Direct viral detection via qPCR
Viral detection directly from biological samples—no RNA purification
We are currently optimizing guidelines for using our PrimeDirect kit with SARS-CoV-2 samples. If you are interested in working with us to optimize performance with unpurified biological samples, please contact us. See below for information on our mix and our current protocol guidelines for detecting SARS-CoV-2 in different sample types.
NOTE: The products featured here are for Research Use Only. They are not for use in diagnostics procedures.
Detection of inactivated influenza A virus H1N1
PrimeDirect Probe RT-qPCR Mix may be used directly with many types of biological samples, without the need for RNA purification, saving valuable time and resources. We performed H1N1 detection directly on biological samples using PrimeDirect Probe RT-qPCR Mix, following the protocol shown below. Inactivated influenza A virus was spiked into mouth swab samples, nasal swab samples, and saliva samples at a concentration of 2 x 100–102 copies/µl.
| Test materials | Instructions |
|---|---|
| Mouth swab | Soak the sampled mouth swab into 200 µl PBS buffer, shake for 5 seconds, and then add 1 µl of supernatant to the reaction. |
| Nasal cavity swab | Soak the sampled nasal swab in 200 µl PBS buffer, shake for 5 seconds, and then add 1 µl of supernatant to the reaction. |
| Saliva | Directly add 1 µl of supernatant to the reaction. |
| Reagent | Volume |
|---|---|
| PrimeDirect Probe RT-qPCR Mix (2X) | 12.5 µl |
| Forward Primer (10 µM) | 0.5 µl |
| Reverse Primer (10 µM) | 0.5 µl |
| qPCR probe (10 µM) | 0.5 µl |
| Test sample | 1 µl |
| Influenza A virus H1N1 (2 x 100–102 copies/µl) | 1 µl |
| RNase Free H2O | 9 µl |
| Total | 25 µl |
PrimeDirect Probe RT-qPCR Mix enabled the detection of viral RNA directly from biological samples. The detection sensitivity from nasal cavity swab samples and saliva samples was 20 copies/μl, and the sensitivity from mouth swab samples was 2 copies/μl.
Jump down to view our protocol guidelines for SARS-CoV-2 detection.
Overview
Protocol guidelines for direct SARS-CoV-2 detection using qPCR
PrimeDirect Probe RT-qPCR Mix has been successfully tested by Takara Bio's R&D team on influenza A virus H1N1 spiked into biological samples, but we have not yet tested it with SARS-CoV-2. However, protocol guidelines for SARS-CoV-2 detection have been developed as a result of our collaborations with other research groups, and we are making them publicly available here. The guidelines cover a range of relevant sample inputs (protocols are different for each sample type), but the protocols have not been validated by Takara Bio and require optimization and validation by SARS-CoV-2 research laboratories.
By its very nature, the sensitivity of RT-qPCR detection performed directly on unpurified samples will not be identical to that of RT-qPCR carried out with extracted/purified RNA. The aim of our collaboration is to develop optimized protocols that are sensitive enough for SARS-CoV-2 testing without using RNA extraction solutions. If you are interested in collaborating with us, please contact us. As a reminder, the use of our products in these protocols has not been validated by external agencies nor optimized by our R&D team. When working with contagious samples, please follow your institution's or local authorities' biosafety laboratory guidelines.
Protocol guidelines are provided below for the following types of samples:
- Oropharyngeal/nasopharyngeal swabs diluted directly in PBS
- Oropharyngeal/nasopharyngeal swabs diluted directly in viral transport medium (VTM)—with prior heat inactivation
- Oropharyngeal/nasopharyngeal swabs diluted directly in VTM—no prior heat inactivation
- Sputum/saliva samples
- Bronchoalveolar lavage (BAL) fluid
- Stool samples
Note: When working with VTM, preliminary data demonstrates that heat inactivation enables better sensitivity.
Oropharyngeal/nasopharyngeal swabs diluted directly in PBS
This protocol can be used as a guideline for SARS-CoV-2 detection. It has not been validated by our R&D for SARS-CoV-2 detection from patient samples.
Sample collection
- Soak the sampled oropharyngeal/nasopharyngeal swab directly in 200 µl PBS.
- Gently agitate the swab to dilute viral particles in PBS.
- Add up to 2.5 µl of supernatant directly to the reaction mix (below).
