Streptococcus pyogenes Probe qPCR Kit

Description

Biofargo’s Streptococcus pyogenes Probe qPCR Kit is designed for both quantitative and qualitative detection of S. pyogenes-specific DNA in a real-time PCR test using hydrolysis probes. During amplification, Taq DNA polymerase amplifies the DNA using two primer-probe sets — one targets an S. pyogenes-specific DNA region and the other targets an endogenous Internal Control (IC) DNA region. At each PCR cycle, the 5’ nuclease activity of Taq DNA polymerase degrades the bound probes, releasing the quenched S. pyogenes-specific FAM signal and the IC-specific Cy5 signal.

The increase in FAM and Cy5 fluorescence corresponds to S. pyogenes-specific and IC-specific DNA amplification, respectively. When the fluorescence signal is confidently detected above the background signal, a Ct value can be determined. This Ct parameter can then be used to quantitatively and qualitatively evaluate S. pyogenes in samples.

Features

• One-tube PCR minimizes carry-over contamination

• High sensitivity with an analytical LOD of 100 copies/reaction

• High specificity with no cross-reaction with DNA from other organisms

• Amplification positive control provided in the kit

• False-positive results can be ruled out using the endogenous IC

• Compatible with various DNA purification methods

• Suitable for both qualitative and quantitative detection; for quantitative use, the linear range covers at least five orders of magnitude

• Sufficient for 50 reactions (20 µL per reaction)

Input Sample Requirements

It is highly recommended to use DNA purified with the Qiagen QIAamp DNA Mini Kit or the Thermo Fisher MagMAX Nucleic Acid Isolation Kit.

Kit Components

Storage and Handling

Reagents are shipped on ice and should be stored at -20℃ upon receipt. The kit is stable for at least one year from the date of receipt. Repeated thawing and freezing of the kit components should be avoided. All components must be thawed at room temperature before use, kept on ice during use, and stored again at -20℃ after use. Thawed components should be mixed well by gentle vortexing or pipetting before reaction setup.

Materials and Equipment Required but not Supplied in the Kit

Unless otherwise indicated, all materials are available through major laboratory suppliers.

Procedural Guidelines

• Use purified DNA as starting material

• Perform all steps at room temperature (20–25℃)

• Use sterile, disposable, nuclease-free pipette tips and microtubes

• Wear disposable gloves while handling reagents and DNA samples

Procedures

Section 1: Prepare 1E1–1E6 Copies/mL Serial Dilutions for Standard Curve (SDSC)

Note: Since the concentration of the S. pyogenes Positive Control is very high, all dilution steps must be performed in an independent area to avoid potential contamination of the samples or kit components.

1. Label six microcentrifuge tubes as 6, 5, 4, 3, 2, and 1, respectively.

2. Add 45 μL of Template Dilution Buffer into each tube. Use filtered pipette tips (applies throughout the procedure).

3. Transfer 5 μL of S. pyogenes Positive Control (provided in the kit, 1E7 copies/μL) into tube No. 6 and vortex vigorously for 1 minute. The concentration in tube No. 6 is 1E6 copies/μL. Place the tube on ice for later use.

4. Change the pipette tip, transfer 5 μL of the solution from tube No. 6 into tube No. 5, and vortex vigorously for 1 minute. The concentration in tube No. 5 is 1E5 copies/μL. Place the tube on ice for later use.

5. Change the pipette tip, transfer 5 μL of the solution from tube No. 5 into tube No. 4, and vortex vigorously for 1 minute. The concentration in tube No. 4 is 1E4 copies/μL. Place the tube on ice for later use.

6. Repeat the above operation until all six serial dilutions (SDSC) are obtained. Place them on ice for later use. These will serve as templates for generating the standard curve.

Section 2: Prepare Samples and Reagents

7. DNA Purification: Perform N + 2 DNA purifications for N specimens following the manufacturer’s instructions for the DNA purification kit. Of the two additional purifications, one is for the Purification Positive Control (PPC) using a known positive specimen as starting material, and the other is for the Purification Negative Control (PNC) using a known negative specimen. Store all purified DNA at -20℃ until use.

8. Prepare Primer-Probe Mixture: When using the kit for the first time, add 220 μL of ultrapure water into the microtube containing the S. pyogenes and IC Primer-Probe Mix Powder. Vortex for 10 seconds and briefly spin down for 10 seconds. The resulting solution is the S. pyogenes and IC Primer-Probe Mix. Keep it on ice for immediate use. Store any remaining solution at -20℃.

