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The Latest Molecular Diagnostics Technologies to Accelerate Development and Stabilize at Room Temperature qPCR, LAMP and NGS Workflows
M. Amasio, T. Fouqueau, L. Gasiūnaitė, K.Y. Chan, K. Kaniuka, M. N. Podaru, S. Leonardia, S. Radic-Hunter
Introduction
Results
Molecular diagnostics testing is widely adopted to answer clinical questions, but challenges remain for its full democratization. Tests should reach users faster, be quickly developed and commercialized, and rely on simpler logistics for storage and shipping. Using glycerol-free reagents containing the correct excipients allows compatibility with lyophilization, enabling ambient temperature stabilization. Preoptimized formulations for specific applications accelerate assay development. This includes chemistries that can tolerate inhibition from different specimen types (e.g., blood, urine), which can be used in assay development for faster results without needing the extraction step. This study evaluates lyophilized, preoptimized PCR and LAMP reagents for blood and urine samples, demonstrating that these innovative reagents have the unique ability to simplify the experimental workflow by eliminating nucleic acid extraction, thereby accelerating assay development. Additionally, we introduce here a novel and unique set of lyophilized reagents for NGS library preparation, proving storage of NGS reagents at ambient temperature while preserving performance is now possible.
qPCR
NGS
CDC2
IGFBP5
Library Size
Library Yield
LEGEND:
Lyophilized |
Liquid
MDK
E. coli libraries for WGS (Whole Genome Sequencing) were prepared using lyophilized (in red) and liquid (in blue) formulations of NGS library preparation reagents as described in the Methods. The obtained libraries show comparable quality and yield. Both Bioanalyzer traces and qPCR amplification plots confirmed consistent profiles and yields for both formats.
LEGEND : BioRad MDX153 T
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 Lyo-Ready™ Direct RNA/ DNA qPCR Urine in lyophilized format (red) or liquid BioRad Reliance One-Step Multiplex Supermix™ (pink) in the presence of 10% Human Urine. The results illustrate that lyophilized, pre-optimized Lyo-Ready™ Direct RNA/DNA qPCR Urine shows high multiplexing capability and higher sensitivity to detect cancer-related mRNA markers from human urine compared to the competitor mix.
Typical Development
Raw Material Sourcing
Reagents Optimization
Test Prototype & Verification
Validation
LAMP
Meridian’s Acceleration
Pre-optimized reagents
Test Prototype & Verification
Validation
VARIABLE:
%G| %A| %T| %C
LEGEND:
Lyophilized |
Liquid
Materials and Methods
Sequencing results showed similar read numbers, mapping rates, GC bias, library diversity, and duplication rates between the liquid and lyophilized format. Specifically, the number of reads before trimming, normalized to those from liquid reagents, indicated good mapping efficiency. Both formulations exhibited uniform GC content, similar duplication rates, and expected sequence base content, as shown in the respective graphs and tables. Overall, the lyophilized NGS Library Preparation prototype performed comparably to liquid reagents, supporting its potential for simplifying logistics and storage without compromising data quality.
qPCR/RT-qPCR reactions: Urine sample: Gender Unspecified, Unfiltered, 5-Donor Pooled Human Urine (BioIVT) was used in this study. Urine-specific Lyo-Ready™ Direct RNA/DNA qPCR Urine (MDX153) from Meridian Bioscience, were lyophilized with primers and probes. The qPCR/RT-qPCR reactions were initiated by the addition of target-containing urine sample and run using the cycling conditions: 50°C for 10 mins; 95°C for 2 mins; 45 cycle of 95°C for 10 sec and 65°C for 30 sec. Reliance One-Step Multiplex RT-qPCR Supermix™ (Bio-Rad) in liquid format was tested in the same reactions using the concentrations recommended by the suppliers. LAMP/RT-LAMP reactions: Blood sample: Human Whole Blood K2EDTA (Cambridge Bioscience), Human Plasma ACCURUN® 315 SERIES 500 Positive Quality Control. Urine-specific Lyo-Ready™ Direct RNA/DNA LAMP Blood (MDX125) from Meridian Bioscience, was lyophilized with primers according to the product’s technical specifications and stored in a sealed pouch with silica. The RT-LAMP reactions were initiated by the addition of target-containing blood or plasma and run using the following cycling conditions: LAMP/RT-LAMP cycle: 65°C for 60 mins. WarmStart® LAMP kit (DNA&RNA) (NEB) and SuperScript™ IV RT-LAMP Master Mix (ThermoFisher) in liquid format were tested in the same reactions using the mastermix concentration recommended by the suppliers. All reactions were detected using intercalating fluorescent dye (SYTO™82, ThermoFisher). NGS library preparation and WGS sequencing: 100 ng of purified genomic DNA was fragmented to 300 bp using a Covaris LE220 ultrasonicator, then end-repaired, A-tailed, ligated with Illumina adapters, and PCR amplified (4 cycles) using the Meridian NGS Library Preparation Kit, either in liquid or lyophilized format. At each required step, the processed fragments were cleaned using AMPure XP beads by Beckman Coulter. Libraries were quantified using the Meridian qPCR Library Quantification Kit, and their size distribution was evaluated using Bioanalyzer. They were then normalized, pooled, denatured, and diluted to 12 pM before loading onto the MiSeq Reagent Kit v2 (500 cycles) cartridge. Sequencing was performed with a 2 × 250 bp configuration. Reads were mapped to the E. coli K12 MG1655 reference genome using BWA-MEM2, with bcftools. Data analysis included base calling, demultiplexing, number of reads, mapping efficiency, GC bias, duplication rate, and sequence base content. Data from libraries obtained from liquid and lyophilized kits were compared.
HIV-1 with plasma extract
Library Mapping
LEGEND:
MDX153 NEB Thermo
Library Lyophilized reagents
Average mapped reads (%) 99.79 ± 0.04
Liquid reagents
99.79 ± 0.12
Library Duplication
Primers for Dengue Type-1, HCV, WNV, Zika, or HIV-1 were incorporated into Lyo-Ready™ Direct RNA/DNA LAMP Blood and lyophilized (in red), or added to the NEB WarmStart® LAMP kit (DNA & RNA) (in orange) and ThermoFisher SuperScript™ IV RT-LAMP Master Mix (in black). Reactions were prepared as described in the Methods. Results show that the pre-optimized, lyophilized, mastermix delivers faster amplification across a broad range of viral RNA targets in the presence of multiple inhibitors.
Library Lyophilized reagents
Average duplication (%) 2.6 ± 0.2
Liquid reagents
2.7 ± 0.1
Conclusions
This study successfully demonstrated how the latest advances in molecular reagent technologies can support further assay developers reaching their goals of faster commercialization and more sustainable products. Firstly, with pre-optimized amplification chemistries for qPCR or LAMP which accelerate the development time of new MDx assays. Not only these specialized chemistries can considerably shorten the reagents optimization step but they can also facilitate the validation activities due to their robustness. Secondly, lyophilized and lyophilization compatible reagents do not have to concede in performance. This study demonstrated how performance is maintained at a market leading level across several technologies such as qPCR, LAMP or NGS while the reagents can be stored at ambient temperature. Through faster development of new assays and their handling without refrigeration, the presented technologies will for sure power the democratization of MDx not only in developed countries but globally.
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