Description
The MycAway™ Mycoplasma qPCR Detection Kit is a product designed for qualitative detection of mycoplasma contamination in various sources, including raw materials, cell banks, virus seeds, viral or cell harvesting solutions, and cells used in clinical treatments, among others. This kit utilizes Taqman fluorescent probes (FAM and VIC) and employs Multiple polymerase chain reaction (PCR) techniques to separately detect the target and internal control. It has been validated for specificity, detection limit, and robustness according to EP <2.6.7> standards, demonstrating high sensitivity, specificity, efficiency, and safety. The detection limit is equal to or below 10 CFU/mL.
For nucleic acid extraction, this product can be used in conjunction with the MolPure® Magnetic Residual DNA Sample Preparation Kit (Cat#18461), which employs manual extraction methods. Alternatively, nucleic acids in samples can be automatically extracted using the AutoPure 32A automatic nucleic acid extractor (Cat#80501) and the MolPure® Mag32 Residual DNA Sample Preparation Kit FA (Cat#18462). It's important to note that kits containing Cat#18461 and Cat#40619 have undergone comprehensive validation; for detailed validation information, please contact our technical support. After pretreating the samples to remove interfering impurities and obtaining purified nucleic acids, a qPCR reaction is performed using a Real-Time PCR amplifier, and the fluorescence signal from the probe is collected and analyzed.
Cat.No. |
40619ES25 / 40619ES60 |
Size |
25 T / 100 T |
Detection Limit |
10 CFU/mL |
Detect method |
qPCR method (Taqman fluorescent) |
Duration |
4 hours |
Fluorescent probe |
FAM (Target channel);VIC (Internal channel) |
Covered mycoplasma |
≥ 100 |
Validation |
Validated according to EP <2.6.7> |
Components
Components No. |
Name |
40619ES25 |
40619ES60 |
40619-A |
4×MyqPCR Reaction Buffer |
250 μL |
1 mL |
40619-B |
MyPrimer & Probe MIX |
25 μL |
100 μL |
40619-C* |
Internal Control (IC) |
25 μL |
100 μL |
40619-D** |
Positive Control (PC) |
500 μL |
2 mL |
40619-E*** |
DNA Dilution buffer |
1 mL |
4×1 mL |
40619-F**** |
Ultrapure water |
500 μL |
2×1 mL |
*IC: Internal control;
**PCS: Positive control solution ,the concentration is 1,000 copies/µL.
***DNA Dilution buffer: used for IC dilution and the template of NTC and NCS.
****Ultrapure water: used for the preparation of qPCR Mix。
Storage
This product should be stored at -25~-15℃ for 2 year.
*Upon receipt of the kit, please check whether all components are complete and immediately store them in -25~-15℃ condition if not perform the assay immediately. Please note 40619-B should be stored away from light.
Instructions
- Preparations before experiment
1) Prepare the required reagents and materials which will be used in the experiment.
2)Confirm the suitability of the qPCR instrument
This kit can be used on the types of qPCR instrument as below:
- Bio-Rad: CFX96
- Thermo Scientific: 7500 Real-Time PCR System;QuantStudio™5;
- Experiment method
1) DNA Extraction
We recommend to use ‘ Magnetic Residual DNA Sample Preparation Kit’ (Cat#18461ES for manual extraction and Cat#18462ES for auto extraction) for the DNA extraction, you can visit ‘ http:/www.yeasenbiotech.com ’ for the
detailed information and the purchasing.
The kit (Cat#40619ES) contains an internal control (IC). If add the IC to the samples prior to DNA extraction, it can verify the complete process (included DNA extraction and qPCR reaction). If add the IC to the qPCR master mix
directly, the IC will acts as a qPCR control only.
2)qPCR Mix preparation
- According to the sample amount which included Positive control (PCS), No template control (NTC), Negative control solution (NCS) and Testsample (TS) to calculate the number of reactions. Prepare 2 reactions in parallel for
each sample in generally.
* PCS:Positive control solution;NTC:No template control;NCS:Negative control solution;TS:Test sample. There is no need to perform
the sample extraction for PCS and NTC, but NCS and TS are needed.
Reaction wells(M1)=(1×NCS + N×TS) ×2
Reaction wells(M2)=(1×PCS + 1×NTC) ×2
Reaction wells(M3)=(1×PCS + 1×NTC+ N×TS) ×2
- Pre-thaw the required amount reagents on ice according to the experiment design and Tables below.
- Calculate the amount of qPCR Mix according to the Number of reactions. Please note, ifthe kit will be used forGMP activities such as product release, we recommended used Table 1 and 2 for the preparation; If the kit will just used for research and there is no need to add IC before the extraction after the evaluation, then follow Table 3 for
the preparation. Please note that not all M1, M2 and M3 are needed to be prepare.
