What to Do if the qPCR Test for SARS-CoV-2 or Other Pathogen Lacks Endogenous Internal Control? A Simple Test on Housekeeping Genes

Aleksandra Kuzan; Ivo Tabakov; Lukasz Madej; Anna Mucha; Lukasz Fulawka

doi:10.3390/biomedicines11051337

What to Do if the qPCR Test for SARS-CoV-2 or Other Pathogen Lacks Endogenous Internal Control? A Simple Test on Housekeeping Genes

Biomedicines. 2023 May 1;11(5):1337. doi: 10.3390/biomedicines11051337.

Authors

Aleksandra Kuzan^{1

2}, Ivo Tabakov^{1

3}, Lukasz Madej^{1

4}, Anna Mucha⁵, Lukasz Fulawka¹

Affiliations

¹ Molecular Pathology Centre Cellgen, 50-353 Wroclaw, Poland.
² Department of Biochemistry and Immunochemistry, Wroclaw Medical University, 50-367 Wroclaw, Poland.
³ Faculty of Biotechnology, University of Wroclaw, Joliot-Curie 14a, 50-383 Wroclaw, Poland.
⁴ Collegium Medicum, Jan Kochanowski University, 25-516 Kielce, Poland.
⁵ Department of Genetics, Wroclaw University of Environmental and Life Sciences, 51-631 Wroclaw, Poland.

Abstract

Some of the products for the molecular diagnosis of infections do not have an endogenous internal control, and this is necessary to ensure that the result is not a false negative. The aim of the project was to design a simple low-cost RT-qPCR test that can confirm the expression of basic metabolism proteins, thus confirming the quality of genetic material for molecular diagnostic tests. Two successful equivalent qPCR assays for the detection of the GADPH and ACTB genes were obtained. The course of standard curves is logarithmic, with a very high correlation coefficient R2 within the range of 0.9955-0.9956. The reaction yield was between 85.5 and 109.7%, and the detection limit (LOD) with 95% positive probability was estimated at 0.0057 ng/µL for GAPDH and 0.0036 ng/µL for ACTB. These tests are universal because they function on various types of samples (swabs, cytology, etc.) and can complement the diagnosis of SARS-CoV-2 and other pathogens, as well as potentially oncological diagnostics.

Keywords: COVID-19; PCR; SARS-CoV-2; diagnostics; false-negative results.

Grants and funding

The work has been prepared as a part of the Support Programme of the Partnership between Higher Education and Science and Business Activity Sector “Mozart” financed by the City of Wroclaw.