SuperSelective primer pairs for sensitive detection of rare somatic mutations

SuperSelective primers, by virtue of their unique design, enable the selective exponential amplification of rare DNA fragments containing somatic mutations in the presence of abundant closely related wild-type DNA fragments. However, when a SuperSelective primer is used in conjunction with a conventional reverse primer, linear amplification of the abundant wild-type fragments occurs, and this may lead to a late arising signal that can be confused with the late arising signal from the rare mutant fragments. We have discovered that the use of a pair of SuperSelective primers, one specific for the target mutation in a plus strand, and the other specific for the same mutation in the complementary minus strand, but both possessing 3′-terminal nucleotides that are complementary to the mutation, significantly suppresses the linear amplification of the related wild-type sequence, and prevents the generation of false mutant sequences due to mis-incorporation by the DNA polymerase. As a consequence, the absence of mutant fragments in a sample does not give rise to a false-positive signal, and the presence of mutant fragments in a sample is clearly distinguishable as a true-positive signal. The use of SuperSelective primer pairs should enhance the sensitivity of multiplex PCR assays that identify and quantitate somatic mutations in liquid biopsies obtained from patients with cancer, thereby enabling the choice of a targeted therapy, the determination of its effectiveness over time, and the substitution of a more appropriate therapy as new mutations arise.

Verification of the “Upward Variation in the Reporting Odds Ratio Scores” to Detect the Signals of Drug–Drug Interactions

The reporting odds ratio (ROR) is easy to calculate, and there have been several examples of its use because of its potential to speed up the detection of drug–drug interaction signals by using the “upward variation of ROR score”. However, since the validity of the detection method is unknown, this study followed previous studies to investigate the detection trend. The statistics models (the Ω shrinkage measure and the “upward variation of ROR score”) were compared using the verification dataset created from the Japanese Adverse Drug Event Report database (JADER). The drugs registered as “suspect drugs” in the verification dataset were considered as the drugs to be investigated, and the target adverse event in this study was Stevens–Johnson syndrome (SJS), as in previous studies. Of 3924 pairs that reported SJS, the number of positive signals detected by the Ω shrinkage measure and the “upward variation of ROR score” (Model 1, the Susuta Model, and Model 2) was 712, 2112, 1758, and 637, respectively. Furthermore, 1239 positive signals were detected when the Haldane–Anscombe 1/2 correction was applied to Model 2, the statistical model that showed the most conservative detection trend. This result indicated the instability of the positive signal detected in Model 2. The ROR scores based on the frequency-based statistics are easily inflated; thus, the use of the “upward variation of ROR scores” to search for drug–drug interaction signals increases the likelihood of false-positive signal detection. Consequently, the active use of the “upward variation of ROR scores” is not recommended, despite the existence of the Ω shrinkage measure, which shows a conservative detection trend.

Testing as Prevention of Resistance in Bacteria Causing Sexually Transmitted Infections—A Population-Based Model for Germany

Prescribed antibiotic treatments which do not match the therapeutic requirements of potentially co-existing undetected sexually transmitted infections (STIs) can facilitate the selection of antibiotic-drug-resistant clones. To reduce this risk, this modelling assessed the potential applicability of reliable rapid molecular test assays targeting bacterial STI prior to the prescription of antibiotic drugs. The modelling was based on the prevalence of three bacterial STIs in German heterosexual and men-having-sex-with-men (MSM) populations, as well as on reported test characteristics of respective assays. In the case of the application of rapid molecular STI assays for screening, the numbers needed to test in order to correctly identify any of the included bacterial STIs ranged from 103 to 104 for the heterosexual population and from 5 to 14 for the MSM population. The number needed to harm—defined as getting a false negative result for any of the STIs and a false positive signal for another one, potentially leading to an even more inappropriate adaptation of antibiotic therapy than without any STI screening—was at least 208,995 for the heterosexuals and 16,977 for the MSM. Therefore, the screening approach may indeed be suitable to avoid unnecessary selective pressure on bacterial causes of sexually transmitted infections.

False Positive Results in SARS-CoV-2 Serological Tests for Samples From Patients With Chronic Inflammatory Diseases

Patients with chronic inflammatory diseases are often treated with immunosuppressants and therefore are of particular concern during the SARS-CoV-2 pandemic. Serological tests will improve our understanding of the infection and immunity in this population, unless they tests give false positive results. The aim of this study was to evaluate the specificity of SARS-Cov-2 serological assays using samples from patients with chronic inflammatory diseases collected prior to April 2019, thus defined as negative. Samples from patients with multiple sclerosis (MS, n=10), rheumatoid arthritis (RA, n=47) with or without rheumatoid factor (RF) and/or anti-cyclic citrullinated peptide antibodies (anti-CCP2) and systemic lupus erythematosus (SLE, n=10) with or without RF, were analyzed for SARS-CoV-2 antibodies using 17 commercially available lateral flow assays (LFA), two ELISA kits and one in-house developed IgG multiplex bead-based assay. Six LFA and the in-house validated IgG assay correctly produced negative results for all samples. However, the majority of assays (n=13), gave false positive signal for samples from patients with RA and SLE. This was most notable in samples from RF positive RA patients. No false positive samples were detected in any assay using samples from patients with MS. Poor specificity of commercial serological assays could possibly be, at least partly, due to interfering antibodies in samples from patients with chronic inflammatory diseases. For these patients, the risk of false positivity should be considered when interpreting results of the SARS-CoV-2 serological assays.

