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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