A Pancoronavirus RT-PCR Assay for Detection of All Known Coronaviruses

Abstract

The recent discoveries of novel human coronaviruses, including the coronavirus causing SARS, and the previously unrecognized human coronaviruses HCoV-NL63 and HCoV-HKU1, indicate that the family Coronaviridae harbors more members than was previously assumed. All human coronaviruses characterized at present are associated with respiratory illnesses, ranging from mild common colds to more severe lower respiratory tract infections. Since the etiology of a relatively large percentage of respiratory tract diseases remains unidentified, it is possible that for a certain number of these illnesses, a yet unknown viral causative agent may be found. Screening for the presence of novel coronaviruses requires the use of a method that can detect all coronaviruses known at present. In this chapter, we describe a pancoronavirus degenerate primer-based method that allows the detection of all known and possibly unknown coronaviruses by RT-PCR amplification and sequencing of a 251-bp fragment of the coronavirus polymerase gene.

No Evidence of Coronavirus Infection by Reverse Transcriptase-PCR in Bats in Belgium

“…Similarly, neither pancoronavirus RT-PCR detected any coronavirus. The pancoronavirus RT-PCR of Vijgen et al (2008) amplified nonspecific fragments of a size similar to the positive control, especially in lungs. The sequence of these fragments corresponded, however, with the signal recognition particle RNA.…”

“…Lungs and intestines were homogenized in b-mercaptoethanol lysis buffer using stainless-steel beads and a tissue homogenizer (Bullet BlenderH, Next Advance Bio-Connect, Huissen, the Netherlands). RNA was extracted with QIAmp Viral RNA Minikit (QIAGEN Benelux BV, Antwerp, Belgium) and examined with a specific MERS CoV real-time RT-PCR (Corman et al 2012) and two pancoronavirus RT-PCRs (Ksiazek et al 2003;Vijgen et al 2008). The specific RT-PCR uses primers targeting elements upstream of the E gene of the MERS CoV; the pancoronavirus RT-PCRs use primers targeting a conserved region in the RdRp gene present in all coronaviruses.…”

Коронавіруси Людини Та Захворювання Органів Дихання

“…До найбільш важливої особливості геному належить висока частота рекомбінацій молекули РНК. Найбільшу генетичну спорідненість SARS-CoV має з коронавірусом котів та бичачим коронавірусом, а також з коронавірусом свиней [8].…”

“…Один з білків (N) з'єднаний з геномною РНК і входить до складу нуклеокапсиду (рис. 2) [7][8][9][10]. …”

Infections with human coronaviruses NL63 and OC43 among hospitalised and outpatient individuals in São Paulo, Brazil

“…Assays to identify HCoVs in patients were performed using a two-step process. First, an initial pancoronavirus reverse-transcription polymerase chain reaction (RT-PCR) screening assay was performed (Vijgen et al 2008). Second, species-specific RT-PCR assays were used to identify the specific HCoV species (Dare et al 2007).…”