SARS-CoV-2 serological tests can generate false positive results for samples from patients with chronic inflammatory diseases

ObjectivesPatients with chronic inflammatory diseases are often treated with immunosuppressants and therefore are of particular concern during the SARS-CoV-2 pandemic. Serological tests will improve our understanding of the infection and immunity in this population, unless the tests give false positive results. The aim of this study was to evaluate the specificity of SARS-Cov-2 serological assays with samples from patients with chronic inflammatory diseases collected before April 2019, thus defined as negative.MethodsSamples from patients with multiple sclerosis (MS, n=10), rheumatoid arthritis (RA, n=47) with or without rheumatoid factor (RF) and/or anti-cyclic citrullinated peptide antibodies (anti-CCP2) and RF +/- systemic lupus erythematosus (SLE, n=10), were tested with 17 commercially available lateral flow assays (LFA), two ELISA kits and one in-house developed multiplex bead-based assay.ResultsSix LFA and the in-house IgG assay gave the correct negative results for all samples. However, the majority of assays (n=13), gave false positive signal with samples from patients with RA and SLE. This was most notable in RF positive RA samples. MS samples did not give any false positive in any of the assays.ConclusionThe majority of the verified serological assays were sensitive to interfering antibodies in samples from patients with chronic inflammatory diseases and therefore may have poor specificity in this context. For these patients, the risk of false positivity should be considered when interpreting results of the SARS-CoV-2 serological assays.

Overcoming multimeric target interference in a bridging immunogenicity assay with soluble target receptor, target immunodepletion and mild acidic assay pH

Background: Soluble multimeric target proteins can generate a target-mediated false-positive signal in bridging anti-drug antibody (ADA) assays. A high background signal due to target interference was observed in our anti-REGN-Y antibody assay, and two different strategies were evaluated to mitigate this false-positive signal. Results: Multiple anti-target antibodies were tested and found to be ineffective at reducing target interference, so soluble target receptor and co-factor proteins were used in combination to inhibit the target-mediated signal. These competitive blockers synergistically inhibited target interference and increased target tolerance levels, especially when the assay was performed under mild acidic conditions. A separate approach, target immunodepletion using magnetic beads conjugated with an anti-target antibody, was also effective at mitigating the target-mediated signal, also in combination with mild acidic assay pH. Both methods allowed detection of a true ADA signal in monkey and human post-dose serum samples. Conclusion: These methods provide alternative strategies for mitigating target interference when standard anti-target antibodies are ineffective, with the competitive blocker method being recommended, if possible, due to its higher throughput and easier execution.

The study of protein recruitment to laser-induced DNA lesions can be distorted by photoconversion of the DNA binding dye Hoechst

A commonly used approach for assessing DNA repair factor recruitment in mammalian cells is to induce DNA damage with a laser in the UV or near UV range and follow the local increase of GFP-tagged proteins at the site of damage. Often these measurements are performed in the presence of the blue DNA dye Hoechst, which is used as a photosensitizer. However, a light-induced switch of Hoechst from a blue-light to a green-light emitter will give a false positive signal at the site of damage. Thus, photoconversion signals must be subtracted from the overall green-light emission to determine true recruitment. Here we demonstrate the photoconversion effect and suggest control experiments to exclude false-positive results.

The study of protein recruitment to UV-induced DNA lesions can be distorted by photoconversion of DNA dyes like Hoechst or DAPI

A common approach used to assess DNA repair factor binding in mammalian cells is to induce DNA damage with a UV laser and follow the movement of GFP-tagged proteins to the site of damage. Often these measurements are performed in the presence of the blue DNA intercalating dye Hoechst or DAPI, which is used to label nuclear DNA. A UV-induced switch of Hoechst and DAPI from a blue-light to a green-light emitter will give a false positive signal at the site of damage. Thus, photoconversion signals must be subtracted from the overall green-light emission to determine true recruitment. Here we demonstrate the photoconversion effect and suggest control experiments to exclude false-positive results.

T7 exo-mediated FRET-breaking combined with DSN–RNAse–TdT for the detection of microRNA with ultrahigh signal-amplification

A DSN–RNAse–TdT–T7 exo probing system allows the detection of miRNA 21 with very high sensitivity (LOD = 2.57 fM) and selectivity—the result of (i) avoiding the false-positive signal from miRNA reacting with TdT polymerase and (ii) signal amplification occurring through a FRET-breaking mechanism involving T7 exo.