Reaction mix
| Reagent | Volume |
|---|---|
| PrimeDirect Probe RT-qPCR Mix (2X) | 12.5 µl |
| Forward Primer | n* µl |
| Reverse Primer | n* µl |
| qPCR probe | n* µl |
| Viral PBS sample | up to 2.5 µl |
| RNase Free H2O | Fill to 25 µl |
| Total | 25 µl |
*Volume depends on the primer/probe set concentration. Please refer to primer/probe concentration recommendations from published SARS-CoV-2 detection protocols.
Thermal cycler program
| 90°C | 3 min | Viral inactivation and lysis | |||
| 60°C | 5 min | Reverse transcription | |||
| Number of cycles: 45–50 | |||||
| 95°C | 5 sec | Denaturation | |||
| 55–65°C | 30 sec | Annealing/extension* | |||
*Annealing/extension temperature depends on the primers used. Please follow current recommendations: 55°C for WHO and USA CDC primers, 60°C for China CDC primers.
Oropharyngeal/nasopharyngeal swabs diluted directly in VTM—with prior heat inactivation
This protocol can be used as a guideline for SARS-CoV-2 detection. It has not been validated by our R&D for SARS-CoV-2 detection from patient samples.
Sample collection and lysis step
- Soak the swab in 1 ml of viral transport medium (VTM; smaller volumes increase viral particle concentration). Some swabs are soaked in 3 ml VTM. This will decrease reaction sensitivity.
- Collect 200 µl of VTM.
- Heat at 99°C for 5 min.
- Centrifuge at 4,000 rpm for 5 min.
- Add up to 10 µl of supernatant directly to the reaction mix (below).
Reaction mix
| Reagent | Volume |
|---|---|
| PrimeDirect Probe RT-qPCR Mix (2X) | 12.5 µl |
| Forward Primer | n* µl |
| Reverse Primer | n* µl |
| qPCR probe | n* µl |
| Viral lysis sample | up to 10 µl |
| RNase Free H2O | Fill to 25 µl |
| Total | 25 µl |
*Volume depends on the primer/probe set concentration. Please refer to primer/probe concentration recommendations from published SARS-CoV-2 detection protocols.
Thermal cycler program
| 95°C | 30 sec | Initial denaturation | |||
| 60°C | 5 min | Reverse transcription | |||
| Number of cycles: 45–50 | |||||
| 95°C | 5 sec | Denaturation | |||
| 55–65°C | 30 sec | Annealing/extension* | |||
*Annealing/extension temperature depends on the primers used. Please follow current recommendations: 55°C for WHO and USA CDC primers, 60°C for China CDC primers. Note that this protocol currently shows a Ct delay of approximately 0–4 Ct according to the sample, compared to extracted RNA samples.
Oropharyngeal/nasopharyngeal swabs diluted directly in VTM—no prior heat inactivation
This protocol can be used as a guideline for SARS-CoV-2 detection. It has not been validated by our R&D for SARS-CoV-2 detection from patient samples.
Sample collection
- Soak the swab in 1 ml of viral transport medium (VTM; smaller volumes increase viral particle concentration). Some swabs are soaked in 3 ml VTM, which will decrease reaction sensitivity.
- Collect 10 µl of VTM and add 40 µl of PBS or RNase Free H2O.
- Add 5 µl of diluted sample directly to the reaction mix (below).
Reaction mix
| Reagent | Volume |
|---|---|
| PrimeDirect Probe RT-qPCR Mix (2X) | 12.5 µl |
| Forward Primer | n* µl |
| Reverse Primer | n* µl |
| qPCR probe | n* µl |
| Diluted VTM sample | 5 µl |
| RNase Free H2O | Fill to 25 µl |
| Total | 25 µl |
*Volume depends on the primer/probe set concentration. Please refer to primer/probe concentration recommendations from published SARS-CoV-2 detection protocols.
Thermal cycler program
| 90°C | 3 min | Viral inactivation and lysis | |||
| 60°C | 5 min | Reverse transcription | |||
| Number of cycles: 50 | |||||
| 95°C | 5 sec | Denaturation | |||
| 55–65°C | 30 sec | Annealing/extension* | |||
*Annealing/extension temperature depends on the primers used. Please follow current recommendations: 55°C for WHO and USA CDC primers, 60°C for China CDC primers. Note that this protocol can be used to detect high viral load samples. Typically, the Ct delay for such samples is approximately 4–6 Ct. We recommend performing 50 PCR cycles and always running negative controls. The initial VTM dilution will need to be optimized for specific workflows.
