Master Mixes for Molecular Ambient-Temperature Stable Assays DNA Polymerases and qPCR/RT-qPCR Master Mixes Formulated for Lyophilization or Oven/Air-Drying
APPLICATION KEY
Food Testing
Water Testing
Environmental
Blood Banking
Genetic Screening
Human Diagnostics
Vet Health
DNA Barcoding
Molecular diagnostic tests are progressively moving towards lyophilized and air-dried formats. There are several advantages for this, including room temperature shipping and storage, extended shelf-life and increased flexibility in sample volume. In order to be compatible with drying however, enzyme preparations must be glycerol-free and include specialized excipients that preserve the mixture as it is exposed to various conditions including freezing, temperature ramps, vacuum and dehydration. An ideal lyophilization formulation for example, should stabilize an enzyme in a freeze- dried format and allow very fast rehydration and reactivation of the enzyme preparations, without impacting its performance post rehydration.
Key Steps in Lyophilization Process
Plates, vials or strips are filled with solution
Solution is frozen solid
Frozen solution undergoes primary (Sublimation) and secondary (Desorption) drying processes under vacuum
Dried product in cake or bead form
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Air-Dryable
*(Wet mix can be dispensed into vials, plates or strips & subsequently air-dried under temperature-controlled conditions) HOW IT WORKS
Air-Dryable ™ qPCR Mix • Simple and easy to use glycerol-free mix containing Taq polymerase, reaction buffer, dNTPs, MgCl 2 , and air-dry excipients • Ideal for multiplex assays on POC diagnostic platforms or automated high-throughput instruments • Compatible with a range of air-drying protocols to produce an ambient temperature stable mix • Reduces cost and complexity PRODUCT HIGHLIGHTS Full enzyme activity following air-drying Activity of Air-Dryable qPCR Mix in both wet and air-dried formats were compared by singleplex qPCR assay on 10-fold dilution mouse cDNA template. The air-dried mix showed no loss of activity and sensitivity when compared to freshly prepared wet mix up to the assay limit of detection. Air-Dryable ™ RT-qPCR Mix • Simple and easy to use • Ideal for multiplex assays on POC diagnostic platforms or automated high-throughput instruments • Compatible with a range of air-drying protocols to produce an ambient temperature stable mix • Reduces cost and complexity
Applications:
PRODUCT
CAT NO. VOLUME REACTIONS
5 mL
1,000 Rxn
Air-Dryable ™ qPCR Mix, 4x
MDX082
50 mL
10,000 Rxn
Wet | Air-dried
Applications:
PRODUCT
CAT NO. VOLUME REACTIONS
5 mL
1,000 Rxn
Air-Dryable ™ 1-Step RT-qPCR Mix, 4x
MDX095
50 mL
10,000 Rxn
0% UTM+Sputum 5% UTM+Sputum 10% UTM+Sputum
PRODUCT HIGHLIGHTS High-tolerance to PCR inhibitors in sputum samples
Amplification profile of a mouse RNA target spiked into samples containing 10%, 5% or 0% artificial sputum in Universal Transport Media (UTM). The data illustrates that the performance of Air-Dryable 1-Step RT-qPCR Mix (MDX095) exhibits high tolerance towards inhibitors present in artificial sputum and UTM.
Air-Dryable Continued
Air-Dryable ™ Direct DNA qPCR Blood • Glycerol-free mix containing Taq polymerase, reaction buffer, dNTPs, MgCl 2 , and air-dry excipients • Specifically designed to overcome PCR inhibitors found in whole blood, plasma and serum • Ideal for developing direct (extraction-free) qPCR assays that are ambient-temperature stable
Applications:
PRODUCT
CAT NO. VOLUME REACTIONS
5 mL
1,000 Rxn
Air-Dryable ™ Direct DNA qPCR Blood, 4x
MDX092
50 mL
10,000 Rxn
PRODUCT HIGHLIGHTS
High reaction efficiency on plasma, serum and whole blood samples containing anticoagulants Plasmid DNA contains the target S. aureus , P. falciparum and Epstein-Barr virus was spiked into 10% serum or 10% K2-EDTA plasma and amplified in a triplex reaction using air-dried MDX092 format ( red ) and kits from supplier R ( orange ) and supplier T ( blue ). The results illustrate higher end florescence and better sensitivity with MDX092 than with mixes from supplier R and T. Staphylococcus aureus Plasmodium falciparum Epstein-Barr Virus
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Lyo-Ready
Lyo-Ready ™ qPCR Mix • Ready-to-use and glycerol-free qPCR master mix formulated with a specialized blend of excipients • Ideal for multiplex assays on automated high-throughput instruments • Compatible with a range of lyophilization protocols to produce freeze-dried beads or cakes
Applications:
PRODUCT
CAT NO. VOLUME REACTIONS
5 mL
500 Rxn
Lyo-Ready ™ qPCR Mix
MDX021
100 mL 10,000 Rxn
4 mL
500 Rxn
Lyo-Ready ™ qPCR Mix 2.6x
MDX023
100 mL 12,500 Rxn
PRODUCT HIGHLIGHTS Powerful multiplexing capacity
Three viral sequences, Cytomegalovirus (CMV) Adenovirus, Epstein-Barr Virus (EBV) and a DNA Extraction Control were amplified with equal efficiency from synthetic DNA templates with Lyo-Ready qPCR Mix in a quadruplex qPCR probe assay.
