Highly sensitive and specific detection of the SARS-CoV-2 Delta variant by double-mismatch allele-specific real time reverse transcription PCR.

BACKGROUND: The highly transmissible Delta variant of SARS-CoV-2 (B.1.617.2), first identified in India, is currently replacing pre-existing variants in many parts of the world. To help guide public health policies it is important to monitor efficiently its spread. Genome sequencing is the gold standard for identification of Delta, but is time-consuming, expensive, and unavailable in many regions. OBJECTIVE: To develop and evaluate a rapid, simple and inexpensive alternative to sequencing for Delta identification. METHODS: A double-mismatch allele-specific RT-PCR (DMAS-RT-PCR) was developed. The technique exploits allele-specific primers, targeting two spike gene mutations, L452R and T478K, within the same amplicon. The discriminatory power of each primer was enhanced by an additional mismatch located at the fourth nucleotide from the 3' end. Specificity was assessed by testing well characterised cell culture-derived viral isolates and clinical samples, most of which had previously been fully sequenced. RESULTS: In all cases the results of viral genotyping by DMAS-RT-PCR were entirely concordant with the results of sequencing, and the assay was shown to discriminate reliably between the Delta variant and other variants (Alpha and Beta), and 'wild-type' SARS-CoV-2. Influenza A and RSV were non-reactive in the assay. The sensitivity of DMAS-RT-PCR matched that of the diagnostic SARS-CoV-2 RT-qPCR screening assay. Several samples that could not be sequenced due to insufficient virus were successfully genotyped by DMAS-RT-PCR. CONCLUSION: The method we describe would be simple to establish in any laboratory that can conduct PCR assays and should greatly facilitate monitoring of the spread of the Delta variant globally.

Molecular detection and genetic characterization of Salivirus in environmental water in Thailand.

Salivirus (SalV), also known as klassevirus, is a newly discovered member of the Picornaviridae family, which has been proposed to be a potential causative agent of acute gastroenteritis. This study aimed to provide further insight into the currently limited epidemiological data of SalV in environmental water in Thailand, which could be a potential source of human infection. A total of 95 water samples were collected from six locations in Chiang Mai province, northern Thailand, between November 2016 and February 2018. The molecular screening for SalV was performed by the nested polymerase chain reaction. The SalV genotypes were then determined through nucleotide sequencing and phylogenetic analysis. SalV was detected in 31 out of the 95 (32.6%) water samples and all belonged to the A1 genotype, based on phylogenetic analysis of the 5'UTR and 3D regions. The SalV-A1 strains detected in the environmental water were closely related to the SalV-A1 detected in a patient with diarrhoea in the same geographical area, based on the nucleotide sequence identities of the 5'UTR and 3D regions ranging from 91 to 99% and 96-99%, respectively. This study reports the prevalence of SalV-A1 contamination in environmental water in Chiang Mai, Thailand.