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.
Advancing Precision Medicine: Innovative qPCR & Genotyping Chemistries for Enhanced Assay Performance
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Over the past decade, cancer treatment has witnessed substantial changes in the way patients are managed, with a departure from a “one-size-fits-all” approach to an increasing focus on precision medicine based on genomic variants. Molecular oncology testing holds versatile applications across various stages of cancer care, offering invaluable insights that significantly impact patient management. Key areas where testing is instrumental include:
Globally, cancer is the second leading cause of death, responsible for one in six deaths. Over the past few decades, the risk of contracting some form of cancer has risen worldwide from 1 in 3 to 1 in 2 people and in 2040, the rate of cancer diagnosis is expected to be 47% higher than today’s current rate. Early detection of cancer combined with prompt treatment can improve the chances of recovery and long-term survival.
• Assessing an individual’s risk of developing cancer
Cancer Biomarkers Cancer biomarkers are substances or characteristics that can be objectively measured and evaluated as indicators of normal biological processes, pathogenic processes, or pharmacologic responses to therapeutic interventions. They play a crucial role in the detection, diagnosis, prognosis, and monitoring of cancer. By identifying specific molecules or changes in the body associated with cancer development and progression, biomarkers enable early detection, personalized treatment strategies, and improved patient outcomes. The most common biomarkers used for molecular cancer testing include point mutations (both genetic and somatic), circulating tumor DNA (ctDNA), DNA methylation, microRNA and long noncoding RNA (lncRNA). Traditional cancer testing relies on tissue biopsies, and several cancers, such as breast cancer, are still diagnosed through immunohistochemistry (IHC), a method that is time-consuming and requires skilled pathologists. However, this diagnosis method has the potential to be replaced by multiplex qPCR analysis using the same biomarkers as those validated for diagnosis by IHC. With the millions of breast cancer screening tests performed each year, high-throughput qPCR technology has the potential to improve breast cancer screening as well as other cancer screens tests, and dramatically decrease the turn-around time and cost, while improving the accuracy of cancer detection. Traditional Biopsy vs Liquid Biopsy Tissue biopsy is considered the gold standard for cancer diagnosis, however, it is also invasive, requires surgery, and may not be suitable for some hard-to-access areas such as lung or brain tissues. New liquid biopsy techniques that use • M onitor disease progression, response to treatment and safety profiling (toxicity) • Residual disease monitoring after initial treatment interventions • D iagnosis and tumor profiling which enables healthcare providers the ability to tailor treatment based on the specific molecular characteristics
Meridian’s Molecular Solution for Oncology Diagnostic Assays
biological fluids (e.g. blood, urine, saliva and stool) to test for cancer biomarkers have emerged as a more accurate, less expensive, and minimally invasive method. They are increasingly being used for cancer profiling, however due to the low abundance of the biomarker in the biological sample, a sensitive and specific technology must be used for analysis to prevent false negative results. NGS vs qPCR for Liquid Biopsy The primary techniques used for liquid biopsy analysis are qPCR and next-generation sequencing (NGS), and each has its own advantages and disadvantages. qPCR is known for its high sensitivity and specificity, allowing for the precise quantification of specific DNA sequences. It is a rapid and cost-effective method, suitable for detecting known mutations. NGS offers a more comprehensive analysis of the entire genomic landscape, allowing for the simultaneous detection of various mutations across multiple genes. NGS is particularly valuable for identifying rare mutations and uncovering broader genomic alterations. However, it tends to be more resource-intensive and time-consuming and may have higher associated costs compared to qPCR. The choice between qPCR and NGS depends on the specific goals of the liquid biopsy analysis, such as the need for targeted mutation detection versus comprehensive genomic profiling. Both techniques can be utilized in high-throughput platforms for large scale-screening, allowing for the efficient analysis of numerous samples in parallel. Or they can be adapted to point-of-care applications that bring diagnostic capabilities closer to the patient.
