SARS-CoV-2 RT-PCR – Cytecs – Expertise of 50 Years in Flow Cytometry

General information:

The SARS-CoV-2 RT-PCR is for in vitro or research use only. This test detects gene sequences of the SARS-CoV-2 genome (NCBI reference: MN908947.3) via Nucleic Acid Amplification Technology (NAAT). It is a 2-step lyophilized RT-PCR. The used primer system is based on the publication of Corman et al. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR from Berlin University Hospital Charité (Drosten group) and Chu et al. Molecular Diagnosis of a Novel Coronavirus (2019-nCoV) Causing an Outbreak of Pneumonia from Hongkong University (Peiris group).

The Cytecs SARS-CoV-2 RT-PCR detects E gene and N gene in a combined assay and ORF1b-nsp14 in a single assay. Test results will be analysed by the means of gel-electrophoresis.

Sensitivity and Specificity:

Detection of beta coronavirus SARS-CoV-2 genes E gene, N gene and ORF1b-nsp14.

Diagnostical sensitivity: 95,45 %

Diagnostical specificity: 98,46 %

Analytical sensitivity:

E N assay: 62.5 copies/ reaction

ORF1b-nsp14 assay: 62.5 copies/ reaction

Analytical sensitivity:

The in silico investigation via BLAST analysis showed no cross reactivity with other species.

An experimental investigation was performed in the publications from Croman et al. and Chu et al.:

HCoV-NL63^1,2

HCoV-229E^1,2

HCoV-OC43^1,2

HCoV-HKU1^1,2

MERS-CoV^1,2

CCoV-HKU23²

Influenza A (H1N1)²

Influenza A (H1N1/09)¹

Influenza A (H3N2)²

Influenza A (H5N1)^1,2

Influenza A (H7N9)²

Influenza B¹

Avian-Influenza (H1)²

Avian-Influenza (H4)²

Avian-Influenza (H6)²

Avian-Influenza (H9)²

Rhinovirus1,2/ Enterovirus^1,2

Respiratory syncytial virus (A/ B)^1,2

Parainfluenza 1 virus¹

Parainfluenza 2 virus¹

Parainfluenza 3 virus^1,2

Parainfluenza A or B virus¹

Human metapneumovirus^1,2

Adenovirus^1,2

Human Bocavirus^1,2

Legionella spp.¹

Mycoplasma spp.¹

¹Corman et al.

²Chu et al.

SARS-CoV-1 does not circulate in the world population – last time in the year 2003 (Wilder-Smith et al., 2020). A cross reactivity with SARS-CoV-1 is nearly impossible.

Procedure:

cDNA synthesis:

The procedure of SARS-CoV-2 RT-PCR starts with unpacking and marking of the cDNA tubes. Rehydrate the pellet with 15 µl of PCR-grade water (included in scope of delivery). Mix gently by pipetting. Incubate the vial for 2 min, ad 5 µl RNA and mix gently.

PCR:

Unpack and mark the PCR-tubes for E N and ORF1b-nsp14 assay and rehydrate the pellet with 23 µl of PCR-grade water (included in scope of delivery). Mix gently by pipetting. Incubate the vials for 2 min, ad 2 µl cDNA in each assay and mix gently.

PCR products will be analysed by the means of gel-electrophoresis. The valuation will be described in the next chapter. Figure 2 shows the possible results in an agarose-gel.

Valuation of SARS-CoV-2 RT-PCR test:

For analysis the PCR products has to be separated in an agarose-gel. The expected result is shown in figure 1.
E N shows a band on the hight of 110 bp and the ORF1b-nsp14 assay shows a band on the hight of 130 bp.

Valuation:

The valuation, if the test is positive or negative will be done corresponding to the band distribution in the agarose-gel. The possible valuations of the SARS-CoV-2 RT-PCR test is shown in figure 2.

Test limitation:

The Cytecs SARS-CoV-2 RT-PCR test belongs to the NAAT tests. This technique could be influenced negatively by the following parameters:

RNA is instable if not handled with care.
Specimen collection must be performed well. The SARS-CoV-2 RT-PCR was evaluated only with naso- or oropharyngeal swabs. Other specimens are not allowed as sample material.
Swabs should be suitable for naso- or oropharyngeal specimen collection.
Sample must be transferred to the laboratory immediately.
Extreme conditions during transport like to high temperatures must be excluded.
Working in accordance with Good Laboratory Practice conformity is necessary.
The SARS-CoV-2 genome can mutate (Forster et al., 2020). This can occur in primer mismatch.
E gene exists also in SARS-CoV-1. SARS-CoV-1 does not circulate in the world population. A confusion is not possible.
The viral load differs during the infection development. False negative results are seen at the beginning and at the end of a SARS-CoV-2 infection. (Kucrika et al., 2020). Test should be repeated if the results are in contrast with the patient’s clinical parameters.
False positive results may happen from cross contaminations due to bad sample or product handling.
Possible causes of false negative results:

Incorrect sample collection
Extreme transport conditions
RNA degradation
Mutation of primer binding site
RT-PCR inhibitors (e.g. Haemoglobin)
Failure to follow user manual

NAAT test results should not be the sole basis of decisions in patient management.

Analyzing PCR products with the gel-electrophoresis system E-CUBE

Storage:

The ready to use reaction tubes should be stored at room temperature. Avoid freezing, high temperatures and high humidity.

Literature:

M. Corman, O. Landt, M. Kaiser et al., Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR, Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin, vol. 25, no. 3, 2020.

K. W. Chu, Y. Pan, S. M. S. Cheng et al., Molecular Diagnosis of a Novel Coronavirus (2019-nCoV) Causing an Outbreak of Pneumonia, Clinical chemistry, vol. 66, no. 4, pp. 549–555, 2020.

Wilder-Smith A., Chiew C. J., Lee V.J., Can we contain the COVID-19 outbreak with the same measures as for SARS?, Lancet Infect Dis 2020; 20: e102–07, doi: https://doi.org/10.1016/ S1473-3099(20)30129-8.

Forster, L. Forster, C. Renfrew, et al., Phylogenetic network analysis of SARS-CoV-2 genomes, Proceedings of the National Academy of Sciences of the United States of America, vol. 117, no. 17, pp 9241-9243, 2020.

L. M. Kucirka, S. A. Lauer,O. Laeyendecker et al., Variation in False-Negative Rate of Reverse Transcriptase Polymerase Chain Reaction-Based SARS-CoV-2 Tests by Time Since Exposure, Annals of internal medicine, DOI: 10.7326/M20-1495, 2020.