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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, they 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 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 (Catalog MDX062) is specifically optimized for amplification of RNA or DNA viruses with a high secondary structure (reverse transcriptase remains active at 55-60°C, refer to Figure 3 for performance data).
mixes have been designed to overcome the inhibitors present in saliva/ sputum, blood, urine and stool. The new Air-Dryable ™ Direct RNA/DNA qPCR Saliva (Catalog MDX131) is a unique alternative for multiplexing flu/COVID/ RSV assays and is optimized for the sensitive detection of RNA targets from crudely processed saliva or sputum samples and only requires the addition of primers and probes to complete the assay (refer to Figure 4). The mix can be used in a liquid format or oven-dried to create a highly sensitive, ambient temperature stable, multiplex assay. Air-Dryable ™ Direct RNA/DNA qPCR Saliva (Catalog MDX131) can tolerate complex inhibitors in crude saliva such as sputum, mucin, UTM swabs. Syndromic respiratory panels have adopted this master mix to detect COVID-19, Flu, RSV, M. pneumoniae , etc), because of its sensitivity to less than 10 copies and its compatibility with liquid or dry assay formats.
MERS-CoV
Influenza A
RSV
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 ™ 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. Figure 3. Multiplex Analysis of DNA and RNA Viral Targets Using Lyo-Ready ™ 1-Step RT-qPCR Virus Mix
Figure 4. Respiratory Multiplex Testing Using Air-Dryable ™ Direct RNA/DNA qPCR Saliva (MDX131) Three respiratory pathogens, Influenza A, Middle East Respiratory syndrome coronavirus (MERS-CoV) and Respiratory Syncytial Virus (RSV) were amplified in a triplex qPCR assay in the presence of 35% Universal Transport Media (UTM) with 50% artificial sputum swab. The results illustrate that a higher performance was achieved with Air-Dryable ™ Direct RNA/DNA qPCR Saliva (blue) compared to an inhibitor-tolerant RT-qPCR mix (TaqPath ™ 1-step Multiplex Mix) from supplier T (red) and supplier Q (grey) (Ultra-Plex ™ 1-Step Tough Mix).
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