Meridian’s molecular chemistries support cancer testing with innovative qPCR master mixes. We have developed specimen-specific formulations for blood, saliva, urine, and stool that are highly inhibitor- tolerant, and enable detection down to single copies of RNA or DNA. They can prevent carryover inhibitors from affecting assay results in workflows using extracted DNA or RNA, or they can be used to amplify DNA or RNA directly from crude samples, skipping the extraction step altogether. For genotyping assays, our genotyping specimen-specific mixes provide clear allelic discrimination and cluster resolution, ideal for difficult SNPs. All of these specimen-specific master mixes are glycerol-free and formulated for air-drying or lyophilization, enabling the downstream flexibility to create ambient-temperature stable assays that do not require cold chain shipping and storage. The future of cancer testing is heading towards improved access, driven by technological advancements and innovative healthcare delivery models. New point-of-care testing solutions that prioritize accessibility, inclusivity and sustainability are sought after to address the inequality in cancer care across geographic, socioeconomic, cultural, and economic backgrounds. Meridian’s air-dryable and lyophilization-ready master mixes present a novel solution, eliminating the necessity for cold-chain shipment and storage which can restrict access to critical diagnostic tools, particularly in resource-limited settings. By democratizing access to cancer testing through innovative point-of-care solutions, Meridian hopes to promote equitable access to timely and effective cancer testing, leading to improved outcomes and ultimately, a reduction in the global burden of cancer. #closethegap
Inhibitor tolerance is a key factor in improving assay sensitivity as it minimizes the negative impact of inhibitory substances found in complex biological samples. Overall, master mixes that have high inhibitor tolerance increase the robustness of the amplification, which is particularly important in oncology testing that aim to detect low-abundance DNA and RNA targets such as point mutations, ctDNA, and lncRNA. Antibody hot start technologies are combined with the latest advances in PCR enzymatic systems to result in master mixes that provide high sensitivity and robustness, even when using crude samples or when the conditions are suboptimal due to the presence of carry-over inhibitors. High inhibitor-tolerance with ultra-sensitive detection
Maximizing Sensitivity - Detection Down to a Single Copy
We demonstrated that by air-drying MDX092 (Air-Dryable Direct DNA qPCR Blood) and maximising sample volume we could achieve sensitivity down to a single copy of an EGFR Exon 19 deletion (Figure 1).
Figure 1. MDX092 - Comparison of Sensitivity & Sample Volume. Plasma extract was spiked with synthetic template that incorporated an EGFR exon 19 deletion. The maximum sample volume that could be used with Roche Kapa Probe Force was 45% in a wet reaction. MDX092 was air-dried with primers and probe and therefore could be reconstituted in 100% of template.
MDX092 - EGFR Ex19 Deletion, 1 copy MDX092 - EGFR Ex19 Deletion, 10 copies MDX092 - EGFR Ex19 Deletion, 100 copies
Roche Kapa Probe Force - EGFR Ex19 Deletion, 1 copy Roche Kapa Probe Force - EGFR Ex19 Deletion, 10 copies Roche Kapa Probe Force - EGFR Ex19 Deletion, 100 copies
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MDX123
MDX123 vs TaqPath
Figure 2. Activity of Lyo-ready ™ Direct RNA/DNA qPCR Blood Mix (MDX123, red ) was compared in the ability to amplify a 10-fold long non-coding RNA target (MALAT-1 or PCA3) (10,000, 1,000, 100 and 10 copies respectively) in the presence of 5% DNase-treated-plasma and, compared against the activity of Quantabio ( grey ) and TaqPath ( black ). The results illustrate that the MDX123 mix shows higher level of sensitivity, end fluorescence and ability to tolerate DNase-treated plasma than suppliers.
