Validation of conventional PCR-like alternative to SARS-CoV-2 detection with target nucleocapsid protein gene in naso-oropharyngeal samples

Abstract

SARS-CoV-2 has spread worldwide and has become a global health problem. As a result, the demand for inputs for diagnostic tests rose dramatically, as did the cost. Countries with inadequate infrastructure experience difficulties in expanding their qPCR testing capacity. Therefore, the development of sensitive and specific alternative methods is essential. This study aimed to develop, standardize, optimize, and validate conventional RT-PCR targeting the N gene of SARS-CoV-2 in naso-oropharyngeal swab samples compared to qPCR. Using bioinformatics tools, specific primers were determined, with a product expected to be 519 bp. The reaction conditions were optimized using a commercial positive control, and the detection limit was determined to be 100 fragments. To validate conventional RT-PCR, we determined a representative sampling of 346 samples from patients with suspected infection whose diagnosis was made in parallel with qPCR. A sensitivity of 92.1% and specificity of 100% were verified, with an accuracy of 95.66% and correlation coefficient of 0.913. Under current Brazilian conditions, this method generates approximately 60% savings compared to qPCR costs. Conventional RT-PCR, validated herein, showed sufficient results for the detection of SARS-CoV-2 and can be used as an alternative for epidemiological studies and interspecies correlations. China was identified as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [1]. Since then, SARS-CoV-2 has spread worldwide and has become a global health problem, causing coronavirus disease 2019 (COVID-19). As of August 7, 2021, more than 562,000 people have died in Brazil due to complications of COVID-19, being the country with the second highest number of deaths. Worldwide, the 20 countries with the highest death counts have collectively more than 3 million deaths [2]. SARS-CoV-2 is a simple positive-sense enveloped RNA virus that encodes the spike (S), envelope (E), membrane (M), and nucleocapsid (N) as structural proteins [1]. Coronavirus nucleocapsid protein has a structural role as it forms a ribonucleoprotein complex with gRNA and also has RNA chaperone activity [3]. This protein also has an important antigenic action in the formation of specific antibodies in natural infection [4]. The World Health Organization (WHO) recommends the use of real-time PCR (qPCR) for the diagnosis of SARS-CoV-2, using different protocols aimed at detecting genes of non-structural and structural proteins, especially the N gene [5]. However, qPCR inputs and equipment are notoriously scarce with the increase in worldwide demand, especially in countries dependent on imports of these resources [6, 7], such as Brazil. This difficulty in accessing diagnostic resources leads to the underreporting of cases [8]. Countries that implemented greater diagnostic coverage in the monitoring of their populations were able to control the dispersion of the disease more efficiently [9]. Some alternatives to qPCR have been proposed by different researchers and may present sufficient performance to increase the supply of diagnostic resources for SARS-CoV-2 in countries with inadequate infrastructure [7], or for studies of interspecies epidemiological correlations that are yet unclear. Conventional PCR preceded by reverse transcription may be one such alternative [7]. Therefore, the present study aimed to develop, standardize, optimize, and validate a conventional RT-PCR method targeting the N gene of SARS-CoV-2 in nasopharyngeal and oropharyngeal swab samples. And in the end, the conventional RT-PCR for the diagnosis of SARS-CoV-2 proved to be an alternative tool for expanding population testing, with significant sensitivity and specificity.