Synthetic long oligonucleotides to generate artificial templates for use as positive controls in molecular assays: drug resistance mutations in influenza virus as an example

<p>Abstract</p> <p>Background</p> <p>Positive controls are an integral component of any sensitive molecular diagnostic tool, but this can be affected, if several mutations are being screened in a scenario of a pandemic or newly emerging disease where it can be difficult to acquire all the necessary positive controls from the host. This work describes the development of a synthetic oligo-cassette for positive controls for accurate and highly sensitive diagnosis of several mutations relevant to influenza virus drug resistance.</p> <p>Results</p> <p>Using influenza antiviral drug resistance mutations as an example by employing the utility of synthetic paired long oligonucleotides containing complementary sequences at their 3' ends and utilizing the formation of oligonucleotide dimers and DNA polymerization, we generated ~170bp dsDNA containing several known specific neuraminidase inhibitor (NAI) resistance mutations. These templates were further cloned and successfully applied as positive controls in downstream assays.</p> <p>Conclusion</p> <p>This approach significantly improved the development of diagnosis of resistance mutations in terms of time, accuracy, efficiency and sensitivity, which are paramount to monitoring the emergence and spread of antiviral drug resistant influenza strains. Thus, this may have a significantly broader application in molecular diagnostics along with its application in rapid molecular testing of all relevant mutations in an event of pandemic.</p

Rapid Detection of the H275Y Oseltamivir Resistance Mutation in Influenza A/H1N1 2009 by Single Base Pair RT-PCR and High-Resolution Melting

Introduction: We aimed to design a real-time reverse-transcriptase-PCR (rRT-PCR), high-resolution melting (HRM) assay to detect the H275Y mutation that confers oseltamivir resistance in influenza A/H1N1 2009 viruses.Findings: A novel strategy of amplifying a single base pair, the relevant SNP at position 823 of the neuraminidase gene, was chosen to maintain specificity of the assay. Wildtype and mutant virus were differentiated when using known reference samples of cell-cultured virus. However, when dilutions of these reference samples were assayed, amplification of nonspecific primer-dimer was evident and affected the overall melting temperature (Tm) of the amplified products. Due to primer-dimer appearance at .30 cycles we found that if the cycle threshold (CT) for a dilution was .30, the HRM assay did not consistently discriminate mutant from wildtype. Where the CT was ,30 we noted an inverse relationship between CT and Tm and fitted quadratic curves allowed the discrimination of wildtype, mutant and 30:70 mutant:wildtype virus mixtures. We compared the CT values for a TaqMan H1N1 09 detection assay with those for the HRM assay using 59 clinical samples and demonstrated that samples with a TaqMan detection assay CT.32.98 would have an H275Y assay CT.30. Analysis of the TaqMan CT values for 609 consecutive clinical samples predicted that 207 (34%) of the samples would result in an HRM assay CT.30 and therefore not be amenable to the HRM assay.Conclusions: The use of single base pair PCR and HRM can be useful for specifically interrogating SNPs. When applied to H1N1 09, the constraints this placed on primer design resulted in amplification of primer-dimer products. The impact primer-dimer had on HRM curves was adjusted for by plotting Tm against CT. Although less sensitive than TaqMan assays, the HRM assay can rapidly, and at low cost, screen samples with moderate viral concentrations

A single dose, BCG-adjuvanted COVID-19 vaccine provides sterilising immunity against SARS-CoV-2 infection.

Global control of COVID-19 requires broadly accessible vaccines that are effective against SARS-CoV-2 variants. In this report, we exploit the immunostimulatory properties of bacille Calmette-Guérin (BCG), the existing tuberculosis vaccine, to deliver a vaccination regimen with potent SARS-CoV-2-specific protective immunity. Combination of BCG with a stabilised, trimeric form of SARS-CoV-2 spike antigen promoted rapid development of virus-specific IgG antibodies in the blood of vaccinated mice, that was further augmented by the addition of alum. This vaccine formulation, BCG:CoVac, induced high-titre SARS-CoV-2 neutralising antibodies (NAbs) and Th1-biased cytokine release by vaccine-specific T cells, which correlated with the early emergence of T follicular helper cells in local lymph nodes and heightened levels of antigen-specific plasma B cells after vaccination. Vaccination of K18-hACE2 mice with a single dose of BCG:CoVac almost completely abrogated disease after SARS-CoV-2 challenge, with minimal inflammation and no detectable virus in the lungs of infected animals. Boosting BCG:CoVac-primed mice with a heterologous vaccine further increased SARS-CoV-2-specific antibody responses, which effectively neutralised B.1.1.7 and B.1.351 SARS-CoV-2 variants of concern. These findings demonstrate the potential for BCG-based vaccination to protect against major SARS-CoV-2 variants circulating globally