Slide 1: 1-1. Why mycoplasma testing is necessary?
1-1. Why mycoplasma testing is necessary? Due to the changes in the laws (Act on Securing Quality, Efficacy and Safety of Products Including Pharmaceuticals and Medical Devices, Act on the Safety of Regenerative Medicine Products Safety, etc.) in 2012, commercial application of regenerative medicine
Slide 2: 1-2. What is mycoplasma?
1-2. What is mycoplasma? Mycoplasma, Ureaplasma, Spiroplasma, Acholeplasma, and other bacteria that belong to Mollicutes. <Characteristics> Fibroblast infected by Mycoplasma ○Widely distributed in the animal and plant kingdoms and infection shows a species-specificity. ○Infect humans through researc
Slide 3: 1-4. The 7 types of strains to be detected under Japanese Pharmacopoeia 17th Edition
1-4. The 7 types of strains to be detected under Japanese Pharmacopoeia 17th Edition 7 types of strains were selected from the following perspectives as mycoplasma strains that may contaminate cell substrates used for development of medical products in Japan. ・Frequency of contamination and phylogen
Slide 4: 1-5. Method A: Culture method
1-5. Method A: Culture method <Test method> Inoculate medium (liquid medium, agar medium) with the sample and culture to detect colonies specific to mycoplasma. Medium Amount of No. of tests per sample Length of incubation sample Liquid medium 10mL or more 1 or more culture bottles For 14 days (100m
Slide 5: 1-6. Method B: DNA staining method
1-6. Method B: DNA staining method <Test method> This method indirectly detects mycoplasma that have grown in cell culture by inoculating the indicator cells (Vero cells) with the test sample and staining the mycoplasma DNA with fluorescent staining dye to observe small extra-nuclear fluorescence sp
Slide 6: 1-7. Method C: Nucleic Acid Amplification Technique (NAT methods)
1-7. Method C: Nucleic Acid Amplification Technique (NAT methods) <Test method> Extract DNAs from the sample and amplify them using mycoplasma-specific primers to detect mycoplasma. Under Japanese Pharmacopoeia 17th Edition… ・Validation methods for NAT technique are presented ⇒As far as validation i
Slide 7: 2-2. Product outline (Packaging specification)
2-2. Product outline (Packaging specification) Paid samples of Prevrsion (product code: 69201), a mycoplasma gene detection kit, are on sale We have agreed on delivery specification of Preversion with the manufacture (NTS) (The manufacturer performs manufacturing, packaging, and labeling of the ki
Slide 8: 2-3. Test method outline
2-3. Test method outline Following is a rough outline of mycoplasma testing using this kit Extraction of nucleic acid Sample: cell culture ⇒The kit by QIAGEN is recommended ⇒Cell substrate used for the development of Centrifugal medical products concentration Cell/tissue-based medical products, etc.
Slide 9: 2-4. Real-time PCR
2-4. Real-time PCR This kit detects mycoplasma nucleic acid by real-time PCR TaqMan probe method Probe 1) Thermal denaturation Primer Fluorescent material Quencher 2) Primer annealing / probe hybridization Polymerase Hybridize Positive 3) Elongation Negative Fluorescent signal Source: TAKARA website
Slide 10: 2-5. How to use the kit (reagent preparation)
2-5. How to use the kit (reagent preparation) Preparation of reagent is not necessary because dried reaction reagent is set in the reaction tube for each test As a potent amplification enzyme is used, detection can complete within one hour from the start of amplification <Usage method (example)> (
Slide 11: 2-6. Product feature ~ Special positive control~
2-6. Product feature ~ Special positive control~ False-positive result due to contamination of negative sample by positive control is identifiable <Example of detection of positive control> Amplification curve of positive control (1000copies/reaction) Amplification curve of mycoplasma fermentans (1
Slide 12: 2-7. Comparison between commercially available kits ~basic specification~
2-7. Comparison between commercially available kits ~basic specification~ Product name MycoFinder (newly marketed kit) MycoTOOL Mycoplasma real-time PCR Kit MycoSEQ Mycoplasma Real-Time PCR Sold by Shimadzu Diagnostics Corporation Roche ABI JP compliance Compliance with JP17 is intended Compliance w
Slide 13: 2-8. Use example of the kit
2-8. Use example of the kit This kit can detect mycoplasma in cell culture ⇒ It can be used not only for the testing of final products, but also for the voluntary inspection during culture. Culture Cell culture starts Take a sample from culture solution Products such as cells Voluntary inspection My
Slide 14: 3. Performance evaluation of the kit and validation to comply with JP
3. Performance evaluation of the kit and validation to comply with JP Copyright © Shimadzu Diagnostics Corporation All rights reserved. Confidential 14
Slide 15: 3-1. Why validation is necessary?
