene assay followed by a more specific ORF-Gene assay. Each is performed in a separate qPCR tube.

In addition we offer a human RNA target assay to proof if all steps of the process have worked, from swab-RNA extraction via reverse transcription to the qPCR.

The test works especially well with GenXPros ReLy buffer for direct lysis of the buccal swab-content.

Requirements: qPCR machine with FAM channel

The CoV-2 specific assay was designed by the Charité, Berlin (Drosten et al. 2020).

 

Sensitivity:

 

All tests detected the presence of approximately 10 RNA molecules per reaction*

 

Specificity

 

Problems of the WHO E-gene assay and false positives

 

The E-gene specific primers of the WHO assay have been reported to generate up to 10% false positives.

We sequenced a PCR product of a false positive sample and found a 70 bps long region matching to on Neisseira subflavia strains (see BLAST results of the amplicon below), a common nasopharyngal bacterium.

 

Our Multiplex Assays

 

Therefore, we adapted the M-gene assay (Toptan and Widera et al. 2020) for high specificity and potentially lower rates of false positives.

Samples that generated false positive results using the E-primers of the WHO/Charité assay were correctly found to be negative using our multiplex assays. The specificity was prior described by Toptan and Widera et al. 2020 and proven with samples that showed false positive results using E-Primers in our lab. We cannot exclude that our assay leads to false positives but this was not the case with samples that led to false positives with the E-gene (WHO) assay.

 

*testet on  ABI-StepOne in 25 µl reactions.

1For Research use only