Component |
Volume(1×40μL Reactions) |
Volume(M1×40μL) |
4× MyqPCR Reaction Buffer |
10 μL |
(M1+2) ×10 μL |
MyPrimer & Probe MIX |
1 μL |
(M1+2) ×1 μL |
ROX |
0.8 μL /0 μL** |
(M1+2) ×0.8 μL /0 μL |
Purified water |
Up to 20 μL |
Up to (M1+2) ×20 μL |
Total |
20 μL |
(M1+2) ×20 μL |
Table 1 qPCR Mix system for M1
*The configuration system in Table 1 is based on the premise that IC is added before extraction for both NCS and TS, so it is not necessary
to add IC when qPCR Mix preparation. IC adding method before extraction: Firstly, dilute IC by 20 times with DNA diluent, and add 1μL
diluted IC into each 100μL test sample for the further extraction.
**This kit does not contain ROX Reference Dye. If ROX reference dye is needed for the Real Time PCR amplifiers that you are currently using, 50×ROX Reference Dye (Cat#10200ES) is recommended for use. In this case, the added volume is 0.8μL, as shown in Table 1. If using other
brands of ROX products, please refer to their instructions for ROX addition. If no ROX reference dye is required, the added volume is 0 μL.
Component |
Volume(1×40μL Reactions) |
Volume(M2×40μL) |
4× MyqPCR Reaction Buffer |
10 μL |
(M2+2) ×10 μL |
MyPrimer & Probe MIX |
1 μL |
(M2+2) ×1 μL |
Internal Control(IC) |
1 μL* |
(M2+2) ×1 μL /0 μL |
ROX |
0.8 μL /0 μL** |
(M2+2) ×0.8 μL /0 μL |
Purified water |
Up to 20 μL |
Up to (M2+2) ×20 μL |
Total |
20 μL |
(M2+2) ×20 μL |
Table 2 qPCR Mix system for M2
*The configuration system in Table 2 is based on the premise that IC is not added in PCS and NTC before extraction, so IC needs to be added during qPCR Mix preparation. IC adding method after extraction: Dilute IC by 100 times with DNA diluent and add 1μL diluted IC into
each qPCR Mix system.
**This kit does not contain ROX Reference Dye. If ROX reference dye is needed for the Real Time PCR amplifiers that you are currently using, 50×ROX Reference Dye (Cat#10200ES) is recommended for use. In this case, the added volume is 0.8μL, as shown in Table 1. If using other
brands of ROX products, please refer to their instructions for ROX addition. If no ROX reference dye is required, the added volume is 0 μL.
Component |
Volume(1×40μL Reactions) |
Volume(M3×40μL) |
4× MyqPCR Reaction Buffer |
10 μL |
(M3+2) ×10 μL |
MyPrimer & Probe MIX |
1 μL |
(M3+2) ×1 μL |
Internal Control(IC) |
1 μL* |
(M3+2) ×1 μL /0 μL |
ROX |
0.8 μL /0 μL** |
(M3+2) ×0.8 μL /0 μL |
Purified water |
Up to 20 μL |
Up to (M3+2) ×20 μL |
Total |
20 μL |
(M3+2) ×20 μL |
Table 3 qPCR Mix system for M3
*The configuration system in Table 3 is based on the premise that IC is not added to samples before extraction, so IC needs to be added during qPCR Mix preparation. IC adding method after extraction: Dilute IC by 100 times with DNA diluent and add 1μL into each qPCR Mix
system.
**This kit does not contain ROX Reference Dye. If ROX reference dye is needed for the Real Time PCR amplifiers that you are currently using, 50×ROX Reference Dye (Cat#10200ES) is recommended for use. In this case, the added volume is 0.8μL, as shown in Table 1. If using other
brands of ROX products, please refer to their instructions for ROX addition. If no ROX reference dye is required, the added volume is 0 μL.
3)Templates adding
- Mix the qPCR Mix with sufficient shaking, centrifuge at low speed and collect the residualliquid from the cap
to the bottom of the tube.
- Add 20 μL qPCR Mix to each reaction tube/well. Please note adding the corresponding qPCR Mix into each
sample tubes and avoid adding errors.
- AddTemplates to the tube/wells which contained the qPCR Mix. See Table 4 for the templates adding.
Test Samples |
In each tube or well … |
TS |
20 μL qPCR Mix+20 μL Samples after extraction |
NTC |
20 μL qPCR Mix+20 μL DNA Dilution buffer |
NCS* |
20 μL qPCR Mix+20 μL Negative sample after extraction* |
PCS |
20 μL qPCR Mix +20 μL Positive control |
Table 4 Templates adding
*We recommend to use DNA Dilution buffer (40619-E) as the template of NCS for the DNA extraction.
**The total reaction volume in each tube/well is 40 μL.
***Cover the tube lid or the plate film. To avoid affecting the fluorescence signal reading, please take care not to mark the tube lid or film
or even rub the film repeatedly with a scraper.