MDX123 | Quantabio | TaqPath ™
Superior Sensitivity and Reproducibility in Samples Containing 10,000 copies to 1 copy of DNA in 10% Plasma
Figure 3. Activity of Lyo-Ready TM Genotyping Direct qPCR Blood ( blue ) was compared to KAPA Probe Force ( green ), TaqPath TM ( black ), and Type-it Fast ( light blue ) kits in a qPCR assay, using a 10-fold serial dilution DNA (10,000, 1000, 100, 10 and 1 copies respectively), in the presence of 10% plasma. The results illustrate that Lyo-Ready TM Genotyping Direct qPCR Blood has significantly higher sensitivity and reproducibility in assays detecting high or low copies of DNA template and in the presence of PCR inhibitors, resulting in tighter, more discrete, well-defined cluster.
MDX128 | KAPA Probe Force | TaqPath ™ | Type-it Fast
HPV Genotyping Directly from Swab
Human papillomavirus (HPV) Type 16 E6 - T350G mutation assay A) B) C)
D)
Homozygous WT (A) | Homozygous Mutant (C) | Heterozygous (A/C) | NTCs or undetermined
Figure 4. Human papillomavirus (HPV) type 16 and 18 has linked with high-risk to cervical, throat and many other types of cancer, an E6-T35OG mutation is used identify HPV type 16 from a throat swab in the presence of 40% UTM. A/ Lyo-Ready Genotyping Direct qPCR Urine, B/ Kapa Probe Force, C/ TaqPath and D/ Type-it Fast Kits. Homozygous allele A ( red ) and allele C ( blue ) and heterozygous allele A/C ( green ) with a NTC ( black ) and x for undetermined. Again, the results illustrate ability of Lyo-Ready Genotyping Direct qPCR Urine to form tighter, more distinct clustering and achieve accurate allelic discrimination of this clinically relevant mutation directly from universal transport media, used in viral swabs. Other suppliers of genotyping mixes show poor clustering and are not able to detect this mutation.
Accurate genetic analysis relies on highly sensitive and specific genotyping assays which can deliver tight fluorescent clusters and clear allele discrimination. A clear separation of fluorescent clusters reduces ambiguity which is important when trying to detect subtle differences in closely related alleles or challenging SNPs. For high-throughput assays, tight fluorescent clusters also streamline data analysis and interpretation while clear allele discrimination allows for efficient automation of genotyping calls, saving time and resources. Meridian’s genotyping mixes are optimized to provide highly specific allelic discrimination as demonstrated by excellent cluster separation, even in the presence of PCR inhibitors found in blood, urine, and stool. Furthermore, they possess excellent room- temperature stability for flexible pre- and post-PCR setup and analysis and can be used in a liquid or lyophilized format to create ambient-temperature stable assays, making them ideal for point-of-care (POC) devices. Tight fluorescent clusters and clear allele discrimination for genotyping assays Tighter Fluorescence Clusters with Clearer Allele Discrimination Compared to Other Commercially Available Mixes with Samples Containing a Range of Inhibitors Found in Blood K2-EDTA Whole Human Blood SNP differences between two strains of Epstein–Barr Virus (EBV) were tested using Lyo-Ready TM Genotyping Direct qPCR Blood Mix (MDX128), Roche Kapa Probe Force, TaqPath TM and Type-it Fast SNP Probe PCR Kits.
A/
B/
C/
D/
Allele A | Allele C | Allele A/C | NTC
Figure 5. 10% K2-EDTA whole human blood was tested with A/ Lyo-Ready TM Genotyping Direct qPCR Blood Mix, B/ Roche Kapa Probe Force, C/ TaqPath TM and D/ Type-it Fast SNP Probe PCR Kits, using EBV targets. Homozygous samples for allele A ( red ) and allele C ( blue ) and heterozygous samples for allele A/C ( green ) were compared with a NTC ( black ) and x for undetermined. The results illustrate ability of Lyo-Ready TM Genotyping Direct qPCR Blood (MDX128) to form tight clustering and so accurate allelic discrimination in the presence of whole blood unlike the other mixes.
