NIST developed a reference material consisting of synthetic fragments of the SARS-CoV-2 virus RNA, which is the target of molecular diagnostic tests. These RNA fragments can assist in the development and validation of RT-qPCR assays for the detection SARS-CoV-2. The RNA fragments are characterized for concentration using digital PCR methods, may be used to assess limits of detection for SARS-CoV-2 assays, and may calibrate other in-house or commercial SARS-CoV-2 controls. This dataset includes data used for RTGM 10169 SARs-Cov-2 Research Grade Test Material validation. The Illumina and ONT sequence data were used to verify the construct sequence.

NCBI Virus is an integrative, value-added resource designed to support retrieval, display and analysis of a curated collection of virus sequences and large sequence datasets. Its goal is to increase the usability of viral sequence data archived in GenBank and other NCBI repositories. This resource includes resources previously included in HIV-1, Human Protein Interaction Database, Influenza Virus Resource, and Virus Variation.

There is a need for development of an analytical method for rapid detection of SARS-CoV-2 virus which is causing the COVID-19 pandemic. Currently available traditional tissue/cell culture-based analytical method is too laborious and takes several days to get the results on the presence/absence of viable/infectious virus in a sample. Such a delay in getting the sample analysis results can be a serious obstacle in rapidly determining the presence of infectious virus in environment which, in turn, can impact environmental epidemiological investigations and studies on surface transmission of this virus. In this manuscript, development of a Rapid Viability Reverse Transcriptase Polymerase Chain Reaction (RV-RT-PCR) method that can significantly reduce the time-to-results for sample analysis from several days to less than a day is described. The RV-RT-PCR method integrates cell-culture based enrichment of the virus with virus-specific RT-PCR analysis. The RTPCR analysis is conducted before and after the cell-culture-virus (sample) incubation. An optimum algorithm is established such that the resultant RT-PCR cycle threshold (CT) value difference between before and after cell-culture-virus incubation RT-PCR analyses determines the presence of viable/infectious virus in the sample. The data set included here is from this research work. A manuscript has also been included here along with the Supplemental Tables for additional data. The Data-Metadata file includes all the data and a glossary to explain the scientific terms used. This dataset is associated with the following publication: Shah, S., S. Kane, M. Elsheikh, and T. Alfaro. Development of a Rapid Viability RT-PCR (RV-RT-PCR) Method to Detect Infectious SARS-CoV-2 from Swabs. JOURNAL OF VIROLOGICAL METHODS. Elsevier Science Ltd, New York, NY, USA, 297: 114251, (2021).

There is a need for development of an analytical method for rapid detection of SARS-CoV-2 virus which is causing the COVID-19 pandemic. Currently available traditional tissue/cell culture-based analytical method is too laborious and takes several days to get the results on the presence/absence of viable/infectious virus in a sample. Such a delay in getting the sample analysis results can be a serious obstacle in rapidly determining the presence of infectious virus in environment which, in turn, can impact environmental epidemiological investigations and studies on surface transmission of this virus. In this manuscript, development of a Rapid Viability Reverse Transcriptase Polymerase Chain Reaction (RV-RT-PCR) method that can significantly reduce the time-to-results for sample analysis from several days to less than a day is described. The RV-RT-PCR method integrates cell-culture based enrichment of the virus with virus-specific RT-PCR analysis. The RTPCR analysis is conducted before and after the cell-culture-virus (sample) incubation. An optimum algorithm is established such that the resultant RT-PCR cycle threshold (CT) value difference between before and after cell-culture-virus incubation RT-PCR analyses determines the presence of viable/infectious virus in the sample. The data set included here is from this research work. A manuscript has also been included here along with the Supplemental Tables for additional data. The Data-Metadata file includes all the data and a glossary to explain the scientific terms used. This dataset is associated with the following publication: Shah, S., S. Kane, M. Elsheikh, and T. Alfaro. Development of a Rapid Viability RT-PCR (RV-RT-PCR) Method to Detect Infectious SARS-CoV-2 from Swabs. JOURNAL OF VIROLOGICAL METHODS. Elsevier Science Ltd, New York, NY, USA, 297: 114251, (2021).

Background Human herpesvirus-8 (HHV-8) is linked to the pathogenesis of Kaposi's sarcoma (KS), and the HHV-8 DNA load in peripheral blood mononuclear cells (PBMC) is associated with the clinical stage of KS. To examine the expression of HHV-8 in PBMC, four HHV-8 mRNA specific NASBA assays were developed Methods We have developed four quantitative nucleic acid sequence-based amplification assays (NASBA-QT) specifically to detect mRNA coding for ORF 73 (latency-associated nuclear antigen, LANA), vGCR (a membrane receptor), vBcl-2 (a viral inhibitor of apoptosis) and vIL-6 (a viral growth factor). The NASBA technique amplifies nucleic acids without thermocycling and mRNA can be amplified in a dsDNA background. A molecular beacon is used during amplification to enable real-time detection of the product. The assays were tested on PBMC samples of two AIDS-KS patients from the Amsterdam Cohort. Results For all four assays, the limit of detection (LOD) of 50 molecules and the limit of quantification (LOQ) of 100 molecules were determined using in vitro transcribed RNA. The linear dynamic range was 50 to 107 molecules of HHV-8 mRNA. We found HHV-8 mRNA expression in 9 out of the 10 tested samples. Conclusion These real-time NASBA assays with beacon detection provide tools for further study of HHV-8 expression in patient material.

The ability of transfected synthetic small interfering (si) RNAs to suppress the expression of specific transcripts has proved a useful technique to probe gene function in mammalian cells. However, high production costs limit this technology's utility for many laboratories and experimental situations. Recently, several DNA-based plasmid vectors have been developed that direct transcription of small hairpin RNAs, which are processed into functional siRNAs by cellular enzymes. Although these vectors provide certain advantages over chemically synthesized siRNAs, numerous disadvantages remain including merely transient siRNA expression and low and variable transfection efficiency.

This dataset includes the standard curves for ICCRTqPCR to convert the assay quantities to the concentrations of infectious viruses and all the calculations on inactivation rate constants. Also, all the figures used in the manuscripts are presented. This dataset is associated with the following publication: Ryu, H., K. Schrantz, N. Brinkman, and L. Boczek. Applicability of integrated cell culture reverse transcriptase quantitative PCR (ICC-RTqPCR) for the simultaneous detection of the four human enteric enterovirus species in disinfection studies. JOURNAL OF VIROLOGICAL METHODS. Elsevier Science Ltd, New York, NY, USA, 258: 35-40, (2018).