Rapid, reliable, large-scale detection of the newly emerged coronavirus SARS-CoV-2 is crucial for controlling the COVID‑19 pandemic. An effective SARS-CoV-2 detection method must be able to detect just a few copies of the virus in a streamlined protocol that simplifies sample preparation and handling to reduce the risk of contamination, delivers fast, precise, and reproducible results, and can process a large number of samples per day.
Real-time quantitative PCR (RT-qPCR), a powerful tool for gene expression analysis, forms the basis for a wide variety of viral detection methods. A number of specialized RT-qPCR-based detection methods are available that can be used to develop effective SARS-CoV-2 detection protocols. One-step RT-qPCR allows efficient, specific cDNA synthesis and amplification in a single tube, reducing sample handling time and minimizing the risk of contamination. Direct RT-qPCR permits viral detection directly from crude biological samples, eliminating the need for time-consuming RNA purification while maintaining accuracy and sensitivity. Automated, high-throughput SARS-CoV-2 detection enables large-scale sample processing with precision, reproducibility, and speed.
When SARS-CoV-2 was first identified, Takara Bio’s one-step PrimeScript RT-qPCR technology was used to develop the first SARS-CoV-2 screening test (Zhu et al. 2020). These high-quality reagents, manufactured under ISO 13485:2016 conditions, have been utilized in multiple Emergency Use Authorizations issued by the Federal Drug Administration (FDA) to Ipsum Diagnostics, LLC, Hackensack University Medical Center, and LifeHope Laboratory for COVID-19 testing. Our RT-qPCR kits, which include the One Step PrimeScript III RT-qPCR Mix, the most sensitive one-step RT-qPCR master mix on the market, continue to be used worldwide with established primers for sensitive detection of SARS-CoV-2 down to five viral copies (Figure 1).
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.
In addition, researchers in two independent studies confirmed the superior performance of One-Step PrimeScript III RT-qPCR Mix compared to other one-step RT-qPCR solutions:
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 three leading competitors in order to test compatibility and sensitivity for SARS-CoV-2 detection. The authors showed that the lower limit of detection for this kit is 10 to 100 times greater than the competitors' solutions. They also demonstrated that the increased sensitivity of PrimeScript III one-step RT-qPCR reduces the number of false negatives. This study also compared the overall reaction speed for all the mixes and demonstrated that the PrimeScript III One Step RT-qPCR Mix outperforms other one-step RT-qPCR solutions in both sensitivity and speed.
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 viral RNA in samples that were previously found negative using a competitor 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.
Our direct RT-qPCR mixes may be used directly with many types of biological samples, without the need for RNA purification, saving valuable time and resources. The Direct One-Step RT-qPCR Mix for SARS-CoV-2 is designed specifically for unpurified saliva samples, enabling accurate one-step RT-qPCR to detect the presence and amount of virus. The entire RNA extraction-free protocol takes less than one hour and can be used to process many samples at once (Figure 2, Panel A). Since this mix is based on the exceptionally sensitive One Step PrimeScript III RT-qPCR Mix, one can expect it to provide comparable sensitivity. The performance of Direct One-Step PrimeScript RT-qPCR Mix for SARS-CoV-2 with unpurified saliva samples was confirmed across a range of target RNA copy numbers (Figure 2, Panel B).
Figure 2. The Direct One-Step PrimeScript RT-qPCR Mix for SARS-CoV-2 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.
Direct RT-qPCR from a variety of biological samples, such as viruses, bacteria, cultured cells, whole blood, and stool samples, is possible with the PrimeDirect Probe RT-qPCR Mix. Many protocol guidelines for SARS-CoV-2 detection have been developed using this technology. 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.
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. This system is a complete solution that includes chips and reagents, allowing the processing of thousands of samples per day. Each well of the 5,184-well chip holds nanoliter-scale reaction volumes, eliminating a preamplification step for reduced variability and decreasing costs.
We have generated proof-of-concept data from assays run on the SmartChip Real-Time PCR System that indicate sensitive and reproducible detection of human RNase P and viral nucleocapsid genes. In addition, we have developed an in-house SmartChip protocol to detect SARS-CoV-2. Sign up to watch a webinar describing the full data set.
The SmartChip Real-Time PCR System has been used to detect and quantify antibiotic resistance genes and SARS-CoV-2 in wastewater, which can reveal important information about COVID-19 transmission, as described in a webinar by Dr. Windi Muziasari, cofounder and CEO of Resistomap. Sign up to watch the webinar and learn how.
When the conventional RNA extraction approach wasn't cutting it—lengthening turnaround time and limiting the capacity of COVID-19 testing at Indiana Regional Medical Center—Dr. Bharathan, chair of the Department of Biology at IUP, and his lab stepped up to develop a fast, sensitive extractionless protocol using Takara Bio's PrimeDirect Probe mix.
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Overcoming roadblocks in the test development process to improve rural hospital COVID-19 testing (5 min)
When the conventional RNA extraction approach wasn't cutting it—lengthening turnaround time and limiting the capacity of COVID-19 testing at Indiana Regional Medical Center—Dr. Bharathan, chair of the Department of Biology at IUP, and his lab stepped up to develop a fast, sensitive extractionless protocol using Takara Bio's PrimeDirect Probe mix.