3-1. Why validation is necessary? Do users have to perform full-validation at their own facilities? ⇒The revision proposal states, “when a commercially available kit is used for NAT testing, the full validation data from the manufacturer can be used as an alternative to the validation data by the us
Slide 16: 3-2. Method C (NAT method) validation outline
3-2. Method C (NAT method) validation outline The JP17 requires a proper validation be performed. <Outline of validation method> (1) Validation of NAT method Specificity: Detection of 7 species Detection sensitivity Mycoplasma reference strains for which characteristic analysis has been perform
Slide 17: 3-3. Validation implementation plan
3-3. Validation implementation plan Details of kit validation is as follows. <Details of validation> Item Result Detail It has to be confirmed that nucleic acids derived from the 7 reference strains for which sequence analysis has Specificity Pass been performed are detectable. Revised JP Detection
Slide 18: 3-4. Validation conditions
3-4. Validation conditions Conditions of the kit validation are as follows. <Materials and devices used> DNA extraction kit: DNeasy Blood & Tissue Kit(QIAGEN) Real-time PCR system: CFX96(Bio-Rad) Cell: CHO-DG44 cells (1 x 10^6 cells/mL) Mycoplasma reference strains: see below <Reference strain infor
Slide 19: 3-5. Specificity test
3-5. Specificity test To confirm that 7 types of test strains of mycoplasma are specifically detected, following two experiments were conducted. Performed PCR using the genomic DNAs from the 7 reference strains as templates Performed sequence analysis of the PCR product It was confirmed that the ki
Slide 20: 3-6. Sensitivity test (Equivalence test)
3-6. Sensitivity test (Equivalence test) Here is the result of the sensitivity test using the Detection limit for M.arginini Spike(cfu/mL) Run 1 Run 2 Run 3 Total serial dilution. 100 8/8 8/8 8/8 24/24 Sensitivity is to be calculated by statistical analysis. 10 8/8 8/8 7/8 23/24 1 4/8 6/8 7/8 17/24
Slide 21: 3-7. Equivalence test (NAT technique)
3-7. Equivalence test (NAT technique) The sensitivity test result showed that the 7 test strains specified by JP17 are detectable at 10-CFU/mL with a probability of 95% or more. ⇒The method meets conditions required of an alternative to Method A (culture method) Sample 1st test 2nd test 3rd test Tot
Slide 22: 3-8. Equivalence test (culture method)
3-8. Equivalence test (culture method) Using the same sample as the sensitivity test (cell suspension + reference strains), detection test was performed by culture method. Incubated for 14 days 0.2mL Agar medium (N=2) Incubated for 14 ① days Agar medium (N=2) Agar medium(N=2) Agar medium (N=2) 10mL
Slide 23: 3-9. Additional data ~Cross reactivity test~
3-9. Additional data ~Cross reactivity test~ No cross-reactivity was observed for 25 types of bacteria, 8 types of funguses, and 3 types of mammalian cells. <Cross-reactivity> 25 types of bacteria 9 types of funguses Bacteroides vulgatus Propionibacterium acnes Cryptococcus neoformans Bacillus subt