Saliva Specimen-specific Master Mixes for Multiplex Assays

Study 1

assays were set-up (5 µL 4x master mix (MDX131, MDX095 or reference mix), 1 µL primers and probe, 4 µL DEPC water) and 5 µL of RNA template or crude lysate was added to the mix and the reactions run 55°C x 10 min (RT step), 95°C x 3 min, and 46 cycles of 95°C x 10 sec 60°C x 45 sec and 69°C x 20 sec. Results: Air-Dryable ™ Direct RNA/DNA qPCR Saliva (MDX131) demonstrated faster Ct than Air-Dryable ™ 1-Step RT-qPCR Mix (MDX095) and the reference mix with both the extracted DNA and crude saliva templates. This suggests that MDX131 has greater inhibitor tolerance to inhibitors found in saliva than the other mixes, including carry-over inhibitors still present in the extracted samples. Air-Dryable ™ Direct RNA/DNA qPCR Saliva (MDX131) is an ideal option for ultra-sensitive saliva POC assays using either direct detection methods or extracted RNA.

Aim: To determine the performance of

Air-Dryable ™ Direct RNA/DNA qPCR Saliva (MDX131) in comparison to Air-Dryable ™ 1-Step RT-qPCR Mix (MDX095) and a reference mix in a multiplex assay detecting SARS-CoV-2 (targets: ORF1ab (FAM), nucleocapsid (N) protein (ROX) and GAPDH control (Cy5)). Both extracted RNA and crude saliva lysate from infected human saliva was used at the Median Tissue Culture Infectious Dose (TCID50) of 1000 Saliva samples taken from SARS-CoV-2 infected individuals were processed using an RNA extraction protocol, or briefly treated with a lysis buffer to generate a crude lysate (50 µL saliva sample, 50 µL lysis buffer heated at 95°C x 5 min and spun at 11,000 rpm x 1 min). The qPCR copies/mL. Method:

SARS-CoV-2 detection in extracted or crude saliva sample













Crude lysate

MDX095 | MDX131 | Reference Mix

Figure 1 . Detection of SARS-CoV-2 from RNA-extracted and crude lysate samples using Air-Dryable™ Direct RNA/DNA qPCR Saliva (MDX131), Air-Dryable 1-Step RT-qPCR (MDX095) and a reference mix. (A) Bar chart representing average assay Ct Values. (B) Corresponding amplification curves for the extracted and crude lysate assays. MDX131 shows faster detection of FAM, ROX and Cy5 probes in both RNA-extracted and crude samples than MDX095 and the reference mix.

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