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

Component Name	Product Number.	Size	Vial	Lid Color
2×Probe qPCR MasterMix	981201	0.5 mL	0.5 mL	Natural
Template Dilution Buffer	180701	1 mL	1.5 mL	Green
SRY and IC Primer-Probe Mix Powder	yp17-18880	50 T	0.5 mL Brown	Brown
SRY Positive Control (1E7 copies/μL)	pc18880	50 μL	0.5 mL	Yellow
Ultrapure Water	210806	1 mL	1.5 mL	Blue
Manual	sc17-18880	1 Set	NA	NA

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.

Class	Items
General laboratory equipment and consumables	Benchtop microcentrifuge, micropipettors, vortex mixer, disposable powder-free gloves, and sterile pipette tips with filters.
Kit-specific laboratory equipment and consumables	Manual or automated DNA purification kits and equipment, real-time fluorescent PCR instruments with FAM and Cy5 channels, consumables, a PCR reaction preparation station, and known positive and negative samples as controls.

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.

Component	Volume for one reaction	Volume for one quantitative assay run	Volume for one qualitative assay run
2×Probe qPCR MasterMix	10 μL	10 μL × (N + 9) × 1.1	10 μL × (N + 9) × 1.1
S. pyogenes and IC Primer-Probe Mix from step 8	4 μL	4 μL × (N + 9) × 1.1	4 μL × (N + 9) × 1.1
Total Volume	14 μL	14 μL × (N + 9) × 1.1	14 μL × (N + 9) × 1.1

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.

Component	(N + 2) tubes	ANC tube	6 SDSC tubes No. 1 – 6
PCR premix from step 10	14 μL each	14 μL	14 μL each
N + 2 DNA Samples	6 μL each	-	-
Ultrapure Water	-	6 μL	-
Six SDSC	-	-	6 μL each

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.

Component	(N + 2) tubes	ANC tube	APC tube
PCR premix from step 10	14 μL each	14 μL	14 μL
N + 2 DNA Samples	6 μL each	-	-
Ultrapure Water	-	6 μL	-
No. 4 from step 5	-	-	6 μL

Section 4: Run PCR

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

Process	Temperature	Duration	Cycle Number
Denature	95℃	10 minutes	1 cycle
PCR	95℃	15 seconds	45 cycles
	52℃	15 seconds
	72℃	60 seconds, collect signal in the FAM channel and in the Cy5 channel. Both quenchers are TAMRA.

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:

Channel	Criteria	Positivity
FAM	Ct is smaller than 40 and the amplification plot is an inverted S-shaped curve	FAM Positive
	Ct is larger than 40	FAM Negative
	No Ct	FAM Negative
Cy5	Ct is smaller than 40 and the amplification plot is an inverted S-shaped curve	Cy5 Positive
	Ct is larger than 40	Cy5 Negative
	No Ct	Cy5 Negative

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.

Documents

MSDS

Manaual