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

Author:

Kuzan Aleksandra12ORCID,Tabakov Ivo13,Madej Lukasz14,Mucha Anna5ORCID,Fulawka Lukasz1ORCID

Affiliation:

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.

Funder

City of Wroclaw

Publisher

MDPI AG

Subject

General Biochemistry, Genetics and Molecular Biology,Medicine (miscellaneous)

Reference22 articles.

1. (2023, February 07). Weekly Epidemiological Update on COVID-19—1 February 2023, Available online: https://www.who.int/publications/m/item/weekly-epidemiological-update-on-covid-19---1-february-2023.

2. Fulawka, L., and Kuzan, A. (2021). COVID-19 Diagnostics Outside and Inside the National Health Service: A Single Institutional Experience. Diagnostics, 11.

3. Estimate false-negative RT-PCR rates for SARS-CoV-2. A systematic review and meta-analysis;Pecoraro;Eur. J. Clin. Investig.,2022

4. Technical and health governance aspects of the External Quality Assessment Scheme for the SARS-CoV-2 molecular tests: Institutional experience performed in all clinical laboratories of a Regional Health Service;Brunacci;Clin. Chem. Lab. Med.,2022

5. Fulawka, L., and Kuzan, A. (2022). Molecular Diagnostic Tools against SARS-CoV-2 in Poland in 2022. Biomedicines, 10.

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