High Inhibitor Tolerance Enables Greater Assay Sensitivity Compared to Universal Mixes
High Inhibitor Tolerance Enables Greater Assay Sensitivity Compared to Universal Mixes Saliva Specimen-specific TM Master Mixes for Multiplex Assays Using Crude Samples
Introduction With the unprecedented demand for molecular diagnostic testing during the COVID-19 pandemic, saliva emerged as a suitable alternative to traditional nasopharyngeal (NP) swabs for the identification of SARS-CoV-2 RNA in children and adults, and for genotyping SARS-CoV-2 variants. NP specimens are usually considered to have the highest detection rate for respiratory viruses
tropical diseases and respiratory diseases 3 . However, saliva is known to contain a range of PCR inhibitors and its DNA/RNA content is generally lower compared that other specimen types such as NP and blood 4 . This creates significant challenges in developing sensitive diagnostic assays, especially if direct detection is desired. As the demand increases for new assays that are more user-friendly, lower in cost
must remain on par with current methods in terms of sensitivity and reliability in order to meet consumer expectations. Meridian’s newest master mixes,
Specimen-Specific ™ Direct Saliva, help overcome the challenges in developing saliva direct detection assays. These mixes are optimized for sensitive and robust performance using crude saliva specimens and are ready-to-use, only
requiring the addition of assay- specific primers and probes. In addition, they are formulated for downstream lyophilization or air-drying to create ambient- temperature stable assays which are ideal for point-of-care applications. In order to evaluate the performance of Meridian’s saliva-specific mixes, a diagnostic company conducted a series of studies comparing the performance of Air-Dryable ™ Direct RNA/DNA qPCR Saliva (MDX131) against general mixes, including Meridian’s Air-Dryable ™
and are often used as the only specimen type in routine clinical practice and in many surveillance studies for the detection of respiratory viruses 1 . However, NP sample acquisition can become a bottleneck as it requires trained personnel for collection. In contrast, saliva specimens can be self-collected using non-invasive techniques which avoids the exposure of healthy persons to infected patients, reduces waiting times, and eliminates the need for personal protective equipment. Efforts to control transmissible
Combining direct detection with non-invasive sample collection, using specimens such as saliva, provides the ideal combination for next-generation screening assays. However, assay performance cannot be compromised and must remain on par with current methods in terms of sensitivity and reliability in order to meet consumer expectations.
infectious diseases rely on the ability to screen large populations and saliva
1-Step RT-qPCR Mix (MDX095). Using SARS-CoV-2 and influenza viral targets, the mixes were analyzed for their ability to efficiently amplify RNA from extracted samples and crude saliva samples. The data indicates a significant performance advantage with the saliva-specific mix, even after the mix has been dried-down and briefly stored at room temperature.
and can deliver faster sample-to-results, molecular methods that use crude samples and do not require DNA or RNA extraction will increase in popularity. Combining direct detection with non-invasive sample collection using specimens such as saliva provides the ideal combination for next-generation screening assays. However, assay performance cannot be compromised and
specimens are an appealing option that could simplify high-throughput sampling in point-of-care settings. Studies have shown that saliva is a suitable specimen type for detecting a range of diseases including STDs 2 ,
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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
50
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FAM
ROX
CY5
FAM
ROX
Extracted
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.
Study 2:
Aim: The performance of Air-Dryable™ Direct RNA/DNA qPCR Saliva (MDX131) was compared to Air-Dryable™ 1-Step RT-qPCR Mix (MDX095) in both air-dried and liquid format, for the detection of Flu B in saliva samples. Method: Influenza B (Flu B) samples were either processed by bead RNA extraction (sample IFB-1), or left as a crude lysate and treated with 100 µL of storage buffer #1 (sample IFB-2), storage buffer #2 (sample IFB-3), lysis buffer #1 (sample IFB-4) or lysis buffer #4. The Flu B samples were then briefly spun to collect the supernatant and human saliva from healthy individuals was added to dilute the samples by 10 3 or 10 4 (refer to Table 1). Samples were tested using standard cycling conditions (55°C x 10 min (RT step), 95°C x 3 min, followed by 5 cycles at 95°C x 10 sec and 60°C for 60 sec, and 40 cycles of 95°C x 10 sec and 60°C x 30 sec) or fast cycling conditions (55°C x 5 min, 95°C x 1 min, followed by 5 cycles 95°C x 1 sec and 60°C x 15 sec, and 38 cycles of 95°C x 1 sec and 60°C x 15 sec).