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Multiplex detection of multiple genetic markers
Multiplexing in oncology assays enables the simultaneous detection and analysis of multiple genetic markers, allowing pathologists to gain a comprehensive profile of the tumor, leading to more accurate diagnostics, targeted therapies, and personalized treatment strategies. However, the challenge with multiplex assays is the inherent variability between the physicochemical characteristics (e.g., length, GC content) of the amplified sequences, flanking regions, and secondary structures which can impact Ct values and create an imbalance in the amplification of certain targets. Meridian’s enzymes and qPCR master mixes are formulated for multiplex amplification under a wide range of temperatures, reaction and cycling conditions, and specimen types (blood, saliva, urine, and stool). They are ideal for multiplex assays, and ensure that amplification is consistent across all the assay targets, allowing for accurate and reliable results for each analyte.
Fast and More Sensitive Detection of Cancer Markers in Urine Samples
CDC2
IGFBP5
MDK
Figure 6. Three tumor-related mRNA markers (CDC2 kinase, IGFBP5 and MDK) were amplified from total RNA (from a bladder cancer patient) in a triplex reaction using lyophilized Lyo-Ready ™ Direct RNA/DNA qPCR Urine (MDX153, red ) or liquid BioRad Reliance One-Step Multiplex Supermix ™ ( purple ) in the presence of 10% Human Urine. The results illustrate that Lyo-Ready ™ Direct RNA/DNA qPCR Urine (MDX153) is able to detect cancer related RNA markers from urine faster and with higher sensitivity compared to the other mix.
MDX153 | BioRad Reliance 1-Step
As the burden of cancer continues to rise, point-of-care (POC) testing offers a promising solution for early cancer detection and treatment leading to better long-term patient survival. POC techniques are already used in primary care for screening patients for gastrointestinal cancer and advances in technology and translational medicine suggest that POC tests may, in the future, be able to diagnose gastrointestinal cancers at the patient’s bedside, in the outpatient clinic or even by the patient self-testing at home. Meridian’s specimen-specific mixes can be used as a liquid or dried down to create ambient-temperature stable assays, ideal for point-of-care (POC) testing. Suitable for POC oncology assays and lyophilization
Ability to Extend Shelf-life to Over 12 Months in Lyophilized Format
Homozygous A/A
Heterozygous A/C
Homozygous C/C
Liquid | Lyophilized
Figure 7. Lyo-Ready TM Genotyping Direct qPCR Blood (MDX128) was lyophilized, and the stability of the dried assays ( red ) was tested after 1 month at 37°C against fresh liquid mix ( blue ) in the presence of 5% EDTA Blood. Results suggest that Lyo-Ready TM Genotyping Direct qPCR Blood mix has a projected stability of more than 18 months at ambient temperature.
No Loss of Enzyme Performance (Sensitivity and Robustness) after Lyophilization
A) Lyo-Ready ™ Direct DNA qPCR Urine (MDX152)
B) Lyo-Ready ™ Direct RNA/DNA qPCR Urine (MDX153)
10,000 copies
10,000 copies
1,000 copies
1,000 copies
100 copies
100 copies
10 copies
10 copies
Liquid | Lyophilized
Figure 8. Activity of A) Lyo-Ready ™ Direct DNA qPCR Urine (MDX152) 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 (MDX153) 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 presence of 10% pooled urine. The results illustrate that the dried mix retains the ability to efficiently amplified to the same level as the wet mix with the same level of sensitivity and reproducibility for both.
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Ordering Information
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 MDX140 Air-Dryable Direct DNA qPCR Stool, 4x MDX141 Air-Dryable Direct RNA/DNA qPCR Stool, 4x MDX128 Lyo-Ready Genotyping Direct qPCR Blood, 4x MDX158 Lyo-Ready Genotyping Direct qPCR Urine, 4x MDX148 Lyo-Ready Genotyping Direct qPCR Stool, 4x MDX168 Lyo-Ready Genotyping Direct qPCR FFPE, 4x
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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