PB2281: HOW TO AVOID FALSE-NEGATIVE AND FALSE-POSITIVE COVID-19 PCR TESTING

Abstract

Background: Since early 2020 the most demanded molecular genetic test is real-time reverse transcription polymerase chain reaction (RT-PCR) for SARS-CoV-2. However, up to 40% of test results for COVID-19 in the presence of clinical manifestations of the disease might be negative. A false-negative analysis means untimely treatment of the patient and the possible spread of infection from a non-isolated person. Despite the fact that there are many highly sensitive RT-PCR test systems for COVID-19 on the market, the search for new reliable approaches to increase the robustness of testing is still relevant. Aims: To develop a PCR testing approach featured to minimize the risk of the false-negative results and to increase assay sensitivity and specificity by the use of multiple targets. Methods: A cotton swab taken from the nasopharynx was placed in an Eppendorf tube with 300 μl of phosphate-buffered saline. The resulting suspension (100 μl) was taken for RNA isolation using the Riboprep Amplisens kit (InterlabService, Russia). Primer sequences for RT-PCR are shown in Table 1. Results: The reason for a false-negative result might originate from any stage of the analysis: poor-quality or empty swab, poor RNA isolation, inactivation of reverse transcriptase or Taq polymerase in the test. To control the entire process usually control DNA or an RNA-containing virus (eg bacteriophage MS-2) is added to the sample before RNA isolation. This is sufficient to control RNA isolation, reverse transcription, and PCR amplification. However, improper swab application, handling, transporting, or even attempts to falsify the sample, cannot be excluded this way. Here we propose to include primers for the detection of human ABL1 gene RNA to control swab quality and integrity. Designed primers work with the cDNA of the ABL1 gene, not genomic DNA. Therefore achieved ABL1 gene amplification ensures that enough material containing host RNA was taken by the swab, RNA was nicely isolated, reverse transcribed, and PCR-amplified. For SARS-CoV-2 detection, we propose to use primers targeted to the conserved region of the nucleocapsid gene and the less conserved region of the spike gene (currently specific for the Omicron strain). The simultaneous appearance of three signals corresponding to the nucleocapsid, spike, and ABL1 gene indicates infection with the Omicron strain. The amplification of ABL1 gene and nucleocapsid only indicate other than Omicron infection. The appearance of ABL1 amplification only indicates a true negative result for SARS-CoV-2. All other variants are null and void. Image:Summary/Conclusion: A system has been developed for multiplex PCR diagnostics of SARS-CoV-2, which makes it possible to eliminate errors leading to false-negative and false-positive results at all stages of analysis. This is accomplished by the presence of specific primers for human RNA, controlling proper swab application, handling, and all the stages of RT-PCR. Targeting diverse regions of SARS-CoV-2 increases the reliability of the results and makes it possible to identify the virus strain.