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.

Human ELISA Detects anti-SARS-CoV-2 Antibodies in Cats: Seroprevalence and Risk Factors for Virus Spread in Domestic and Stray Cats in Bulgaria

“…Unlike these reports, we observed a positive seroprevalence among stray cats. Cats with active infection can emit the virus [ 2 , 5 , 7 , 10 , 11 ]. In addition, another study detected the virus in stray dogs from the Amazon rainforest [ 36 ].…”

“…RNA was extracted from the ELISA-negative serum samples (to detect pre-symptomatic cats) using an ISOLATE II RNA Mini kit (Bioline, Meridian Bioscience, Memphis, TN, USA) according to the manufacturer’s instructions. RT-PCR was run as described by [ 7 ].…”

“…The symptoms in cats are similar to other diseases caused by Feline herpesvirus (FHV), Chlamydophila felis , Feline calicivirus (FCV) and Mycoplasma felis . Therefore, it is recommended to include SARS-CoV-2 in the panel for differential diagnosis [ 7 ].…”

A Comparative Analysis of Molecular Biological Methods for the Detection of SARS-CoV-2 and Testing the In Vitro Infectivity of the Virus

“…We also applied the method in one reaction as a conventional One-Step RT PCR in the manner described above [ 51 ], but using only the inner primers and reducing the elongation temperature from 40 to 30 s, due to the smaller fragment size expected with these primers.…”

“…Our previous study validated and applied nested (n) One-Step RT PCR for SARS-CoV-2 detection in swab samples [ 51 ] and sera from cats [ 52 ]. The assay showed high sensitivity, as it was able to detect low concentrations of virus RNA.…”