- Background
The data summarized in this report are performed by Yeasen Biotechnology (Shanghai) Co., Ltd. for MycAwayTM Mycoplasma Real-time qPCR Detection Kit (2G) product.
The summary data is only for the user's reference, and the user needs to verify the mycoplasma method by using their own samples to confirm the method can meet the user's requirements. All laboratories who using this kit are recommended to verify the following parameters: Detection limit, Specificity, and Robustness.
Yeasen Biotechnology (Shanghai) Co., Ltd. can provide the detailed qualification report for this kit. If the user uses this report, then only the suitability (which included Detection limit and Specificity) should be verified.
Validation Content：
|
Performance |
Validation Parameters |
Validation Results |
|
Detection Limit |
Mycoplasma Strain Detection Limit |
Detection limit for 10 types of pharmacopeial mycoplasma: 10 CFU/mL, each tested 24 times with a detection rate of ≥95%. |
|
Specificity |
Sample Matrix Interference |
Tested 9 different sample matrices and DNA dilution solutions, no mycoplasma detected. |
|
Cross-reactivity |
Tested 14 strains and 6 commonly used engineering cells in biopharmaceuticals, no mycoplasma detected. |
|
|
Durability |
Freeze-Thaw Stability |
Can withstand 20 repeated freeze-thaw cycles without affecting kit performance. |
|
Thermal Acceleration Stability |
Unaffected under conditions of 37°C for 14 days and 4°C for 30 days. |
|
|
Instrument Compatibility |
Compatible with ABI7500, ABI QuantStudio™5, Bio-Rad CFX96, and Roche LightCycler®480. |
|
|
Coverage |
Coverage of Mycoplasma DNA Types |
Database comparison using mycoplasma 16S rRNA sequences covers 183 species within the class Mollicutes. |
- Experimental materials and methods
2.1 MycAwayTM Mycoplasma Real-time qPCR Detection Kit (2G), Vendor: Yeasen, Cat No.: 40619ES60.
2.2 MolPureTM Magnetic Residual DNA Sample Preparation Kit, Vendor: Yeasen, Cat No.: 18461ES60.
2.3 The Mycoplasma sensitivity standards are purchased from Minerva Biolabs (MB), whose strains are from the National Collection of Type Cultures (NCTC). The sensitivity standards are inactivated mycoplasma and used for the methodological verification of nucleic acid amplification method (qPCR/PCR method) for mycoplasma detection. Total 10 kinds of mycoplasmas are purchased which including the requirements of EP 2.6.7 and JP G3. The information of the standards are as follows:
|
Vendor |
Cat No. |
NCTC No. |
ATCC No. |
Material Name |
|
Minerva Biolabs (MB) |
102-1003 |
10129 |
23838 |
10CFU™ Sensitivity Standard Mycoplasma arginini |
|
102-2003 |
10112 |
23714 |
10CFU™ Sensitivity Standard Mycoplasma orale |
|
|
102-3003 |
10115 |
19610 |
10CFU™ Sensitivity Standard Mycoplasma gallisepticum |
|
|
102-4003 |
10119 |
15531 |
10CFU™ Sensitivity Standard Mycoplasma pneumoniae |
|
|
102-5003 |
10124 |
25204 |
10CFU™ Sensitivity Standard Mycoplasma synoviae |
|
|
102-6003 |
10117 |
19989 |
10CFU™ Sensitivity Standard Mycoplasma fermentans |
|
|
102-7003 |
10130 |
17981 |
10CFU™ Sensitivity Standard Mycoplasma hyorhinis |
|
|
102-8003 |
10116 |
23206 |
10CFU™ Sensitivity Standard Acholeplasma laidlawii |
|
|
102-9003 |
10164 |
27556 |
10CFU™ Sensitivity Standard Spiroplasma citri |
|
|
102-11003 |
10113 |
23064 |
10CFU™ Sensitivity Standard Mycoplasma salivarium |
2.4 Original data: the electronic version were recorded in the 'Experimental Report' the subfile of the parent file 'MycAwayTM Mycoplasma Real-time qPCR Detection Kit (2G)'.
