Meridian is a primary manufacturer of specialized high-quality molecular reagents and offers solutions to a wide range of industries to diagnose and treat diseases, discover new therapeutics or develop tests for environmental, food and cosmetic safety.
Accelerating Efficiency: A Comprehensive Guide to Essential Reagents for High-Throughput Molecular Diagnostic Assays (MDx)
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High-throughput molecular diagnostic platforms require specific reagent solutions that are highly sensitive and specific, fast, stable, and enable a simplified workflow.
In the fast-paced world of healthcare, advancements in diagnostic technologies are shaping the way we identify and combat diseases. Traditional diagnostic testing is commonly characterized by costly and time-consuming processes, from sample collection to result interpretation, leading to delays in critical decision-making for patient care. High- throughput platforms, however, prioritize speed without compromising accuracy and can process large volumes of samples in a relatively short timeframe. As technology continues to advance, high-throughput platforms will evolve and reshape the landscape of healthcare, making diagnostics more cost-efficient, efficient, and precise.
High-throughput molecular diagnostic platforms require specific reagent solutions that are highly sensitive and specific, fast, stable, and enable a simplified workflow. Some key features beneficial for high throughput assays are glycerol-free and high-concentration formulations that maximize sample input for greater assay flexibility and ensure precision in liquid handling. In addition, reagent stability at room-temperature enables benchtop reaction set-up and storage, improving the overall workflow efficiency of an assay.
Benefits of Glycerol-Free Formats • Compatible with lyophilization • Compatible with air-drying • Ambient temperature and on-board instrument stability
Advantages to Glycerol-free and High-Concentration Enzymes
Benefits of High-Concentration Reagents • Maximize sample input • Smaller reaction volumes
Meridian has developed high-concentration, glycerol-free enzymes and master mixes to streamline high-throughput assay development, minimizing manufacturing time and enhancing user-friendliness. These optimized mixes aim to reduce sample variation and background noise, accelerate reactions, and maintain sensitivity levels. They feature glycerol-free enzymes and mastermixes for consistent performance, even in the presence of PCR inhibitors. Some mixes include dUTP to insure compatibility with UDGase system when required. Moreover, Meridian’s glycerol- free reagents are compatible with automated liquid handling and can be lyophilized or air-dried for stable ambient shipment and storage. Additionally, high-concentration enzyme formats are available, enabling greater sensitivity and flexibility in assay development while maximizing sample utilization.
• Greater flexibility in assay optimization
Benefits of Preoptimized Master Mixes • Robustness, minimizing sample to sample variation • Speed, faster development and faster time to results
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Sensitivity & Inhibitor-Tolerance Enzymes and Master Mixes with Superior Efficiency,
One of the key advantages of high-throughput diagnostic platforms is their ability to break down barriers to testing. The increased speed and efficiency make it possible to conduct large-scale screenings, monitor outbreaks more effectively, and address public health challenges with agility. Meridian’s DNA polymerases are designed to have a high affinity for DNA and deliver significantly faster amplification. Antibody, aptamer, or chemical-mediated hot start technologies are combined with the latest advances in buffer chemistry and PCR enhancers to result in enzymes and mixes that provide higher sensitivity and robustness, even when using crude samples where the conditions are suboptimal due to the presence of PCR inhibitors.
A. Lyo-Ready ™ Master Mixes demonstrate consistent high performance in fast cycling conditions
Lyo-Ready ™ Direct RNA/DNA qPCR Blood (MDX123)
Lyo-Ready ™ Direct DNA qPCR Blood (MDX122)
Standard cycling (54 min)
Fast cycling (37 min)
Standard cycling (54 min)
Fast cycling (37 min)
Standard vs fast cycling conditions. The performance of Lyo-Ready ™ Direct DNA (MDX122) and RNA/DNA qPCR Blood (MDX123) were tested on DNA and RNA targets respectively, in the presence of 5% EDTA whole human blood and compared using standard qPCR cycling conditions ((10 min @ 42 º C), 3 min @ 95 º C, 40 cycles of 10 sec @ 95 º C and 25 sec @ 60 º C) against the fastest cycling conditions available on a QuantStudio 7 Flex Real-Time PCR System ((5 min @ 42 º C), 3 min @ 95 º C, 40 cycles of 1 sec @ 95 º C and 1 sec @ 60 º C).