Patient Sample
Sample preparation
1 IFB-1-1 Extracted RNA from Flu B diluted 10 3 1 IFB-1-2 Extracted RNA from Flu B diluted 10 4
2 IFB-2-1 10 3 diluted Flu B in human saliva, treated with storage buffer #1 2 IFB-2-2 10 4 diluted Flu B in human saliva, treated with storage buffer #1 3 IFB-3-1 10 3 diluted Flu B in human saliva, treated with storage buffer #2 3 IFB-3-2 10 4 diluted Flu B in human saliva, treated with storage buffer #2 4 IFB-4-1 10 3 diluted Flu B in human saliva Diluted Flu B in human saliva, treated with lysis buffer #1 4 IFB-4-2 10 4 diluted Flu B in human saliva treated with lysis buffer #1 5 IFB-5-1 10 3 diluted Flu B in human saliva, treated with lysis buffer #2 5 IFB-5-2 10 4 diluted Flu B in human saliva, treated with lysis buffer #2 NC DEPC water Table 1 . Samples used for Flu B detection in human saliva samples using Air-Dryable ™ Direct RNA/DNA qPCR Saliva (MDX131) and Air-Dryable 1-Step RT-qPCR (MDX095) mixes. Samples were spiked with Influenza B viruses 1-5 as described in the table notes.
A. Standard Cycle
40
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IFB-1-1 IFB-1-2
IFB-2-2 IFB-3-1 IFB-3-2 IFB-5-1 IFB-5-2 IFB-NC IFB-NC IFB-2-1
Sample
B. Fast Cycle
40
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IFB-1-1 IFB-1-2
IFB-2-2 IFB-3-1 IFB-3-2 IFB-5-1 IFB-5-2 IFB-NC IFB-NC IFB-2-1
Sample
Air Dried (MDX095) | Wet (MDX095) | Air Dried (MDX131) | Wet (MDX131) | Reference Mix
Figure 2. Detection of Flu B from human saliva RNA-extracted and crude lysate samples using the Air-Dryable™ Direct RNA/DNA qPCR Saliva (MDX131) and Air-Dryable™ 1-Step RT-qPCR (MDX095) Mixes and either (A) standard or (B) fast cycling conditions. Air-dried MDX131 was the most efficient at detecting Flu B, followed by wet MDX131, then wet MDX095, dry MDX095 and the reference mix in both the fast and standard cycles.
Results: In comparison to Air-Dryable ™ 1-Step RT-qPCR Mix (MDX095), Air-Dryable ™ DIRECT RNA/DNA qPCR Saliva (MDX131) demonstrated faster Cts across all sample, for both standard and fast cycling conditions (Figure 2). This suggests that MDX131 has greater inhibitor tolerance to inhibitors found in saliva, including carry-over inhibitors still present in the extracted samples. Conclusion Air-Dryable ™ Direct RNA/DNA qPCR Saliva Mix (MDX131) and Air-Dryable ™ 1-Step RT-qPCR Mix (MDX095) were both able to perform better under fast cycling conditions, in contrast to a universal inhibitor tolerant reference mix, however Air-Dryable ™ Direct RNA/DNA qPCR Saliva Mix (MDX131) performed much better than Air-Dryable ™ 1-Step RT-qPCR Mix in the presence of both purified RNA from infected human saliva and RNA in crude infected human saliva lysate. The results show that universal inhibitor tolerant reference mix will work with saliva, but they are not as fast or as sensitive as a mix designed specifically for crude saliva lysates, making Air-Dryable ™ Direct RNA/DNA qPCR Saliva Mix ideal for SARS-CoV-2 and influenza viral targets, even after the mix has been dried-down and stored at ambient temperature.
References: 1. To, K.K., et. al. Additional molecular testing of saliva specimens improves the detection of respiratory viruses. Emerg Microbes Infect. 2017 Jun 7;6(6):e49. doi: 10.1038/ emi.2017.35. 2. Wang, C., et. al. A New Specimen for Syphilis Diagnosis: Evidence by High Loads of Treponema pallidum DNA in Saliva, Clinical Infectious Diseases . 2021 Nov 1; 73(9), e3250–e3258. doi:10.1093/cid/ciaa1613 3. Valinetz, E. and Cangelosi, G. A Look Inside: Oral Sampling for Detection of Non-oral Infectious Diseases. Journal of Clinical Microbiology . 2021;59(10). doi:10.1128/ JCM.02360-20 4. Oberkanins, C., et al. Use of saliva and salivary DNA for comprehensive genotyping based on RealFast and StripAssays. Ann Transl Med . 2017 Sep;5(Suppl 2):AB079. doi: 10.21037/atm.2017.s079.
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