Pushing the boundaries of COVID-19 testing
As the COVID‑19 pandemic swept across the globe in 2020, requirements for clinical assays evolved. Initially, the focus was on primers and targets specific to SARS‑CoV‑2. However, supply chain issues put constraints on a variety of key testing materials such as collection swabs, transport media, RNA purification kits, one-step RT‑qPCR reagents, cyclers, and plasticware for plates and pipette tips. Concurrent were significant increases in throughput demands as patient samples backed up. Accordingly, diagnostic tests were altered to focus on aspects that could address some of these obstacles, like multiplexing and extraction-free reverse transcription quantitative PCR (RT‑qPCR).
We have and continue to provide a number of specialized RT‑qPCR-based detection methods used to develop effective COVID‑19 tests.
Avoid false negatives with the most sensitive mix
For sensitive, fast, and cost-effective detection of SARS‑CoV‑2 variants, you can count on our One-Step PrimeScript III RT‑qPCR Mix (PrimeScript III mix). In independent studies, two research groups confirmed the superior performance and value of PrimeScript III mix compared to other one-step RT‑qPCR solutions.
- In a comparison between PrimeScript III mix and three leading products, QuantiFast Multiplex PCR and R master mix (Qiagen), TaqMan Fast Virus 1‑Step Master Mix (Thermo Fisher Scientific), and TaqPath One Step RT‑qPCR Master Mix (Thermo Fisher Scientific), Brown et al. 2020 showed that PrimeScript III mix outperforms the other mixes in sensitivity, speed, and detection limit (10–100X greater LoD).
- Reijns et al. 2020 also reported that PrimeScript III mix was able to detect viral RNA in patient samples that were previously found negative using the TaqPath COVID‑19 Combo Kit (Thermo Fisher Scientific). The mix demonstrated superior sensitivity for multiplex reactions and was 1/10th the price.
Save time and report results faster
The pandemic led to the development of a wide range of RT‑PCR based diagnostic assays. However, with a global population to monitor for viral spread, test turnaround times were lengthy and costly for many regions.
- Given the need to develop a fast and cost-effective diagnostic assay for SARS‑CoV‑2 that provided an assessment of viral load, Bustin et al. 2020 developed a <20 min assay using the PrimeScript III mix.
- This fast protocol was applicable to multiple types of equipment due to the single-tube reaction format, premixed reagents, and ability to handle 1‑min reverse transcription followed by 1‑sec denaturation and 1-sec annealing/extension steps.
Reduce time and costs using multiplex RT‑qPCR for known variant tracking
Authors from the WHO Influenza Center of Turkey and Bioeksen R&D Technologies reported the development of a rapid RT‑qPCR assay for identified SARS‑CoV‑2 mutations.
- The assay utilized Bioeksen’s Bio-Speedy SARS‑CoV‑2 N501Y Mutation Detection Kit, containing the PrimeScript III mix paired with multiple hydrolysis probes and primers specific to the Spike N501Y and HV69-70del mutations (UK Variant, Alpha; South African variant, Beta).
- The new variant detection method was assessed in over 1,000 clinical samples and results were 100% concordant with next-generation sequencing and Sanger sequencing technologies, indicating specificity and accuracy.
- This protocol supports screening of a much higher number of positive SARS‑CoV‑2 samples for known mutations and could be applied to the Delta (India), Gamma (Japan and Brazil), Epsilon (United States) or any other identified variants of concern that may emerge.
Simplify workflows and reduce costs with direct RT‑qPCR on crude samples
Our PrimeDirect Probe RT‑qPCR Mix was designed to work with a wide range of sample types, including blood, stool, and cultured cells, for detection of viruses and bacteria. Multiple groups utilized this solution to streamline their workflows:
- Lübke et al. described their simple SARS‑CoV‑2 detection protocol for respiratory samples using PrimeDirect Probe RT‑qPCR Mix in a Journal of Clinical Virology paper and a guest webinar
- Dr. Bharathan, in conjunction with Takara Bio, Indiana University of Pennsylvania, and the Indiana Regional Medical Center, successfully deployed a direct SARS‑CoV‑2 test that was utilized in their underserved rural community: watch video
We also developed the Direct One-Step RT‑qPCR Mix for SARS‑CoV‑2 in the midst of the pandemic for unpurified saliva samples, enabling accurate one-step RT‑qPCR to detect the presence and amount of virus. The entire RNA extraction-free multiplex protocol takes less than one hour and can be used to process many samples at once (Figure 1, Panel A). The performance of the Direct one-step mix for SARS‑CoV‑2 with unpurified saliva samples was confirmed across a range of target RNA copy numbers (Figure 1, Panel B).
