Optimized master mixes formulated for direct amplification of crude samples (e.g. sputum, stool, saliva, urine and blood).
Inhibitor-Tolerant qPCR & RT-qPCR Mixes Optimized master mixes formulated for direct amplification of crude samples (e.g. sputum, stool, saliva, urine and blood)
Traditionally, sample processing and nucleic acid extraction are used to mitigate the effects of inhibitors. However, extraction methods are not 100% efficient, often resulting in loss of target nucleic acid and potential co-purification of inhibitors that can interfere with amplification, leading to false-negative results. Meridian’s universal Inhibitor-Tolerant qPCR/RT-qPCR formulations provide a superior solution for developing qualitative multiplex assays that require minimal sample processing and deliver rapid turnaround times (TAT). These advanced master mixes enable direct amplification from crude lysates and inhibitor-rich samples such as urine, cerebrospinal fluid (CSF), blood, sputum, saliva, and stool, eliminating the need for complex extraction steps. By streamlining workflows and improving assay robustness, our formulations help accelerate molecular diagnostics, ensuring reliable results even in the most challenging sample types. Inhibitors present in clinical and environmental samples can significantly impact the sensitivity and accuracy of molecular assays, leading to unreliable results.
Direct Amplification Workflow
Standard Workflow
Reaction setup
Data Analysis
Sample 1
2
3
4
5
6
Result
Lysis
Capture
Washing
Elution
Direct Workflow
Data Analysis
Sample
1
Result
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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, cerebrospinal fluid (CSF), milk and blood • Eliminates need for sample extraction and speeds up assay TAT • Lyo-ready master mix can be lyophilized, just add primers and probe to create an ambient temperature stable assay
PRODUCT
CAT NO.
VOLUME REACTIONS
5 mL 500 Rxns 100 mL 10,000 Rxns 500 Rxns 40 mL 10,000 Rxns 2 mL 5 mL 500 Rxns 50 mL 10,000 Rxns
Inhibitor-Tolerant qPCR Mix
MDX013
Inhibitor-Tolerant qPCR Mix, 5x
MDX073
Lyo-Ready Inhibitor- Tolerant qPCR Mix 4x
MDX184
High qPCR efficiency across a range of inhibitors
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.
WHOLE BLOOD SPECIMENS
Amplification Plot A
Amplification Plot B
40.0
32.0 34.0 36.0 38.0
30.0
28.0
Amplification Plot C
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%
Percent Blood
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
Amplification Plot
Amplification Plot
Amplification Plot
Cerebrospinal fluid
Urine
Cow milk
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 tube, one-step mix. Only add primers, probes and clinical samples • Lyo-ready master mix can be lyophilized, with primers and probe to create an ambient temperature stable assay • Suitable for single and multiplex detection of RNA and DNA pathogens
PRODUCT
CAT NO.
VOLUME REACTIONS
5 mL 1,000 Rxns 50 mL 10,000 Rxns 1,000 Rxns 50 mL 10,000 Rxns 5 mL 5 mL 1,000 Rxns 50 mL 10,000 Rxns
Inhibitor-Tolerant RT-qPCR Mix, 4x
MDX016
Inhibitor-Tolerant RT-qPCR Low-ROX Mix, 4x Lyo-Ready Inhibitor-Tolerant RT-qPCR Mix, 4x
MDX105
MDX185
Inhibitor-Tolerant RT-qPCR Mix exhibits a high tolerance to PCR inhibitors from clinical samples
SALIVA SPECIMENS
10% Saliva
20% Saliva
MDX016 (red) and a standard RT-qPCR Mix (black) amplification traces of Influenza A spiked into saliva swab matrix (COPAN ESwab 359C), used at final concentrations of 10% (left) and 20% (right). 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 for the detection of viral RNA in presence of saliva swab resuspension in UTM.
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SPUTUM SPECIMENS
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 as the RT-qPCR Mix RT-qPCR Mix with no sputum (blue). In contrast, the sensitivity and performance of the standard RT-qPCR Mix significantly decreases in the presence of 5% artificial sputum (black) compared to no sputum or compared to MDX016 with sputum.
1.25% Sputum
2.5% Sputum
5% 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 RT-qPCR Mix (black).
STOOL SPECIMENS
Adenovirus
Norovirus GII
Campylobacter jejuni
Rotavirus A
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).
Ordering information: USA 5171 Wilfong Road Memphis, Tennessee 38134 Phone: +1 901-382-8716 Fax: +1 901-333-8223
Email: info@meridianlifescience.com Orders: orders@meridianlifescience.com www.MeridianBioscience.com/lifescience
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