Product Design and Validation References:
- Chinese Pharmacopoeia 2020 Edition: 1101 Sterility Test Method
- Chinese Pharmacopoeia 2020 Edition: 9201 Pharmaceutical Microbiological Test Alternative Method Validation Guidelines
- “Human Stem Cell-derived Pharmaceutical Research and Evaluation Technical Guidelines (Trial)”
- “Immune Cell-derived Pharmaceutical Research and Evaluation Technical Guidelines (Trial)”
- American Pharmacopoeia USP44: Sterility Tests
- Japanese Pharmacopoeia JP17: Sterility Tests
- Polymerase Chain Reaction (PCR) Laboratory Inspection Guidelines (2016 Edition)
- For rapid qualitative detection of fungal contamination in culture media, cell culture products, and biological products. Results are available in 2 hours.
- It has been verified that the detection limit for Candida albicans and Aspergillus niger is 50 CFU/mL. It can also detect approximately 500 types of fungi, including Candida tropicalis, Candida glabrata, Penicillium, Trichoderma, etc.
- Using FAM and HEX two fluorescent probes to detect fungal target sequences and internal controls respectively. It has good specificity and can eliminate false-negative results caused by PCR inhibitors in samples. There is no cross – reaction with stem cells, NK cells, and 293 cells.
Product Name | Catalog No. | Product Specification | Storage Conditions |
Fungi Detection Kit (qPCR Method) | FU0001-50 | The test kit contains 5×Fu reaction solution, enzyme, Fu positive control, Fu internal control, PCR dilution buffer, and 50 tests/ kit. | Transport and store at -20℃ Shelf life: 12 months. |
Performance Verification I: Specificity
After extracting the internal control plasmid with different culture media using the Yocon magnetic bead extraction kit, the detection was carried out through the fungal qPCR system. The detection Ct values of the internal control HEX channel fluctuated between 29-31, and there was no detection in the positive FAM channel except for the positive control.
Detection Results after Co – extraction of Internal Control and Different Samples(Amplification Ct Values) | ||
| Different Samples | Internal Control | Fungi |
| GMP-grade Mesenchymal Stem Cell Serum-free Medium | 29.24 | NoCt |
| 29.23 | NoCt | |
| Primary Mesenchymal Stem Cell Serum-free Medium | 31.33 | NoCt |
| 31.1 | NoCt | |
| DEPC Water | 30.22 | NoCt |
| Positive Control | 29.58 | 35.67 |
| NTC1 | NoCt | NoCt |
| NTC2 | NoCt | NoCt |
Performance Verification II: Limit of Detection and Sensitivity
Candida albicans and Aspergillus niger were prepared at concentrations of 10 CFU/mL and 50 CFU/mL, respectively, in accordance with pharmacopoeia requirements. Extraction was performed using different batches of Yocon magnetic bead extraction kits, and detection was conducted using different batches of fungal qPCR systems. Both fungi at the two concentrations could be stably detected. The limit of detection for both fungi was 50 CFU/mL.
Limit of Detection Verification Results (Ct Values for Fungal Channel Amplification) | |||||||||
Verification for Fungi | Well 1 | Well 2 | Well 3 | Well 4 | Well 5 | Well 6 | Well 7 | Well 8 | NTC |
50 CFU/mL Candida albicans | 33.41 | 34.62 | 34.24 | 34.97 | 33.72 | 34.22 | 33.89 | 33.78 | NoCt |
50 CFU/mL Aspergillus niger | 33.64 | 33.65 | 33.22 | 34.08 | 34.01 | 34.10 | 34.42 | 33.61 | NoCt |
Limit of Detection Verification Results (Ct Values for Fungal Channel Amplification) | ||||||||
Verification for Fungi | Well 2 | Well 3 | Well 4 | Well 5 | Well 6 | Well 7 | Well 8 | NTC |
10 CFU/mL Candida albicans | 34.41 | 34.89 | 34.22 | 34.91 | 35.39 | 36.60 | NoCt | NoCt |
10 CFU/mL Aspergillus niger | 35.45 | 35.53 | NoCt | NoCt | 35.35 | 38.74 | 36.41 | NoCt |
Performance Verification III: Repeatability and Stability Testing
The fungal detection system was set up and tested at corresponding concentrations by different personnel at three different time points. The results showed stable detection of Candida albicans and Aspergillus niger at a concentration of 50 CFU/mL.
Tester 1
Fungal Detection | Concentration | Number of Wells Tested | Test Batch | Detection Status |
Candida albicans | 100 CFU/mL | 8 well | 1 | 8/8 positive |
Aspergillus niger | 100 CFU/mL | 8 well | 1 | 8/8 positive |
Tester 2
Fungal Detection | Concentration | Number of Wells Tested | Test Batch | Detection Status |
Candida albicans | 50 CFU/mL | 8 well | 1 | 8/8 positive |
Aspergillus niger | 50 CFU/mL | 8 well | 1 | 8/8 positive |
Tester 3
Fungal Detection | Concentration | Number of Wells Tested | Test Batch | Detection Status |
Candida albicans | 10 CFU/mL | 8 well | 1 | 6/8 positive |
Aspergillus niger | 10 CFU/mL | 8 well | 1 | 6/8 positive |
*For other related experimental validations, please refer to the “Technical White Paper” of Yocon Biotechnology’s products.
