Real-time qPCR for COVID-19 research

The recent emergence of the coronavirus SARS-CoV-2 (the virus that causes COVID-19) has led to a need for rapid and reliable detection. Takara Bio offers solutions to support researchers and clinicians studying this novel coronavirus, such as viral RNA purification kits, NGS kits, and RT-qPCR kits that can be used with established primers for sensitive detection of SARS-CoV-2 down to five viral copies (Figure 1).

Takara Bio reagents in FDA Emergency Use Authorizations 

Our high-quality reagents, manufactured under ISO 13485:2016 conditions, have been utilized in multiple Emergency Use Authorizations issued by the Federal Drug Administration (FDA) for COVID-19 testing:

• Ipsum Diagnostics, LLC
• Hackensack University Medical Center
• LifeHope Laboratory

Publications

Fast, cost-effective detection of SARS-CoV-2 variants with PrimeScript III one-step RT-qPCR

In a January 2021 bioRxiv paper, Korukluoglu and other authors from the WHO Influenza Center of Turkey and Bioeksen R&D Technologies report 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 One Step PrimeScript III RT-qPCR Mix paired with multiple hydrolysis probes and primers.

Their assay focuses on the recently emerged Spike N501Y and HV69-70del mutations, which are linked with reports of higher transmission rates and the potential for escape from neutralizing antibodies. Current methods for detecting SARS-CoV-2 variants are reliant on sequencing-based technologies, which are costly and time-consuming. Quick and accurate variant prevalence estimations are critical for epidemiology, as specific measures need to be rapidly implemented to prevent a surge in SARS-CoV-2 cases.

The assay’s cycling conditions are based upon an earlier publication from Bustin et al., which reported a <20 min assay for SARS-CoV-2 using the One Step PrimeScript III RT-qPCR Mix. This fast protocol using the Takara Bio mix 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.

Critically, 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. Now, a much higher number of positive SARS-CoV-2 samples can be screened for known mutations, and potential future variants can be added to this methodology as the virus continues to evolve.

Highly sensitive, fast SARS-CoV-2 detection with PrimeScript III one-step RT-qPCR

In an April 2020 bioRxiv paper, Brown et al., from the Great Ormond Street Hospital (NHS, London, UK), compared One Step PrimeScript III RT-qPCR Mix to QuantiFast Multiplex PCR +R master mix (Qiagen), TaqMan Fast Virus 1-Step Master Mix (Thermo), and TaqPath One Step RT-qPCR Master Mix (Thermo) in order to test compatibility and sensitivity for SARS-CoV-2 detection.  

In the initial experiment to test the limit of detection, One Step PrimeScript III RT-qPCR Mix showed superior sensitivity by consistently detecting 1 viral copy/µl with all three primer/probe assays. The authors showed that the lower limit of detection for this kit is 10 to 100 times greater than the competitors' solutions.

Next, the authors tested 74 real-life samples from nose and/or throat swabs from patients with suspected cases of SARS-CoV-2 infection. They compared PrimeScript III mix to the Quantifast multiplex kit (Qiagen). Of the 74 swab samples tested, 67/74 showed concordant results between both master mixes. However, among the discordant results, PrimeScript III mix could detect 7 patient samples with low RNA sample inputs (median Ct = 40.9 [range 38.5–41.9]), whereas these samples were undetected by Quantifast, showing that the increased sensitivity of PrimeScript III one-step RT-qPCR reduces the number of false negatives.

Furthermore, the authors compared the overall reaction speed for all mixes. The PrimeScript III one-step RT-qPCR experiment can be run in less than 1 hour (33% faster than Quantifast Multiplex PCR +R master mix), meaning even faster detection of SARS-CoV-2 infection. Faster reaction speeds will help increase the throughput for COVID-19 diagnostic labs.

Brown et al. conclusively showed that the One Step PrimeScript III RT-qPCR Mix outperforms other one-step RT-qPCR solutions in both sensitivity and speed. In a time where screening for SARS-CoV-2 infection is of utmost importance, the efficiency and rapidity of One Step PrimeScript III RT-qPCR Mix play a critical role in the optimal management and control of this pandemic.

In a July 2020 medRxiv paper, Reijns et al., from the University of Edinburgh, also found that One-Step PrimeScript III RT-qPCR Mix demonstrated superior sensitivity when performing a multiplex reaction on patient samples. Critically, they report that this mix was able to detect positive samples that were previously found negative using the TaqPath COVID-19 Combo Kit. Moreover, they reported that the assay developed using the Takara Bio mix was 1/10th the price of the other kits. These data reinforce the exceptional performance and value of our mixes for SARS-CoV-2 detection.

Compatibility with primer/probe sets from published SARS-CoV-2 detection protocols

We tested qPCR detection of SARS-CoV-2 using the One Step PrimeScript III RT-qPCR Mix and PrimeDirect Probe RT-qPCR Mix in publicly available protocols with primer sets from the US and China CDCs, WHO, and Japan NIID. The sensitivity of the assay varies depending on the nature of the template. The template used in the Japan NIID protocol was provided by the NIID, and the templates used in the other protocols were synthesized RNA prepared by Takara Bio. You can download our results by clicking on the PDF thumbnail images below.

The China CDC has also published a paper using our kits for SARS-CoV-2 detection (Zhu et al. 2019).

We have bulk formats of our one-step RT-qPCR kits. If you are interested, please contact us with your inquiry.

Explore industry-leading products that can advance your COVID-19 research: