Benefits of Direct Detection for Molecular Respiratory Assays
The number of Food and Drug Administration (FDA)–cleared molecular diagnostics for acute respiratory tract infections has increased significantly over the last decade. Studies have shown that multiplex molecular testing for multiple respiratory viruses can be more cost-effective than traditional antigen- or culture-based methods. However, respiratory specimen samples, such as sputum, nasal swabs and saliva, contains inhibitors that can interfere with PCR amplification by interacting directly with the target DNA and blocking the activity of the polymerase or other PCR mixture components. In order to prevent this interference, RNA or DNA is typically first extracted from the sample before PCR is performed.
Point-of-Care Using Saliva-Specific
Saliva has been extensively studied as a diagnostic specimen due to its non-invasive and simple sample collection/logistics. Similar to serum, saliva contains hormones, antibodies, growth factors, enzymes, and microbes that can be used as biomarkers for disease detection. Combining direct detection with non-invasive sample collection, using specimens such as saliva, provides the ideal combination for next-generation screening assays. However, there have been challenges in adopting saliva for molecular diagnostics due to the low concentration of analytes and high concentration of PCR inhibitors. Meridian’s newest master mixes, Specimen-Specific™ Direct Saliva, help overcome the challenges in developing saliva direct detection assays. These mixes are optimized for sensitive and robust performance using crude saliva specimens and are ready-to-use, only requiring the addition of assay-specific primers and probes. In addition, they are formulated for downstream lyophilization or air-drying to create ambient temperature stable assays which are ideal for point-of-care applications.
Sensitive qPCR detection in multiplex assays using samples with Universal Transport Media (up to 35% UTM)
Air-Dryable™ Direct RNA/DNA qPCR Saliva (MDX131) was tested for its amplification efficiency in a multiplex reactions using samples that contained up to 35% Universal Transport Media (UTM). Three respiratory pathogens, Influenza A, Middle East Respiratory syndrome coronavirus (MERS-CoV) and Respiratory Syncytial Virus (RSV) were amplified in a triplex qPCR assay in the presence of 35% Universal Transport Media (UTM) with artificial sputum swab. The results illustrate that a higher performance was achieved with Air-Dryable™ Direct RNA/DNA qPCR Saliva (red) compared to the inhibitor-tolerant RT-qPCR mixes TaqPath™ (Thermo, black) or Ultraplex™ (QuantBio, grey).
Lyo-Ready™ Direct RNA/DNA LAMP Saliva: High inhibitor tolerance and fast amplification to deliver quicker time-to-results (TTR) in saliva, sputum and samples containing Universal Transport Media (UTM)
Primers for RSV were added to Lyo-Ready™ Direct RNA/DNA LAMP Saliva (MDX135) and lyophilized (blue) or added to NEB WarmStart® (orange) and kept as a liquid. The mixes were amplified using inactivated respiratory syncytial virus (RSV) RNA prepared with 30% human saliva, 30% Universal Transport Medium (UTM) with/without an artificial sputum swab, and Viral Transport Medium (VTM). All reactions were incubated at 65 °C for 60 minutes, and the average time to results (TTR) was measured at 1:10 of end fluorescence. Error bars represent the standard deviation across six technical replicates. Results show that MDX135 (blue) has a shorter TTR than NEB WarmStart® (orange) amplification across all sample types. The data suggests that MDX135 performs better with faster amplification across the range of inhibitors.
|Lyo-Ready™ 1-Step RT-qPCR Virus Mix||MDX062|
|Air-Dryable™ 1-Step RT-qPCR Mix||MDX095|
|Air-Dryable™ RT-LAMP 1-Step Mix||MDX118|
|Lyo-Ready™ RT-LAMP 1-Step Mix||MDX108|
|Glycerol-Free Taq SH 50U/mL||MDX011|
|High Conc. Glycerol-Free BST (100U/μL)||MDX018|
|Lyo-Ready™ qPCR Buffer, 2.5x||MDX022|
|Lyo-Ready™qPCR Buffer w/o Excipient, 4x||MDX061|