B. Lyo-Ready ™ Master Mixes exhibit high sensitivity in crude urine samples across a range of template concentrations in both liquid and dried-down assay formats
Lyo-Ready ™ Direct RNA/DNA qPCR Blood (MDX123)
Lyo-Ready ™ Direct DNA qPCR Blood (MDX122)
10,000 copies
10,000 copies
1,000 copies
1,000 copies
100 copies
100 copies
10 copies
10 copies
Lyophilized | Liquid
Performance of lyophilized vs liquid mix. Activity of A) Lyo-Ready ™ Direct DNA qPCR Urine in lyophilized ( red ) and liquid ( blue ) format was compared in the ability to amplify a 10-fold serial dilution of synthetic DNA (10,000, 1,000, 100 and 10 copies respectively) and B) Activity of Lyo-Ready ™ Direct RNA/DNA qPCR Urine in lyophilized ( red ) and wet ( blue ) format was compared in the ability to amplify a 10-fold synthetic RNA target (10,000, 1,000, 100 and 10 copies respectively) in the presence of 10% pooled human urine. The results illustrate that the dried mix retains the same performance as the wet mix, with equivalent sensitivity and reproducibility.
Room-temperature Stability for Benchtop Reaction Set-up & Storage Benchtop reaction setup and storage is crucial to the efficiency, flexibility, and cost-effectiveness of high-throughput operations. It enhances the overall workflow efficiency, reduces turnaround times, simplifies logistics, minimizes dependency on specialized equipment, and contributes to the overall reliability of assays in large-scale testing environments. Merdian’s enzymes undergo rigorous functional stability testing, including stability at room temperature in different formats (e.g. liquid or lyophilized) to ensure their compatibility with various applications including high-throughput platforms.
A. Performance of Air-Dryable ™ Direct RNA/DNA qPCR Saliva when stored at room temperature for one year
B. γ -actin
A. GAPDH
C. B2mg
Room-Temp | -20°C
Stability of Air-Dryable ™ Direct RNA/DNA qPCR Saliva (MDX131). The performance of Air-Dryable ™ Direct RNA/DNA qPCR Saliva (MDX131) in a triplex reaction on A) GAPDH, B) γ -actin and C) β 2mg DNA targets at two different concentrations, after being stored at -20°C ( blue ) and room temperature ( red ) for one month. The results illustrate that the stability of the Air-Dryable ™ Direct RNA/DNA qPCR Saliva is unaffected, with no loss of functional activity, even at room temperature for one month.
B. Comparison of lyophilized Lyo-Ready ™ Direct DNA qPCR Blood Mix
A) Lyo-Ready ™ DNA qPCR Blood (MDX122)
(B) Lyo-Ready ™ RNA/DNA qPCR Blood (MDX123)
Lyophilized | Liquid
(A) Lyo-Ready ™ Direct DNA qPCR Blood (MDX122) and (B) Lyo-Ready ™ Direct RNA/DNA qPCR Blood (MDX123) were lyophilized and the stability of the dried assays ( red ) was tested after 1 month at 37°C against fresh liquid mix ( blue ) in triplex assays in the presence of 5% EDTA blood. The lyophilized mixes retained the same performance as the liquid mix. Results suggest that Lyo-Ready Direct Blood mixes have a projected stability of more than 18 months at ambient temperature.
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Reagents Ideal for High-Throughput Multiplex Assays
High-throughput platforms, when combined with multiplexing capabilities, offer a formidable toolkit for diagnosticians, enhancing the depth of information obtained from each sample. Molecular multiplex assays can significantly lower the cost of testing multiple targets at one time. Compared to singleplex reactions, multiplex requires less reagents, less time, and less labor to achieve the same result. However, in mutiplex assays, the inherent variability between the physicochemical characteristics (e.g. length, GC content) of the amplified sequences , flanking regions and secondary structures can impact Ct values and create an imbalance in the amplification of certain targets. Meridian’s enzymes and master mixes are formulated for robust amplification under a wide range of temperatures, reaction and cycling conditions, and specimen types (blood, saliva, urine, and stool). They are ideal for multiplexing assays, which typically require diverse reaction conditions, and ensure that amplification is consistent across all the assay targets, allowing for accurate and reliable results for each analyte.
A. 55C MMLV-RT (MDX117) exhibits robust performance in multiplexed assays with samples containing sputum and Universal Transport Media (UTM)
Flu A
MERS-CoV
RSV
55C MMLV-RT
Performance of 55C MMLV-RT in multiplex RT-qPCR. 55C MMLV-RT was tested in a triplex one-step RT-qPCR assay on respiratory RNA virus targets (Influenza A, MERS-CoV and RSV). The results demonstrate the sensitivity and reproducibility of 55C MMLV-RT even in challenging conditions such as multiplex RT-qPCR reactions where targets may differ greatly in abundance.
