Establishment of a new method for rapid detection of SARS-CoV-2 by visual LAMP (without nucleic acid extraction)

A new type of coronavirus (SARS-CoV-2) infection caused acute or fatal pneumonia. The virus is another coronavirus that is transmitted from animal to human and capable of transmitted from human to human, following the severe acute respiratory syndrome coronavirus (SARS-CoV) and the Middle East respiratory syndrome coronavirus (MERS CoV). In order to control the epidemic as soon as possible, there is an urgent need, for rapid detection and confirmation of infected patients. In this study, according to the SARS CoV-2 whole genome published in GenBank as target gene, LAMP Desiner 2.0 software was used to screen efficient and highly specific combinatorial loop primers. The amplification characteristics of Bst 4.0 DNA polymerase relys RNA as template for DNA synthesis. Viral RNA-positive test results showed that 5 to 20 copies of virus nucleic acid could be detected. The inactivated virus was directly used as amplification template for clinical detection. The amplified nucleic acid molecules are combined with OG (Orange-Green) dye. Positive samples are green and negative samples are orange yellow.. The established SARS-CoV-2 one-step visual constant temperature rapid detection method realizes rapid detection of nucleic acids with high sensitivity. This study provides a new method for SARS-CoV-2 detection.

Epidemiological monitoring to ascertain if SARS-CoV-2 infects dogs and cats

2019 novel coronavirus (SARS-CoV-2) is a new strain of coronavirus that has never been found in humans. SARS-CoV-2 is a beta coronavirus. whereas the coronaviruses infecting pet dogs and cats arise mainly from a-coronaviruses. Whether SARS-CoV-2 infects cats, dogs and other pets is an important public-health issue during this time. In the present study, respiratory-tract symptoms in 20 pet cats and 4 pet dogs (especially with obvious fever and cough symptoms) in Beijing, China, were detected by fluorescence quantitative polymerase chain reaction (PCR) of SARS-CoV-2 and established diagnostic methods. Throat swabs were collected to detect the nucleic acids of SARS-CoV-2 using fluorescence quantitative PCR and to detect other pathogens. The nucleic acids of SARS-CoV-2 were not present in the 24 pets that we evaluated.

Development of a TaqMan loop-mediated isothermal amplification assay for the rapid detection of pigeon paramyxovirus type 1.

Pigeon paramyxovirus-1 (PPMV-1) is a strain of Newcastle disease virus (NDV) that has adapted to infect pigeons and poses a constant threat to the commercial poultry industry. Early detection via rapid and sensitive methods, along with timely preventative and mitigating actions, is important for reducing the spread of PPMV-1. Here, we report the development of a TaqMan loop-mediated isothermal amplification assay (TaqMan-LAMP) for rapid and specific detection of PPMV-1 based on the F gene. This system makes use of six novel primers and a TaqMan probe that targets nine distinct regions of the F gene that are highly conserved among PPMV-1 isolates. The results showed that the limit of detection was 10 copies µL-1 for PPMV-1 cDNA and 0.1 ng for PPMV-1 RNA. The reaction was completed within 25 min and was thus faster than conventional RT-PCR. Moreover, no cross-reactions with similar viruses or with peste des petits ruminants virus (PPRV) or NDV LaSota vaccine strains were observed under the same conditions. To evaluate the applicability of the assay, the TaqMan-LAMP assay and a commercial RT-PCR assay were compared using 108 clinical samples, and the concordance rate between two methods was found to be 96.3%. The newly developed PPMV-1 TaqMan-LAMP assay can therefore be used for simple, efficient, rapid, specific, and sensitive diagnosis of PPMV-1 infections.

Establishment of an indirect ELISA-based method involving the use of a multiepitope recombinant S protein to detect antibodies against canine coronavirus.

Here, we report the development of an indirect enzyme-linked immunosorbent assay (ELISA) method that involves using multiepitope recombinant S protein (rSP) as the coating antigen to detect antibodies against canine coronavirus (CCoV). rSP was designed by arranging its four S fragments (91-135 aa, S1 gene; 377-434 aa, S2 gene; 647-671 aa, S3 gene; 951-971 aa, S4 gene; 207-227 aa) and two T-cell epitopes in tandem: T-E1-E2-E3-E4-T. This multiepitope antigen, which has a molecular weight of approximately 25 kDa and contains a His-tag, was recognized by a CCoV-positive serum in a Western blot assay. The optimal concentration of rSP as a coating antigen in the ELISA was 2 µg/mL, and the optimal dilution of enzyme-labeled secondary antibody was 1:10,000. The cutoff OD450 value was established at 0.2395. No reactivity was observed with antisera against canine distemper virus, canine parvovirus, or feline calicivirus, indicating that this assay is highly specific. We also tested 64 clinical serum samples using our newly established method, and the positive rate was found to be 82.8%. In conclusion, our assay was found to be highly sensitive and specific for the detection of antibodies against CCoV, and it can therefore serve as a new, efficient diagnostic method.

Animal coronaviruses and SARS-CoV-2.

COVID-19 is a highly contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It has rapidly spread to 216 countries and territories since first outbreak in December of 2019, posing a substantial economic losses and extraordinary threats to the public health worldwide. Although bats have been suggested as the natural host of SARS-CoV-2, transmission chains of this virus, role of animals during cross-species transmission, and future concerns remain unclear. Diverse animal coronaviruses have extensively been studied since the discovery of avian coronavirus in 1930s. The current article comprehensively reviews and discusses the current understanding about animal coronaviruses and SARS-CoV-2 for their emergence, transmission, zoonotic potential, alteration of tissue/host tropism, evolution, status of vaccines and surveillance. This study aims at providing guidance for control of COVID-19 and preventative strategies for possible future outbreaks of zoonotic coronavirus via cross-species transmission.