Figure 1. Direct One-Step PrimeScript RT‑qPCR Mix uses a simple 3-step protocol to detect SARS‑CoV‑2 in saliva samples across a range of target RNA copy numbers. Panel A. Our simple protocol takes less than an hour to complete. Panel B. Unpurified saliva samples spiked with synthetic RNA of the N1 and N2 regions of SARS‑CoV‑2 were processed according to the Direct One-Step PrimeScript RT‑qPCR Mix for SARS‑CoV‑2 protocol, and positive detection was achieved for these targets as well as human RNase P (RP), the positive control.
Maximize your throughput with the SmartChip system
The SmartChip Real-Time PCR System automates SARS‑CoV‑2 detection, allowing processing of a large number of samples with precision, reproducibility, and speed. It minimizes false positives and negatives, accurately captures transcripts down to single-digit copy numbers, reduces hands-on time, and maximizes the number of samples processed per day. Moreover, it is a flexible platform that enables the rapid addition, subtraction, and modification of assays within panels and tests to streamline and speed up validation studies.
The system was deployed at many sites throughout the pandemic to handle sample backlogs and manage costs. Learn how the SmartChip system can enhance your diagnostic testing and development workflows.
- We developed an in-house SmartChip protocol to detect SARS‑CoV‑2 and generated proof-of-concept data using CDC assays run on the SmartChip Real-Time PCR System. Sign up to watch a webinar describing the full data set.
- Cofounder and CEO of Resistomap Dr. Windi Muziasari used the SmartChip system to detect and quantify antibiotic resistance genes and SARS‑CoV‑2 in wastewater, which can reveal important information about COVID‑19 transmission. Sign up to watch her webinar.
Frequently asked questionsWhich other applications do you currently support?
We have supported a countless range of applications using our wide array of techniques and tools. The applications covered in this section are highlights of some of the areas we have supported over time. Contact us if you would like to discuss your specific application.
Do you provide assays, primer sets, or panels for COVID-19, antibiotic-resistance surveillance, or pathogen detection?
Aside from our complete clinical testing kits, we currently do not offer any assays for these applications. In many cases, our partners already have their own panels defined or supplied. However, we have multiple relationships with providers of primers and panels and can help you find the resources and suppliers you need to build a panel for your applications.
Can you help with nucleic acid purification or sample collection?
We have a large selection of nucleic acid purification products that we would be glad to discuss with you and help to implement into your workflows. We do not currently provide tools for sample collection.
References
CDI Enhanced COVID‑19 Test EUA Summary, Hackensack University Medical Center (HUMC) Molecular Pathology Laboratory (April 15, 2020). Available at: https://www.fda.gov/media/137036/download
COV-19 IDx assay EUA Summary, Ipsum Diagnostics, LLC (April 1, 2020). Available at: https://www.fda.gov/media/136621/download
Bustin, S. et al. CoV2-ID, a MIQE-compliant sub-20-min 5-plex RT‑PCR assay targeting SARS‑CoV‑2 for the diagnosis of COVID‑19. Sci. Rep. 10, 22214 (2020). Available at: https://www.nature.com/articles/s41598-020-79233-x
Brown, J. R. et al. Comparison of SARS-CoV2 N gene real-time RT‑PCR targets and commercially available mastermixes. bioRxiv 2020.04.17.047118. Available at: https://doi.org/10.1101/2020.04.17.047118
Lübke, N. et al. Extraction-free SARS‑CoV‑2 detection by rapid RT‑qPCR universal for all primary respiratory materials. Clin. Virol. 130, 104579 (2020). Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7405857/
Korukluoglu, G. et al. 40 minutes RT‑qPCR Assay for Screening Spike N501Y and HV69-70del Mutations. bioRxiv (2021). Available at: https://doi.org/10.1101/2021.01.26.428302
Reijns, M. A. M. et al. A sensitive and affordable multiplex RT‑qPCR assay for SARS‑CoV‑2 detection. medRxiv 2020.07.14.20154005. Available at: https://doi.org/10.1101/2020.07.14.20154005
Zhu, N. et al. A novel coronavirus from patients with pneumonia in China, 2019. N. Engl. J. Med. NEJMoa2001017 (2020). Available at: https://www.nejm.org/doi/full/10.1056/NEJMoa2001017
Zhu, N. et al. supplementary appendix: RT‑qPCR protocol and primers used
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