CMV
EBV
Adenovirus Amplification Plot
DNA Extraction Control
Amplification Plot
Amplification Plot
Amplification Plot
Cycle
Cycle
Cycle
Cycle
Lyo-Ready ™ 1-Step RT-qPCR Mix • Glycerol-free RT-qPCR master mix formulated with a specialized blend of excipients • Suited for multiplex assays and low-copy number targets • Virus mix (MDX062) is optimized for amplification of RNA or DNA viruses with high secondary structure from either extracted or intact virus samples PRODUCT HIGHLIGHTS High sensitivity amplification from both DNA and RNA templates Rotavirus A (dsRNA) and Adenovirus (dsDNA) were amplified in a single multiplexed RT-qPCR assay using inactivated crude viral lysates and Lyo-Ready 1-Step RT-qPCR Mix. The result illustrates that both viruses were amplified with high sensitivity, demonstrating the ability of Lyo-Ready 1-Step RT-qPCR Mix to detect low-copy number RNA and DNA targets simultaneously from a single sample.
Applications:
PRODUCT
CAT NO. VOLUME REACTIONS
10 mL
1,000 Rxn
Lyo-Ready ™ 1-Step RT-qPCR Mix
MDX024
100 mL 10,000 Rxn
10 mL
1,000 Rxn
Lyo-Ready ™ 1-Step RT-qPCR Virus Mix
MDX062
100 mL 10,000 Rxn
Adenovirus Rotavirus A
Cycle
Lyo-Ready ™ LAMP Mix • Ideal for POC diagnostic platforms or automated high-throughput instruments • Optimized for Loop-Mediated Isothermal DNA Amplification (LAMP) • Concentrated 4x master mix • Contains all the required excipients for subsequent lyophilization
Applications:
PRODUCT
CAT NO. VOLUME REACTIONS
5 mL
800 Rxn
Lyo-Ready™ LAMP Mix
MDX097
50 mL
8,000 Rxn
MDX097 | Supplier O | Supplier N
PRODUCT HIGHLIGHTS Better Sensitivity with Lower Sample Input The average time to results (TTR) for Lyo-Ready LAMP Mix (orange) and a mix from supplier O (tan) and supplier N (blue) were compared using a 10-fold serial dilution for the of BRCA1 gene (1,000, 100 and 10 copies). The relative concentrations of LAMP oligos were optimized to obtain earlier TTR compared to other experiments. The results demonstrate the increased sensitivity and greater reproducibility of the Lyo-Ready LAMP Mix, with earlier TTR values and a lower SD when compared to other suppliers. Reactions were incubated at 65°C for 60 min and TTR were measured at 1:10 of end fluorescence.
20
15
10
5
0
1,000 copies
100 copies
10 copies
DNA Polymerase
Aptamer Taq HS (Glycerol Free) • Highly suited to multiplex, high-throughput viral detection assays requiring high specificity • No activation step - reduce an assay run time by up to 15 minutes • Convenient room temperature reaction set-up • High enzyme concentration (50 U/μL) compatible
Applications:
PRODUCT
CAT NO. VOLUME REACTIONS
20 µL
50 mL
Aptamer Taq HS (glycerol free) (50 U/μL)
MDX015
500 µL
500 mL
with lyophilization protocols PRODUCT HIGHLIGHTS Fast Hot-Start
Comparison of qPCR performance of Aptamer Taq HS (glycerol free) vs an antibody hot-start Taq. PCR reactions were run with and without an initial 2 minute high temperature activation step for both polymerases and Ct values were compared. No difference was observed in the Ct values between the reactions for the Aptamer Taq HS (glycerol free), indicating that an activation step does not have an effect on polymerase performance. In contrast, the antibody hot-start Taq had a lower Ct value in the reaction with a 2 min high-temperature denaturation step indicating a improvement in performance over the reaction with no activation step. Overall, the data illustrates the immediate activation of the Aptamer Taq HS (glycerol free) allowing for faster hot-start and faster reaction protocols.
Condition
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Glycerol-Free Taq HS 50 U/µL • Lyophilization-compatible DNA polymerase ideal for automated high-throughput testing • Provided as separate ‘tubes’ of Taq, hot-start antibody, and enzyme dilution buffer • Can be used with Lyo-Ready qPCR Buffer (Cat #MDX022) to develop lyophilized qPCR mixes
Applications:
PRODUCT
CAT NO. VOLUME REACTIONS
20 µL
1,000 Units
Glycerol-Free Taq HS 50 U/µL
MDX011
500 µL 25,000 Units
4 mL
1,000 Rxn
Lyo-Ready qPCR Buffer 2.5x
MDX022
100 mL 12,500 Rxn
PRODUCT HIGHLIGHTS Comparison of freshly prepared and
lyophilized glycerol-free Taq HS master mixes A 10-fold serial dilution of template DNA was used to set up two sets of qPCR assays. One set was lyophilized (blue) and the other left as a wet mix (black). Results illustrate that lyophilization of Glycerol-Free Taq HS does not have an effect on the quality of the qPCR, as both the assays demonstrate identical efficiency and sensitivity.
Cycle
Reverse Transcriptase
Lyo-compatible MMLV-RT • High-concentration MMLV-RT suitable for incorporation into lyophilized RT-PCR assays • Demonstrates high efficiency in RT-qPCR assays and high sensitivity detection of low copy number RNA targets
Applications:
PRODUCT
CAT NO. VOLUME REACTIONS
8 µL
1,000 Rxn
Lyo-compatible MMLV-RT
MDX042
80 µL
10,000 Rxn
To test a sample or to ask additional questions, E: info@meridianlifescience.com
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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