****Centrifuge the reaction tube or plate briefly at low speed after templates adding. After sufficient shaking and mixing, repeat centrifuge
at low speed to collect the liquid from the lid or wall to the bottom. Avoid bubbles when operation. The baseline will be impacted if the mix
is not mixed well, so this step is very important to a good experiment result.
4)qPCR programs setting
- Program file Settings
Example (7500 Real-Time PCR System instrument and Real-Time PCR Software v2.4):
Instrument type:7500 (96 Wells)
Experiment type:Quantitation-Standard Curve;
Chemistry:Taqman® Reagents
Ramp Speed:Standard (~2 hours to complete a run)
- Target channel Settings
In "Define Targets and Samples" of "Plate Setup", create a Target 1 channel (FAM), select FAM as the reporting fluorescence group and MGB or none as the quenching fluorescence group. Create a Target 2 channel (VIC), select the reporting fluorescence group as VIC and the quenching fluorescence group as none. In "Assign Targets and Samples" of "Plate Setup", if no additional ROX dye is added, select "none"; If an additional ROX is added, select
ROX.
- Standard amplificationprogram Settings
S/N |
Reaction Stage |
Temperature |
Time |
Cycle(s) |
1 |
Initial denaturation |
95℃ |
5 min |
1 |
2 |
Denaturation |
95℃ |
15 sec |
45 |
3 |
Annealing/Extension (fluorescence signal collection) |
62℃ |
30 sec |
Table 5 Standard amplification program Settings
- Baseline and threshold setting:
Principle of baseline adjustment: use the automatic baseline generally. If need to adjust in manual, choose the cycle before the exponential growth period as the start cycle, and avoid the fluctuation zone of initial fluorescence collection. Choose the cycle which is 1-2 cycles before the Ct of the earliest exponential amplification sample as the
end point.
Principle of threshold adjustment: use the automatic threshold generally. If need to adjust in manual, the threshold should be set higher than the Negative sample or the baseline noise, it’s generally set the threshold in the late
stage of the exponential amplification, relative independent and suitable threshold is needed for each tunnel.
5) Result Analysis
- Resultjudgement for PCS, NTC and NCS:
If IC added: the specification of each control samples should be satisfied in Table 6:
Control sample |
FAM Signal |
VIC Signal |
PCS |
Ct < 40, and has obvious amplification curve |
Ct < 40 and has obvious amplification curve |
NTC |
Ct ≥ 40 or no obvious amplification curve |
Ct < 40 and has obvious amplification curve |
NCS |
Ct ≥ 40 or no obvious amplification curve |
Ct < 40 and has obvious amplification curve |
Table 6 Result judgement of PCS, NTC and NCS
If IC not added: each quality control sample shall meet the specification of FAM signal column in Table 6, and no
need to analyze VIC channel.
- Resultjudgement for TS
Prerequisite: It is necessary to determine whether PCS, NTC and NCS passed the specification in Table 6 before TS results analysis. If passed, then can proceed to the next step. If not passed, the TS results may not be reliable, and
the reason needs to be investigated.
If IC added: find the corresponding results judgement according to the result information of FAM and VIC in Table 7:
FAM Signal |
VIC Signal |
Result judgement |
Ct<40 and has obvious amplification curve |
Ct<40 and has obvious amplification curve |
Positive |
Ct≥40 or no obvious amplification curve |
An inhibition is existed, the experiment need to be repeated |
|
Ct≥40 or no obvious amplification curve |
Ct<40 and has obvious amplification curve |
Negative |
Ct≥40 or no obvious amplification curve |
An inhibition is existed, the experiment need to be repeated |
Table 7: Result judgement of TS (IC added)
*If there is inhibition for VIC signal,treatment is needed to eliminate the inhibitors or repeat the test.
If IC not added: find the corresponding results judgement according to the result information of FAM in Table 8 , and it is no need to analyze the VIC signal.
FAM Signal |
Result judgement |
Ct<40 and has obvious amplification curve |
Positive |
Ct≥40 or no obvious amplification curve |
Negative |
Table 8: Result judgement of TS (IC not added)
Notes
- Please read thismanual carefully before usingthis kit. The experiment should be conducted in a standardized manner, including sample handling, preparation of reaction system and sample addition.
- Keep operations ofsample adding and reagents preparing on ice if possible.
- Vortex and mix well for each reagents before use.
- Please operatewith lab coats and disposable gloves,for your safety.
- This product is for research use only.
[1] Zhao F, Wang X, Li Y, Chen X, Geng Z, Zhang C. Effects of Dietary Supplementation with Epigallocatechin Gallate on Meat Quality and Muscle Antioxidant Capacity of Broilers Subjected to Acute Heat Stress. Animals (Basel). 2021;11(11):3296. Published 2021 Nov 18. doi:10.3390/ani11113296(IF:2.752)