Development of Nested PCR for SARS-CoV-2 Detection and Its Application for Diagnosis of Active Infection in Cats

Abstract

SARS-CoV-2 emerged in 2019 and found diagnostic laboratories unprepared worldwide. To meet the need for timely and accurate virus detection, laboratories used rapid Ag tests and PCR kits based on costly multi-channel real-time techniques. This study aimed to develop a conventional nested PCR based on the SARS-CoV-2 N gene, validate it against some approved assays, and apply it to samples from six cats with respiratory symptoms obtained in early 2020 during the first COVID-19 wave in humans in Bulgaria. The nested PCR technique showed 100% sensitivity and specificity; it could detect extracted SARS-CoV-2 RNA at concentrations as low as 0.015 ng/μL. The results identified the six tested cat samples as positive. Sequence analysis performed in two of them confirmed this. The presented technique is reliable, easy to implement and inexpensive, and can be successful in strategies for the prevention and control of SARS-CoV-2 in humans, cats and other susceptible species.

Keywords: SARS-CoV-2; cats; humans; nested PCR; validation.

Figure 1

Nested PCR amplification products separated via electrophoresis in agarose gel. (a) Specificity of nested PCR assay determined using six cDNA samples from cats. 1—DNA Ladder 1 kb (Bioline, Meridian Bioscience, Memphis, TN, USA); 2–7—samples from cats with respiratory symptoms; 8—negative control. Determination of the specificity of nested PCR assay. 9—FCoV positive sample; 10—FCV positive; 11—Chlamydia and Mycoplasma spp. positive; 12—FHV positive; 13—IVD real-time RT-PCR SARS-CoV-2 negative human sample; 14—IVD LAMP SARS-CoV-2 negative human sample; 15—IVD real-time RT-PCR SARS-CoV-2 positive human sample; 16—IVD LAMP SARS-CoV-2 positive human sample. Bottom line I: external primers, expected size of product 335 bp and; top line II: internal primers, expected size of products 212 bp. (b) Sensitivity of nested PCR assay determined using different quantities of extracted RNA from inactivated SARS-CoV-2 USA/WA1/2020 isolate. 1—DNA Ladder 1 kb; top line, external primers: 2—45.6 ng (final concentration in the reaction mixture 2.28 ng/μL), 3—11.4 ng (0.57 ng/μL), 4—5.7 ng (0.285 ng/μL), 5—3.7 ng (0.185 ng/μL), 6—1.3 ng (0.065 ng/μL), 7—0.8 ng (0.04 ng/μL), 8—0.3 ng (0.015 ng/μL), 9—negative control of external primers. Bottom line internal primers, 2—external primers, 9—first negative control (from the first reaction) primers; 10—negative control of internal primers.

Figure 2

Results of six samples from cats by using real-time RT-PCR 1−positive control; 2−7−samples from cats with respiratory symptoms; 8−negative control.