Sputum/saliva samples
This protocol can be used as a guideline for SARS-CoV-2 detection. It has not been validated by our R&D for SARS-CoV-2 detection from patient samples.
Sample collection
- Collect 200 µl of sample in a sample collection tube.
- Heat at 99°C for 5 min.
- Centrifuge at 4,000 rpm for 5 min.
- Add up to 2.5 µl of sample directly to the reaction mix (below).
Reaction mix
| Reagent | Volume |
|---|---|
| PrimeDirect Probe RT-qPCR Mix (2X) | 12.5 µl |
| Forward Primer | n* µl |
| Reverse Primer | n* µl |
| qPCR probe | n* µl |
| Sputum/saliva sample | up to 2.5 µl |
| RNase Free H2O | Fill to 25 µl |
| Total | 25 µl |
*Volume depends on the primer/probe set concentration. Please refer to primer/probe concentration recommendations from published SARS-CoV-2 detection protocols.
Thermal cycler program
| 90°C | 3 min | Viral inactivation and lysis | |||
| 60°C | 5 min | Reverse transcription | |||
| Number of cycles: 45–50 | |||||
| 95°C | 5 sec | Denaturation | |||
| 55–65°C | 30 sec | Annealing/extension* | |||
*Annealing/extension temperature depends on the primers used. Please follow current recommendations: 55°C for WHO and USA CDC primers, 60°C for China CDC primers.
Bronchoalveolar lavage (BAL) fluid
This protocol can be used as a guideline for SARS-CoV-2 detection. It has not been validated by our R&D for SARS-CoV-2 detection from patient samples.
Sample collection
- Collect 200 µl of sample in a sample collection tube.
- Heat at 99°C for 5 min.
- Centrifuge at 4,000 rpm for 5 min.
- Add up to 2.5 µl of sample directly to the reaction mix (below).
Reaction mix
| Reagent | Volume |
|---|---|
| PrimeDirect Probe RT-qPCR Mix (2X) | 12.5 µl |
| Forward Primer | n* µl |
| Reverse Primer | n* µl |
| qPCR probe | n* µl |
| BAL sample | up to 2.5 µl |
| RNase Free H2O | Fill to 25 µl |
| Total | 25 µl |
*Volume depends on the primer/probe set concentration. Please refer to primer/probe concentration recommendations from published SARS-CoV-2 detection protocols.
Thermal cycler program
| 90°C | 3 min | Viral inactivation and lysis | |||
| 60°C | 5 min | Reverse transcription | |||
| Number of cycles: 45–50 | |||||
| 95°C | 5 sec | Denaturation | |||
| 55–65°C | 30 sec | Annealing/extension* | |||
*Annealing/extension temperature depends on the primers used. Please follow current recommendations: 55°C for WHO and USA CDC primers, 60°C for China CDC primers.
Stool samples
This protocol can be used as a guideline for SARS-CoV-2 detection. It has not been validated by our R&D for SARS-CoV-2 detection from patient samples.
Sample collection
- Dilute the stool sample to 5–10% in PBS or RNase Free H20.
- Centrifuge for 5 min at 10,000–15,000 rpm.
- Add up to 2.5 µl of supernatant directly to the reaction mix.
Reaction mix
| Reagent | Volume |
|---|---|
| PrimeDirect Probe RT-qPCR Mix (2X) | 12.5 µl |
| Forward Primer | n* µl |
| Reverse Primer | n* µl |
| qPCR probe | n* µl |
| Stool sample | up to 2.5 µl |
| RNase Free H2O | Fill to 25 µl |
| Total | 25 µl |
*Volume depends on the primer/probe set concentration. Please refer to primer/probe concentration recommendations from published SARS-CoV-2 detection protocols.
Thermal cycler program
| 90°C | 3 min | Viral inactivation and lysis | |||
| 60°C | 5 min | Reverse transcription | |||
| Number of cycles: 45–50 | |||||
| 95°C | 5 sec | Denaturation | |||
| 55–65°C | 30 sec | Annealing/extension* | |||
*Annealing/extension temperature depends on the primers used. Please follow current recommendations: 55°C for WHO and USA CDC primers, 60°C for China CDC primers.
More 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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