Fig. 2 Efficient multiplex reactions in the presence of inhibitors (20% stool) using Inhibitor-Tolerant RT-qPCR Mix (MDX016)
Another major concern for multiplexing assays, especially ones on high-complexity platforms that involve the manipulation of amplified PCR products, is the potential for false-negative results from PCR inhibition or false-positive results from PCR amplicon carryover. Most assay protocols require DNA or RNA extraction prior to testing in order to remove PCR inhibitors found in clinical samples such as nasopharyngeal or sputum specimens. However, these purification methods are problematic, can cause sample loss, and are not completely effective at removing all inhibitors. In addition, during the height of the first wave of the COVID-19 pandemic, the huge demand for molecular testing created an RNA extraction reagent shortage. Faced with the challenge of overcoming this supply shortage, assay manufacturers looked for alternative methods that rely on direct detection, entirely avoiding the need to purify the DNA or RNA. To address these issues, Meridian focused on creating inhibitor-tolerant molecular master mixes that allow clinical crude specimens to be run directly on a PCR machine, without performing purification or extraction first. Meridian’s first direct detection mix, Inhibitor-Tolerant RT-qPCR Mix (Catalog MDX016) was designed for qualitative multiplex assays using crude lysates or inhibitor-rich samples from different sources. The mixes can be used for direct amplification to substantially reduce steps in the assay workflow and
Adenovirus
Norovirus GII
Campylobacter jejuni
Rotavirus A
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 Inhibitor-Tolerant RT-qPCR Mix (MDX016) in the presence of inhibitors found in stool (up to 20% final volume).
Fig. 3 Multiplex Analysis of DNA and RNA Viral Targets Using Lyo-Ready ™ 1-Step RT-qPCR Virus Mix
improve assay turn-around times. POCT Molecular Assays & Multiplexing
Molecular testing methods require skilled technicians, sophisticated lab equipment, and cold shipment and storage to maintain the shelf-life of the product. Although these challenges are manageable in a centralized large laboratory, there are not suitable in the field where point of care (POCT) assays are needed. Meridian has developed two reagent POCT solutions for molecular assays. The first is a group of lyophilization compatible mixes (Lyo-Ready ™ ) that are glycerol-free and fully optimized with a specialized blend of lyo-excipients and which only require the addition of primers and probes to complete the assay. The formulation can be used as a liquid mix for an instant multiplex RT-qPCR diagnostic test or lyophilized into cakes or beads to create an ambient-
Respiratory Syncytial Virus (RSV) (green), Rotavirus A (purple) and Dengue Type 2 Virus (DENV-2) (red) were amplified in a single multiplexed RT-qPCR assay using inactivated crude viral lysates and lyophilized Lyo-Ready ™ 1-Step RT-qPCR Virus Mix (MDX062). The result illustrates that Lyo-Ready ™ 1-Step RT-qPCR Virus Mix can be used in a multiplex RT-qPCR assay to detect several low-copy number RNA targets simultaneously from a single sample.
temperature stable assay. The Lyo-Ready ™ 1-Step RT-qPCR Mix (Catalog MDX024) and Lyo-Ready ™ 1-Step RT-qPCR Virus Mix (Catalog MDX062) are widely used formulations in regulated assays around the world. The virus mix (Cat #MDX062) is specifically optimized for amplification of RNA or DNA viruses with a high secondary structure (reverse transcriptase remains active at 55-60°C).
The second POCT solution is Meridian’s new Air-Dryable ™ Direct RNA/DNA product line which combines the benefit of inhibitor- tolerance with the ability to air-dry the liquid mix to create an ambient-temperature stable assay. Unlike lyophilization, air-drying is relatively quick, inexpensive, and can be performed in-house with an oven. Novel sample-specific solutions of Air-Dryable ™ mixes have been designed to
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