Detect SARS-CoV-2 in crude saliva samples—without RNA extraction

Note: We recommend using our newer PrimePath Direct Saliva SARS-CoV-2 Detection Kit—a complete kit for SARS-CoV-2 RT-qPCR detection from saliva.

Direct One-Step RT-qPCR Mix for SARS-CoV-2 (Cat. # 638329 & 638330) will be phased out soon. Contact your local sales representative for more information.

Note: We recommend using our newer PrimePath Direct Saliva SARS-CoV-2 Detection Kit—a complete kit for SARS-CoV-2 RT-qPCR detection from saliva.

Direct One-Step RT-qPCR Mix for SARS-CoV-2 (Cat. # 638329 & 638330) will be phased out soon. Contact your local sales representative for more information.

Direct One-Step RT-qPCR Mix for SARS-CoV-2 enables accurate one-step RT-qPCR on unpurified saliva samples to detect the presence and amount of virus. No RNA extraction step is needed; just mix the sample and pretreatment reagent, heat treat, and then combine with the RT-qPCR master mix. The whole protocol, from pretreatment through detection, takes less than one hour, enabling fast time to results. This protocol provides guidelines for multiplexed SARS-CoV-2 detection in saliva samples, but no primers are included (see Overview section below for more information).

Recent research from the Yale University School of Medicine has found a direct correlation between high levels of SARS-CoV-2 in saliva to an increased likelihood of hospitalization with severe disease and death (Silva et al. 2021). This correlation between viral load and severity of infection was not present in nasopharyngeal samples. These results suggest that monitoring viral load in saliva provides a better understanding of disease progression and can be used to stratify and effectively treat patients with SARS-CoV-2.

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Cat. #	Product	Size
638330	Direct One-Step RT-qPCR Mix for SARS-CoV-2	4,000 Rxns	USD $6287.00
Takara Bio’s Direct One-Step RT-qPCR Mix for SARS-CoV-2 allows the detection of SARS-CoV-2 in unpurified saliva samples, following pretreatment with the included Sample Prep Solution (not sold separately). Cat. No. 638330 provides enough reagents for 4,000 25-µl reactions. Notice to purchaser Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval. Documents Components
638329	Direct One-Step RT-qPCR Mix for SARS-CoV-2	200 Rxns	USD $404.00
Takara Bio’s Direct One-Step RT-qPCR Mix for SARS-CoV-2 allows the detection of SARS-CoV-2 in unpurified saliva samples, following pretreatment with the included Sample Prep Solution (not sold separately). Cat. No. 638329 provides enough reagents for 200 25-µl reactions. Notice to purchaser Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval. Documents Components

Overview

Our one-step PrimeScript III RT-qPCR mix contains the most sensitive reverse transcriptase on the market, outperforming Qiagen/TaqPath. PrimeScript III mix is widely trusted for use with a variety of sample types to detect low viral loads at the beginning of infection when only one test will be performed. Seeing the great need for rapid, accurate SARS-CoV-2 detection in saliva samples, we adapted and optimized our same gold-standard PrimeScript III technology for extractionless RT-qPCR detection from crude saliva samples, resulting in the Direct One-Step RT-qPCR Mix for SARS-CoV-2.

One-step PrimeScript technology has been used in two FDA emergency use authorizations (EUAs) for COVID-19 diagnostic tests. View more information on these EUAs, recent publications using this technology for SARS-CoV-2 detection, and data on compatibility with primer/probe sets from the US CDC, WHO, China CDC, and Japan NIID.

The Direct One-Step RT-qPCR Mix for SARS-CoV-2 protocol was optimized using the primers and probes recommended by the US CDC for N1, N2, and RNase P targets in a multiplex reaction. These custom primers and probes will need to be ordered separately.

Application data

The performance of Direct One-Step PrimeScript RT-qPCR Mix for SARS-CoV-2 for its intended application was confirmed across a range of target RNA copy numbers (Figure 1).

Synthetic RNA of the N1 and N2 regions of SARS-CoV-2 was spiked into unpurified saliva samples. The samples were processed according to the Direct One-Step PrimeScript RT-qPCR Mix for SARS-CoV-2 protocol, with or without the included Sample Prep Solution (SPS). Positive detection of SARS-CoV-2 N1 and N2 targets and human RNase P (RP) target was achieved from the samples treated with SPS. On the other hand, no Ct values for N1 and N2 targets were obtained (ND) from the samples that were not treated with SPS, although the Ct values of the RP target were still obtained due to the remaining human genomic DNA. The higher reaction volume that allowed higher sample input showed more consistent results, especially for detecting low copy numbers of the viral genome.

images/550-qPCR/CC-TechFigs/Direct_Covid-19_saliva_test_8984_T.jpg

20-µl reaction (total volume)
Sample prep solution	No	Yes
Target	N1	N2	RP	N1	N2	RP
Target RNA (copies)	0	ND	ND	27.6	ND	ND	24.6
10	ND	ND	27.5	38.1	36.4	24.8
100	ND	ND	27.8	34.1	32.7	24.5
1,000	ND	ND	27.9	31.0	29.7	24.7
10,000	ND	ND	27.9	27.3	26.6	24.3

50-µl reaction (total volume)
Sample prep solution	No	Yes
Target	N1	N2	RP	N1	N2	RP
Target RNA (copies)	0	ND	ND	27.7	ND	ND	24.9
10	ND	ND	27.7	36.7	36.3	25.0
100	ND	ND	27.5	34.5	32.9	24.7
1,000	ND	ND	27.6	30.6	29.7	24.8
10,000	ND	ND	27.7	27.1	26.5	24.9

More Information

Applications

Detection of SARS-CoV-2 in unpurified saliva samples (protocol provided)

References

Silva, et al. Saliva viral load is a dynamic unifying correlate of COVID-19 severity and mortality. medRxiv 10, doi: 10.1101/2021.01.04.21249236 (2021).

Additional product information

Please see the product's Certificate of Analysis for information about storage conditions, product components, and technical specifications. Please see the Kit Components List to determine kit components. Certificates of Analysis and Kit Components Lists are located under the Documents tab.

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