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Real-time qPCR for COVID-19 research

14 Jul 2020

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 kitsNGS 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:


 

COVID-19 detection using PrimeDirect RT-qPCR

Figure 1. Reliable detection of SARS-CoV-2 at 5, 50, 500, 5,000, and 50,000 copies with our one-step RT-qPCR kits. These data were generated by Takara Bio using publicly available protocols from the CDC. These data have not been validated by the CDC.

 


Publication: 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 PrimeScript III One Step 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 plays a critical role in the optimal management and control of this pandemic.


 

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-PCR Kit and PrimeDirect Probe RT-qPCR Mix in publicly available protocols with primer sets from the US and China CDCs, WHO, and Japan NIID. 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).

US CDC

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WHO

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China CDC

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Japan NIID

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The results were generated by Takara Bio by following the publicly available protocols. Use of our products in these protocols has not been validated by external agencies nor optimized by our R&D team. These data are provided for illustrative purposes only. Please refer to published literature for more information about these protocols.

Takara Bio Blog Team

Takara Biotech

14 Jul 2020

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