9. Calculate the Total Number of PCR Reactions:

• • For N samples, if quantitative analysis is performed with one replicate per sample, the recommended total reaction number is (N + 2) × 1 + 7 = N + 9.
•  
  • Among them, N + 2 reactions are for the N + 2 purified samples (from Step 7), one is for the Amplification Negative Control (ANC) using water as the template, and six are for the six SDSC dilutions.
•  
  • For qualitative analysis with one replicate per sample, the recommended total reaction number is (N + 2) × 1 + 2 = N + 4.
•  
  • Among them, N + 2 reactions are for the N + 2 purified samples (from Step 7), one is for the Amplification Negative Control (ANC) using water as the template, and one is for the Amplification Positive Control (APC) using the dilution obtained in Step 5 as the template.
•  
  • If more than one replicate is tested for each sample, recalculate the total reaction numbers accordingly.

Section 3: PCR Reaction Setup

10. Prepare the PCR Premix: In a 1.5 mL microtube, add the following two components according to the table below.

11. Set Up PCR Reactions for Quantitative Analysis (One Replicate per Sample):
Add each component into the N + 9 labeled tubes according to the table below. The six SDSC samples should be added after all other samples have been added and the tubes have been capped.

12. Alternatively, Set Up PCR Reactions for Qualitative Analysis (One Replicate per Sample): Add each component into the N + 4 labeled tubes according to the table below. The positive control should be added after all other samples have been added and the tubes have been capped.

Section 4: Run PCR

13. Perform PCR: Run the PCR using the parameters described in the table below.

Note: For some PCR machine models (e.g., Bio-Rad CFX96), if the three-step protocol does not yield satisfactory results, a two-step protocol can be used: 95°C for 10 minutes, followed by 45 cycles of 95°C for 15 seconds and 60°C for 60 seconds. Fluorescence signals should be collected during the second step of each cycle. The annealing temperature may need to be optimized.

Section 5: Data Analysis and Interpretation

14. Determine the Positivity or Negativity of All Control Reactions based on the following table:

15. Validation of the Purification Experiment: If the PPC reaction is FAM positive and the PNC reaction is FAM negative, the DNA purification process is valid and you may proceed to the next step. If the PPC reaction is FAM negative or the PNC reaction is positive, the DNA purification process is invalid. There is no need to further analyze the data, and the DNA purification must be repeated. Possible reasons for invalid purification include dysfunction of the DNA purification kit, mistakes during experimental procedures, failure of the PCR machine, or carry-over contamination of the PNC samples from previous experiments or from other positive samples.

16. Validation of the Amplification Experiment: If the No.2–No.6 SDSC reactions are FAM positive and the ANC is negative, the PCR amplification process is valid and you may proceed to the next step. If any of the No.2–No.6 SDSC reactions is FAM negative or the ANC is positive, the PCR amplification experiment is invalid. There is no need to further analyze the data, and the cause of invalid amplification must be identified. Possible reasons for invalid amplification include dysfunction of the PCR reagents, failure of the PCR instruments, or carry-over contamination of the ANC samples from previous experiments or from other positive samples.

17. Analysis of Quantitative Samples: If both purification and amplification experiments are valid, then the results of the samples can be analyzed. For quantitative analysis, take the log value of the concentrations of the six SDSC dilutions as the horizontal axis and the valid FAM Ct values as the vertical axis to draw a standard curve and obtain its regression equation. Then, use the valid FAM Ct value of each sample to calculate the log value of DNA concentrations from the regression equation, which is subsequently converted to DNA concentration in copies/mL. If the FAM signal of a reaction is not detected or its FAM Ct exceeds 40, the positivity of the corresponding sample depends on the positivity of its IC. If the IC is positive, the FAM result is valid and indicates a negative FAM reaction. If the IC is negative, the FAM result is invalid, and the purification and amplification process of that particular sample must be repeated.

18. Analysis of Qualitative Samples: For qualitative analysis, if the FAM signal is S-shaped and the Ct is below 40, the reaction of the sample is positive regardless of whether the Cy5 signal is positive or negative. If the FAM signal is not detected or the FAM Ct exceeds 40, the reaction of the sample is negative only if the Cy5 signal is positive. If both the FAM and Cy5 signals are negative, the reaction is invalid and must be repeated.

Limited Product Warranty

• Biofargo, Inc. warrants its products as set forth in the Biofargo’s General Terms and Conditions of Sale at www.biofargo.com

• If you have any questions, please contact us contact@biofargo.com

• The information in this guide is subject to change without notice.

• This product is intended for research purposes only. This product is not intended to be used for therapeutic and diagnostic purposes in humans or animals.

• DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, BIOFARGO INC. WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.

Q: Can this kit be used for digital droplet PCR (ddPCR)?

A: Yes.

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