2.5 Methods: The materials and operation methods used for the experiment were basically produced or set by Yeasen Biotechnology (Shanghai) Co., Ltd., which were assessed and verified for a long time, the kit development and manufacture are followed the ISO 13485 system.
- Qualification contents and results
3.1 Detection Limit (LOD)
Nucleic acid extraction as well as detection of the 10 CFU/mL Mycoplasma standard strain was performed according to the instructions for the standard strain as well as the instructions for the extraction and detection kit. A total of 24 assays were obtained for the 10 CFU/mL Mycoplasma standard strain. NCS (Negative Control Sample) and NTC (No Template Control) samples were tested simultaneously in each experiment.
The results showed that the NCS and NTC results were satisfactory, and all 24 10 CFU/mL samples tested positive, meeting the requirement of having at least 23 positive results out of 24 tests.
|
No. |
Material Name |
Concentration of bacterial liquid |
|
1 |
Mycoplasma arginini |
10 CFU/mL |
|
2 |
Mycoplasma orale |
10 CFU/mL |
|
3 |
Mycoplasma gallisepticum |
10 CFU/mL |
|
4 |
Mycoplasma pneumoniae |
10 CFU/mL |
|
5 |
Mycoplasma synoviae |
10 CFU/mL |
|
6 |
Mycoplasma fermentans |
10 CFU/mL |
|
7 |
Mycoplasma hyorhinis |
10 CFU/mL |
|
8 |
Acholeplasma laidlawii |
10 CFU/mL |
|
9 |
Spiroplasma citri |
10 CFU/mL |
|
10 |
Mycoplasma salivarium |
10 CFU/mL |
Table 1 Information on validated strains
|
10 CFU/mL |
Experiment 1 |
Experiment 2 |
Experiment 3 |
|
Detection Rate |
8/8 |
8/8 |
8/8 |
|
Total |
24/24 |
||
Table 2 Detection limit results
3.2 Specificity
3.2.1 Mycoplasma test range
The primer probe sequences in the kit were compared with the 16S rRNA genome in the database. Through bioinformatics analysis, it was obtained that the MycAwayTM Mycoplasma qPCR Detection Kit (Probe Method) of NextSense Biologicals can cover at least 183 species of Zooxanthellae, including 120 Mycoplasma, 9 Ureaplasma, 33 Spiroplasma, and 7 Cholestrongyla, and so on.
3.2.2 Sample matrix interference
9 substrates (Dulbecco’s MEM, Ham's F-10 Medium, M-199 Medium, Ham's F-12K Kaighn's, RPMI-1640 Medium, McCoy's 5A, Ham's F-12K Kaighn's+1.0mM/L Glutamine, L-15 LEIBOVITZ MEDTA +2.05 mM L-Glutamine, DMEM-low glucose Standard Medium) are used as samples to verify the sample matrix interference, the results showed that the medium have no interference to the kit reagents.
3.2.3 Flexibacteria detection range
Extraction and detection of inactivated mycoplasma (Mycoplasma arginini, Mycoplasma orale, Mycoplasma gallisepticum, Mycoplasma pneumoniae, Mycoplasma synoviae, Mycoplasma fermentans, Mycoplasma hyorhinis, Acholeplasma laidlawii, Spiroplasma citri, Mycoplasma salivarium) at 10 CFU/mL is required along with NCS and NTC sample testing.
The results showed that NCS and NTC were not detected and the results were negative; all 10 mycoplasma species were detected and the results were positive.
