Optimized master mixes formulated for direct amplification of crude samples (e.g. sputum, stool, saliva, urine and blood).
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
Crude Lysate
Master Mix
Crude Lysate
qPCR/ RT-qPCR
COLLECT PATIENT SWAB
Primers Probes
SAMPLE PRE-TREATMENT
5 min
Requires RNA/DNA Extraction
Primers Probes
Lysis
Wash
Elution
Master Mix
qPCR/ RT-qPCR
COLLECT PATIENT SWAB
Extracted DNA or RNA
SAMPLE EXTRACTION
20 - 60 min
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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, cerebral spinal fluid (CSF), milk and blood
PRODUCT
CAT NO.
VOLUME REACTIONS
5 mL
500 Rxn
Inhibitor-Tolerant qPCR Mix
MDX013
100 mL
10,000 Rxn
• 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
100
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.
80
60
40
20
0
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
A
B
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 ).
C
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.
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SPINAL FLUID, URINE AND MILK SPECIMENS
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 use one-step mix. Only add primers, probes and clinical samples • Suitable for single and multiplex detection of RNA and DNA pathogens
PRODUCT
CAT NO.
VOLUME REACTIONS
5 mL
1,000 Rxn
Inhibitor-Tolerant RT-qPCR Mix
MDX016
50 mL
10,000 Rxn
Inhibitor-Tolerant RT-qPCR Mix exhibits a high tolerance to PCR inhibitors from clinical samples
SALIVA SPECIMENS
10% Saliva
20% Saliva
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.
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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 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.
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 MDX032 ( black ).
STOOL SPECIMENS
Adenovirus
Norovirus GII
Rotavirus A
Campylobacter jejuni
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 Fax: +1 901-333-8223 Toll Free: +1 800 327 6299
Email: info@meridianlifescience.com Orders: orders@meridianlifescience.com www.MeridianLifeScience.com
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