Optimized master mixes formulated for direct amplification of crude samples (e.g. sputum, stool, saliva, urine and blood) Inhibitor-Tolerant qPCR & RT-qPCR Mixes
A variety of different inhibitors inherent to clinical and environmental samples can negatively impact the sensitivity and accuracy of a molecular assay. Conventionally, to overcome this, specimens undergo processing prior to testing. However, extraction technologies are not 100% efficient which can impact the amount of target nucleic acid available for testing, and some inhibitors can co-elute following purification and induce false-negative assay results. Meridian’s Inhibitor-Tolerant qPCR/RT-qPCR formulations are designed for developing qualitative multiplex assays that require minimal sample processing and fast turn-around times (TAT). The mixes can be used for direct amplification from crude lysates or inhibitor-rich samples such as urine, cerebral spinal fluid (CSF), blood, sputum, saliva and stool.
Direct Amplification Workflow
No need for RNA/DNA Extraction with Meridian’s Inhibitor-Tolerant Mix
COLLECT PATIENT SWAB
Requires RNA/DNA Extraction
COLLECT PATIENT SWAB
Extracted DNA or RNA
20 - 60 min
Inhibitor-Tolerant qPCR Mix
• Ready-to-use qPCR mix engineered with a proprietary buffer system designed for overcoming common PCR inhibitors • Allows for DNA amplification direct from crude lysate or unprocessed samples such as urine, cerebral spinal fluid (CSF), milk and blood
Inhibitor-Tolerant qPCR Mix
• Ideal for developing qualitative assays where sample is limited • Eliminates need for sample extraction and speeds up assay TAT
High qPCR efficiency across a range of inhibitors
Inhibitor-Tolerant qPCR Mix Reaction Efficiencies
Reaction efficiencies were determined from reactions containing a variety of known PCR inhibitors ranging from whole blood to biofluids (20% in reaction). The results demonstrate that the reaction efficiency of the Inhibitor- Tolerant qPCR Mix (MDX013) remained within 90-110% in the presence of a wide range of common PCR inhibitors.
Efficient amplification from various liquid sample types (spinal fluid, urine, milk and blood).
WHOLE BLOOD SPECIMENS
40.0 32.0 34.0 36.0 38.0 30.0 28.0 26.0
Direct detection of (A) 25% (B) 46% and (C) 64% GC-rich DNA from 20% whole blood using MDX013 ( green ) and mixes from suppliers R ( red ) and T ( blue ).
20% 10% 1%
Direct detection of GC-rich (64%) DNA from 1% to 20% whole blood using MDX013 ( green ) and mixes from other suppliers.
SPINAL FLUID, URINE AND MILK SPECIMENS
A 10-fold serial dilution of genomic DNA was spiked into cerebrospinal fluid, human urine or cow whole milk and the DNA was amplified, using MDX013 ( green ) and supplier R ( red ). The results illustrate that MDX013 is more sensitive than supplier R mix, as lower dilutions could be detected with better efficiencies, across all three sample types.
Inhibitor-Tolerant RT-qPCR Mix • Designed for amplification from crude samples without the need for complex or time-consuming extraction • 4x concentration master mix allows for greater sample volume • Single use one-step mix. Only add primers, probes and clinical samples • Suitable for single and multiplex detection of RNA and DNA pathogens
Inhibitor-Tolerant RT-qPCR Mix
Inhibitor-Tolerant RT-qPCR Mix exhibits a high tolerance to PCR inhibitors from clinical samples
MDX016 ( red ) and MDX032 ( black ) amplification traces of Influenza A spike in presence of saliva swabs (COPAN ESwab 359C) at 10% (left) and 20% (right) final concentration. With earlier Ct (approx. 4 Ct) and higher fluorescence (approx. +50%), the results demonstrate the superiority of Inhibitor-Tolerant RT-qPCR Mix (MDX016) against a standard RT-qPCR Mix (MDX032) for the detection of viral RNA in presence of saliva swab resuspension in UTM.
Amplification profile of inactivated influenza virus spiked into samples containing 5% sputum or no sputum. The data illustrates that the performance of Inhibitor-Tolerant RT-qPCR Mix (MDX016) in the presence of 5% artificial sputum ( red ) is the same Fast One-Step RT-qPCR Mix (MDX032) with no sputum ( blue ). In contrast, the sensitivity and performance of MDX032 significantly decreases in the presence of 5% artificial sputum ( black ) compared to no sputum or compared to MDX016 with sputum.
Amplification profiles of inactivated influenza virus spiked into samples containing various concentrations of sputum (1.25% - 5%). Results demonstrate that MDX016 ( red ) exhibits a higher tolerance to inhibitors present in artificial sputum (Kirchner, S. et al. J Vis Exp (64) 2012) compared to the standard MDX032 ( black ).
Amplification profiles of 4 pathogens (RNA: Norovirus and Rotavirus, DNA: Adenovirus, C. jejuni ) in a multiplex reaction containing 5% ( yellow ), 10% ( red ) and 20% ( black ) stool extract. The results demonstrate the multiplexing capability of MDX016 in the presence of inhibitors found in stool (up to 20% final volume).
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