Multiplexing Molecular Tests

Inhibitor-tolerant qPCR & LAMP Master Mixes and Enzymes for Respiratory Testing

Multiplexing Molecular Tests

Inhibitor-tolerant qPCR & LAMP Master Mixes and Enzymes for Respiratory Testing

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.

As the demand increases for new assays that are more user-friendly, lower in cost, and can deliver faster sample-to-results, molecular methods that use crude samples and do not require DNA or RNA extraction is expected to become common practice. Combining direct detection with non-invasive sample collection using specimens such as saliva, provides the ideal combination for next-generation screening assays. However, assay performance must remain on par with current methods in terms of sensitivity and reliability in order to improve patient care and clinical outcomes.

Point-of-Care MDx Using Saliva-Specific Mixes

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.

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Air-Dryable Direct RNA/DNA qPCR Saliva: Sensitive qPCR detection in Multiplex Assays Using Samples Containing 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). Two respiratory pathogens, Influenza A 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).

MDX   FluA


Lyo-Ready Direct RNA/DNA LAMP Saliva: High Inhibitor Tolerance and Fast Amplification Delivers Quicker Time-to-Results (TTR) Using Saliva, Sputum and Samples Containing Universal Transport Media (UTM)

Saliva LAMP Mix RSV data  x
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.

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Additional Mixes & Enzymes for POC Respiratory Tests

Point-of-care molecular assays utilize various techniques including PCR, LAMP and NASBA to rapidly and accurately detect genetic material (DNA or RNA) from pathogens. These techniques are designed to be efficient, sensitive, and suitable for on-site testing, and when combined with microfluidics, miniaturization, and automation, enable the development of portable and user-friendly point-of-care molecular assays for rapid and accurate diagnosis of respiratory infections and other diseases.
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