B. Lyo-Ready ™ Direct DNA qPCR Saliva (MDX132) demonstrates sensitive detection in multiplex assays with samples containing sputum
Mycoplasma pneumoniae
ADV
CMV
0% Artificial sputum | 10% Artificial sputum | 20% Artificial sputum | 60% Artificial sputum
Performance of Lyo-Ready ™ Direct DNA qPCR Saliva. Mycoplasma pneumoniae genomic DNA, adenovirus (ADV) and cytomegalovirus (CMV) were amplified in a triplex qPCR assay in the presence of 0% ( Black ), 10% ( red ), 20% ( blue ) and 60% ( green ) artificial sputum. The results illustrate that the performance of the Lyo-Ready ™ Direct DNA qPCR Saliva is not affected by increasing concentrations of sputum.
Ordering Information
Glycerol-free & High-Concentration
Polymerases and Ligases MDX011 Glycerol-Free Taq HS, 50 U/ µ L
Glycerol-Free and dUTP Master Mixes MDX031 Hi-throughput dUTP qPCR Mix MDX060 Hi-throughput dUTP qPCR Lo-ROX MDX021 Lyo-Ready qPCR Mix MDX024 Lyo-Ready 1-Step RT-qPCR Mix MDX122 Lyo-Ready Direct DNA qPCR Blood, 4x MDX123 Lyo-Ready Direct RNA/DNA qPCR Blood, 4x MDX132 Lyo-Ready Direct DNA qPCR Saliva, 4x MDX133 Lyo-Ready Direct RNA/DNA qPCR Saliva, 4x MDX142 Lyo-Ready Direct RNA/DNA qPCR Stool, 4x MDX143 Lyo-Ready Direct RNA/DNA qPCR Stool MDX152 Lyo-Ready Direct DNA qPCR Urine, 4x MDX153 Lyo-Ready Direct RNA/DNA qPCR Urine, 4x MDX092 Air-Dryable Direct DNA qPCR Blood, 4x MDX121 Air-Dryable Direct RNA/DNA qPCR Blood, 4x MDX130 Air-Dryable Direct DNA qPCR Saliva, 4x MDX131 Air-Dryable Direct RNA/DNA qPCR Saliva, 4x MDX150 Air-Dryable Direct DNA qPCR Urine, 4x MDX151 Air-Dryable Direct RNA/DNA qPCR Urine, 4x MDX113 Lyo-Ready dUTP 1-Step RT-qPCR Mix MDX140 Air-Dryable Direct DNA qPCR Stool MDX141 Air-Dryable Direct RNA/DNA qPCR Stool
MDX170 Glycerol-Free Taq HS, 50 U/ µ L (premixed) MDX015 Aptamer Taq HS (Glycerol-Free, 50 U/ µ L) MDX018 High Conc. Glycerol-Free Bst (100 U/ µ L) MDX201 Glycerol-Free T7 RNA Polymerase (HC, 1,000 U/ µ L) MDX203 Glycerol-Free High-Fidelity Pfu (HC, 20 U/ µ L) Reverse Transcriptases MDX042 Lyo-Compatible MMLV-RT MDX117 55C MMLV-RT (200 U/ µ L) MDX205 Glycerol-Free HS Tth DNA Polymerase (HC, 200 U/ µ L)
MDX120 RNase Inhibitor (Glycerol Free, 40 u/ µ L) MDX202 Glycerol-Free RNase H (HC, 50 U/ µ L) Nucleotides and dUTP mixes
MDX058 dUTP Mix, 50 mM (Lithium) MDX059 dUTP, 100 mM (Lithium) MDX098 dATP, 100mM (Lithium) MDX099 dCTP, 100mM (Lithium) MDX100 dGTP, 100mM (Lithium) MDX101 dTTP, 100mM (Lithium) MDX051 dNTP Mix, 100mM (Lithium) MDX085 dNTP Mix, 40mM (Lithium)
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Meridian’s commitment to providing reliable and superior quality supply Meridian is committed at all levels of our organization to provide the highest quality enzymes that deliver value to our partners. Each of our facilities is certified to ISO 13485 and we operate with a high commitment to quality with an ongoing customer focus. Sourcing bulk quantities of products from Meridian offers: We provide bulk quantities as single batches to support you through the commercialization of robust, reliable assays. CONSISTENCY:
QUALITY:
We perform rigorous quality testing to ensure consistency across our product batches and that our reagents remain free of DNase contamination.
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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03/24
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