3.2.4 Cross reaction
For 14 non-flexibacterial strains including Clostridium (Clostridium perfringens, Clostridium acetobutylicum), Lactobacillus (Lactobacillus acidophilus), Streptococcus (Streptococcus mutans, Streptococcus pneumoniae), a number of other common bacillus-coccus-fungi microorganisms (Staphylococcus epidermidis, Acinetobacter baumannii, Enterobacter aerogenes, Micrococcus garciniae, Pseudomonas aeruginosa, Pseudomonalbacterium leucocephala, Salmonella enterica enteritidis subsp. enterica, Bacillus subtilis, Bacillus subtilis, and Bacillus sp. Bacillus cereus, Bacillus cereus), and six engineered cells (HEK293, 293T, Vero, CHO, E.coli, SF9) were used for mycoplasma detection using the MolPureTM Magnetic Bead Method for Residual DNA Sample Preprocessing Kit (bottles) and the MycAwayTM Mycoplasma qPCR Detection Kit (Probe Method) (2G), and each experiment was performed simultaneously with the PCS, NCS, and NTC sample testing.
The results showed that the PCS, NCS, and NTC results were satisfactory; the test samples were negative and no cross-reactivity occurred.
3.3 Robustness
3.3.1 Freeze-thaw stability
The Mycoplasma test kit was subjected to freeze-thawing experiments under the conditions of -25℃~-15℃, and the results showed that the kit was able to withstand 20 times of repeated freezing and thawing, and its performance was not affected.
3.3.2 Accelerated Stability
The Mycoplasma detection kit was heat-accelerated at 37℃ for 14 days and at 4℃ for 30 days, respectively, and the results showed that the product performance was not affected.
3.3.3 Instrument applicability
For the mycoplasma detection kit, different concentrations of positive control and Mycoplasma pneumoniae DNA in the kit were used as samples to be tested, and the experiments were performed on four different brands and models, namely, Thermo Scientific ABI7500, Thermo Scientific ABI Q5, Shanghai Hongshi SLAN-96S, and Roche LightCycler®480, respectively. The experiments were performed on the qPCR instruments of four different brands and models, Shanghai Hongshi SLAN-96S and Roche LightCycler®480.
The results showed that the samples to be tested were detected on qPCR instruments of different manufacturers' models and the negative control results were qualified, and the performance of the kit was not affected.
- 4.Reference
4.1 Mycoplasma examination method <3301>. Chinese Pharmacopoeia, Part III [S]. Beijing: China Science and Technology Press, 2020: 542-543.
4.2 Guidelines for validation of analytical methods <9101>. Chinese Pharmacopoeia, Part III [S]. Beijing: China Science and Technology Press, 2020: 682.
4.3 Guidelines for validation of alternative methods for microbiological testing of pharmaceuticals <9201>. Chinese Pharmacopoeia, Part III [S]. Beijing: China Science and Technology Press, 2020: 684.
4.4 Mycoplasmas. European Pharmacopoeia[S]. 10th Edition, 2020: Section 2.6.7, 194-199.
4.5 Mycoplasma Testing for Cell Substrates used for the Production of Biotechnological/Biological Products <G3-14-170>. The Japanese Pharmacopoeia[S]. 18th Edition, 2021: 2678-2682.
4.6 Erica J. Fratz-Berilla, Phillip Angart, Ryan J.Graham, et al. Impacts on product quality attributes of monoclonal antibodies produced in CHO cell bioreactor cultures during intentional mycoplasma contamination events. Biotechnology and Bioengineering. 2020;117(9): 2802-2815.
4.7 Dmitriy V. Volokhov, Laurie J Graham, Kurt A Brorson, Vladimir E Chizhikov. Mycoplasma testing of cell substrates and biologics: Review of alternative non-microbiological techniques. Mol Cell Probes. 2011 Apr-Jun;25(2-3):69-77.
4.8 A.L. Ingebritson, C.P. Gibbs, C. Tong, G.B. Srinivas. A PCR detection method for testing Mycoplasma contamination of veterinary vaccines and biological products. Lett in Applied Microbiology. 2015 Feb;60(2):174-180.
Ordering information
|
Description |
Part Number |
|
18461ES |
|
| MycAway™ Mycoplasma qPCR Detection Kit (2G) | 40619ES |