Inhibitor-Tolerant qPCR & RT-qPCR Mixes 0525

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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