Real-Time fast PCR amplification using designated and conventional real time thermal cycler systems: COVID-19 perspective.

The study aimed to shorten multiplex RT-PCR run time for detection of SARS CoV-2 N1 and N2 sequences and human RNase P (RP) sequence as internal mRNA control using conventional and designated real time thermal cycler systems. Optimization of Fast PCR protocol using plasmid-based N1 and N2 positive control and synthetic version of human RP was done on Applied Biosystems (ABI) QuantStudioTM5 (conventional), ABI 7500 Fast Dx (designated), and CFX96 Touch Real Time Detection System, Bio-Rad (conventional). Finally, a performance evaluation of Fast PCR was performed in terms of sensitivity, specificity, and precision. For a 40-cycle PCR with optimized Fast PCR protocols on QuantStudioTM5, ABI 7500 Fast Dx, and CFX96 Touch (conventional), standard/regular versus Fast PCR run times (min) were 84 vs. 49, 96 vs. 48, and 103 vs. 61, thereby saving 35, 48, and 43 min, respectively. For each thermal cycler, Standard and Fast PCR generated identical shapes of fluorescence curves, Ct values, and (3) R2 (0.95 to 0.99) for 5 10-log dilution panels of each positive control. The fast PCR approach generated results with 100% sensitivity and specificity. Median test comparisons between standard PCR and Fast PCR Cts of COVID-19 samples did not produce significance (p>0.5), suggesting that Fast PCR and Standard PCR were comparable. Also, the median and mean of each target had closely-related values, further suggesting that the two approaches were comparable. That is, there is an equivalency between Conventional and Fast PCR instruments for detection of COVID-19.

Development and performance evaluation of the first in-house multiplex rRT-PCR assay in Bangladesh for highly sensitive detection of SARS-CoV-2.

BACKGROUND: The emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic is posing a great threat to global health and economy. Due to the lack of broad diagnostic setup, consistent reagent supply lines, and access to laboratory instruments and equipment, it is undoubtedly an enormous burden for developing countries to face the crisis. OBJECTIVES: To develop a cost-effective, reliable and sensitive multiplex assay for SARS-CoV-2 screening which would expand the testing capacities of a developing and low-income country like Bangladesh. STUDY DESIGN: Initially a singleplex and then a multiplex real-time reverse-transcriptase PCR assays were developed targeting 2 nucleocapsid genes of SARS-CoV-2, and the human RNase P gene as an internal control using laboratory-made mastermixes. Three sets of primer- probes were designed for each of the target genes and one set was optimized for the final reaction set-up. Limit of detection, cross-reactivity and reproducibility were checked in order to assess the sensitivity and specificity of the assays, and validation was done using clinical specimens. RESULTS: Clinical evaluation of the new assays using 240 nasopharyngeal swabs showed 100 % sensitivity, specificity, and accuracy in detecting SARS-CoV-2 infection in human. Equal efficiency and concordant results were observed between the singleplex and multiplex approaches. Notably, the kit was able to detect SARS-CoV-2 RNA at very low concentration upto 5 copies/reaction. CONCLUSION: This is the first locally developed multiplex rRT-PCR kit in Bangladesh providing rapid and low-cost screening of COVID-19 which would be valuable for infection prevention and clinical management in the perspective of Bangladesh.

Lateral flow immunoassay for diagnosis of Trypanosoma cruzi infection with high correlation to the radioimmunoprecipitation assay.

The incidence of blood donors seropositive for Trypanosoma cruzi in North America has increased with population migration and more rigorous surveillance. The United States, considered nonendemic for T. cruzi, could therefore be at risk to exposure to parasite transmission through blood or organ donations. Current tests show variable reactivity, especially with Central American sera. Here we describe the development of a lateral flow immunoassay for the rapid detection of T. cruzi infection that has a strong correlation to the radioimmunoprecipitation assay (RIPA) "gold standard" in the United States. Such a test could have utility in small blood banks for prescreening donors, as well as in cardiac transplantation evaluation. T. cruzi consensus and/or RIPA-positive sera from Central and South America were evaluated in enzyme immunoassays (EIAs). These included commercial panels from Boston Biomedica, Inc. (BBI) (n = 14), and HemaBio (n = 21). Other sources included RIPA-positive sera from the American Red Cross (ARC) (n = 42), as well as from Chile. Sera were tested with the multiepitope recombinant TcF. All but one of the BBI samples were positive and 7 of 21 HemaBio samples and 6 of 42 ARC samples were low positive or negative. This observation indicated the need for additional antigens. To complement TcF reactivity, we tested the sera with peptides 30, 36, SAPA, and 1.1, 1.2, and 1.3 His fragments of 85-kDa trans-sialidase. We identified a promising combination of the tested antigens and constructed a single recombinant protein, ITC6, that enhanced the relative sensitivity in U.S. blood donor sera compared to that of TcF. The data on its evaluation using RIPA-confirmed positive sera in EIA and lateral flow immunoassay studies are presented, along with an additional recombinant protein, ITC8.2, with two additional sequences for peptide 1 and Kmp-11. The latter, when evaluated in a dipstick assay with consensus positive sera, had a sensitivity of 99.2% and a specificity of 99.1%.