No abstract

The Delta variant of SARS‐CoV‐2 has now become the predominant strain in the global COVID‐19 pandemic. Strain coverage of some detection assays developed during the early pandemic stages has declined due to periodic mutations in the viral genome. We have developed a real‐time RT‐PCR (RT‐qPCR) for SARS‐CoV‐2 detection that provides nearly 100% strain coverage, and differentiation of highly transmissible Delta variant strains. All full or nearly full (≥28 kb) SARS‐CoV‐2 genomes (n = 403,812), including 6422 Delta and 280 Omicron variant strains, were collected from public databases at the time of analysis and used for assay design. The two amino acid deletions in the spike gene (S‐gene, Δ156‐157) that is characteristic of the Delta variant were targeted during the assay design. Although strain coverage for the Delta variant was very high (99.7%), detection coverage for non‐Delta wild‐type strains was 93.9%, mainly due to the confined region of design. To increase strain coverage of the assay, the design for CDC N1 target was added to the assay. In silico analysis of 403,812 genomes indicated a 95.4% strain coverage for the CDC N1 target, however, in combination with our new non‐Delta S‐gene target, total coverage for non‐Delta wild‐type strains increased to 99.8%. A human 18S rRNA gene was also analyzed and used as an internal control. The final four‐plex RT‐qPCR assay generated PCR amplification efficiencies between 95.4% and 102.0% with correlation coefficients (R2) of >0.99 for cloned positive controls; Delta and non‐Delta human clinical samples generated PCR efficiencies of 93.4%–97.0% and R2 > 0.99. The assay also detects 98.6% of 280 Omicron sequences. Assay primers and probes have no match to other closely related human coronaviruses, and did not produce a signal from samples positive to selected animal coronaviruses. Genotypes of selected clinical samples identified by the RT‐qPCR were confirmed by Sanger sequencing.

A duplex real-time reverse transcription-polymerase chain reaction (qRT-PCR) assay was developed for a simple and rapid diagnosis of Peste des petits ruminants (PPR). qRT-PCR primers and TaqMan probe were designed on a conserved region of nucleocapsid protein (Np) of PPR virus (PPRV) genome. An in vitro transcript of the target region was constructed and tested to determine analytical sensitivity. Commercial heterologous Armored RNA(®) was used as an internal positive control (IPC) for either RNA isolation or RT-PCR steps. The detection limit of the newly designed duplex real-time RT-PCR (qRT-PCR PPR_Np) was approximately 20 copies/μl with a 95% probability. No amplification signals were recorded when the qRT-PCR PPR_Np was applied to viruses closely related or clinically similar to PPRV- or to PPR-negative blood samples. A preliminary evaluation of the diagnostic performance was carried out by testing a group of 43 clinical specimens collected from distinct geographic areas of Africa and Middle East. qRT-PCR PPR_Np showed higher sensitivity than the conventional gel-based RT-PCR assays, which have been used as reference standards. Internal positive control made it possible to identify the occurrence of 5 false-negative results caused by the amplification failure, thus improving the accuracy of PPRV detection.

BackgroundRift Valley fever (RVF) is classified as viral hemorrhagic fever and is endemic in East and West Africa. RVF is caused by an arthropod borne virus (RVFV); the disease is zoonotic and affects human, animal health as well as international trade. In livestock it causes abortions, while human infection occurs through close contact with infected animals or animal products.MethodsA quantitative observational study using stratified sampling was conducted in the western region of Uganda. Blood samples and abortion events from 1000 livestock (goats, sheep and cattle) was collected and recorded. Serum was analyzed for RVFV IgG reacting antibodies using competitive ELISA test.ResultsThe overall RVFV seroprevalence was of 10.4% (104/1000). Cattle had the highest seroprevalence (7%) followed by Sheep (2.2%) then goats (1.2%). Species specific RVFV seroprevalence was highest in cattle (20.5%) followed by sheep (6.8%) then goats (3.6%). RVFV seroprevalence in northern highlands (21.8%) was significantly higher (p < 0.001) than in the southern lowlands (3.7%). Overall prevalence of abortion was (17.4%), sheep had the highest prevalence of abortion (7.8%) followed by goats (6.3%) and then cattle (3.3%). Species specific abortion prevalence was highest in Sheep (24.1%) followed by goats (18.8%) and then 9.7% in cattle.ConclusionRVFV is endemic in Kisoro district and livestock in the highland areas are more likely to be exposed to RVFV infection compared to those in the southern lowlands. Out breaks in livestock most likely will lead to zoonotic infection in Kisoro district.

The SARS‐CoV‐2 virus is the causative agent of COVID‐19 and has undergone continuous mutations throughout the pandemic. The more transmissible Omicron variant has quickly spread and is replacing the Delta variant as the most prevalent strain globally, including in the United States. A new molecular assay that can detect and differentiate both the Delta and Omicron variants was developed. A collection of 660,035 SARS‐CoV‐2 full‐ or near‐full genomes, including 169,454 Delta variant and 24,202 Omicron variant strains, were used for primer and probe designs. In silico data analysis predicted an assay coverage of >99% of all strains, including >99% of the Delta and >99% of Omicron strains. The Omicron variant differential test was designed based on the Δ31‐33 aa deletion in the N‐gene, which is present in the original B.1.1.529 main genotype, BA.1, as well as in BA.2 and BA.3 subtypes. Therefore, the assay should detect the majority of all Omicron variant strains. Standard curves generated with human clinical samples indicated that the PCR amplification efficiencies were 104%, 90.7% and 90.4% for the Omicron, Delta, and non‐Delta/non‐Omicron wild‐type genotypes, respectively. Correlation coefficients of the standard curves were all >0.99. The detection limit of the assay was 14.3, 32.0, and 21.5 copies per PCR reaction for Omicron, Delta, and wild‐type genotypes, respectively. The assay was designed to specifically detect SAR‐CoV‐2 strains. Selected samples with Omicron, Delta and wild‐type genotypes identified by the RT‐qPCR assay were also confirmed by sequencing. The assay did not detect any animal coronavirus‐positive samples that were tested. Human nasal swab samples that previously tested positive ( n = 182) or negative ( n = 42) for SARS‐CoV‐2 by the ThermoFisher TaqPath COVID‐19 Combo Kit, produced the same result with the new assay. Among positive samples, 55.5% (101/182), 23.1% (42/182), and 21.4% (39/182) were identified as Omicron, Delta, and non‐Omicron/non‐Delta wild‐type genotypes, respectively.