Slide 1: Proposal of the New Rapid Sterility Test
Proposal of the New Rapid Sterility Test Shimadzu Diagnostics Corporation
Slide 2: Contents
Contents 1 Background 2 BactFinder /FungiFinder 3 Rapid Test Method 4 High Sensitivity Test Method 5 Conclusion Copyrights © Shimadzu Diagnostics Corporation. All rights reserved. 1
Slide 3: Contents
Contents 1 Background 2 BactFinder /FungiFinder 3 Rapid Test Method 4 High Sensitivity Test Method 5 Conclusion Copyrights © Shimadzu Diagnostics Corporation. All rights reserved. 2
Slide 4: About our Products
About our Products Kits for quality testing of ATMP and similar products 01 02 03 NEW Myco Finder VirFinder Type-A/Type-B BactFinder /FungiFinder ✓ PCR kit for mycoplasma ✓ PCR kit for virus detection ✓ PCR kit for rapid microbial detection. Compatible with EP, testing detection testing USP, and JP.
Slide 5: Alternative Microbiological Test Methods
Alternative Microbiological Test Methods EP 2.6.27 and 5.1.6, and USP <1071> list various detection techniques as alternative rapid microbiological test methods. We have developed a real-time PCR kit that Examples of alternative microbiological detects microorganisms by targeting DNA. test methods ✓
Slide 6: Contents
Contents 1 Background 2 BactFinder /FungiFinder 3 Rapid Test Method 4 High Sensitivity Test Method 5 Conclusion Copyrights © Shimadzu Diagnostics Corporation. All rights reserved. 5
Slide 7: BactFinder/FungiFinder Features
BactFinder /FungiFinder Features ✓ PCR kit that detects microbial nucleic acids ✓ Clean kit with reduced background nucleic acids ✓ Target-specific detection ✓ Detects a wide range of environmental and human- derived microorganisms Copyrights © Shimadzu Diagnostics Corporation. All rights reserved.
Slide 8: BactFinder/FungiFinder
BactFinder /FungiFinder BactFinder FungiFinder DNA pol MgCl2 Buffer dNTP PPmix Water PC IC DNA pol MgCl2 Buffer dNTP PPmix Water PC IC 1.5 mL reagent 1.5 mL reagent preparation tube preparation tube 8-cap strips for PCR 8-cap strips for PCR 8-tube strips for PCR 8-tube strips for PCR Capacity: Bacte
Slide 9: BactFinder and FungiFinder Performance Attributes
BactFinder and FungiFinder Performance Attributes Expected Samples Culture supernatant, cell suspensions Detection Sensitivity 100 CFU/sample Kit-derived Background Very little Noise Specificity Target-specific detection Between-device Variation Verified compatible with three different real-time PCR
Slide 10: Contents
Contents 1 Background 2 BactFinder /FungiFinder 3 Rapid Test Method 4 High Sensitivity Test Method 5 Conclusion Copyrights © Shimadzu Diagnostics Corporation. All rights reserved. 9
Slide 11: Rapid Test Method
Rapid Test Method Developed for reduced microbial background noise in three steps. Pre-treatment Extraction PCR Remove Enzyme Bead BactFinder Sample treatment Crushing Nucleic acid the supernatant FungiFinder extraction DNA Extraction Kit Centrifuge Add enzime Steps to remove non-target nucleic acid
Slide 12: Method
Method Objectives ⚫ Investigate test sensitivity and background noise to verify the suitability of the rapid test method ⚫ Tested 1-mL samples of 6 microorganism species at 100 CFU combined with Jurkat cells Sensitivity evaluation at 1.0 × 106 cells/mL (N = 8 of each microorganism) with 6 microorgan
Slide 13: Sensitivity Evaluation
Sensitivity Evaluation Microorganism strain Result Aspergillus brasiliensis ATCC 16404 8/8 Candida albicans ATCC 10231 8/8 Bacillus subtilis subsp. spizizenii ATCC 6633 8/8 Clostridium sporogenes ATCC 11437 8/8 Pseudomonas aeruginosa ATCC 9027 8/8 Staphylococcus aureus ATCC 6538 8/8 Shows number of
Slide 14: Background Noise Evaluation
Background Noise Evaluation BactFinder FungiFinder 1 35.31 9 N/A 1 N/A 9 N/A 2 N/A 10 N/A 2 N/A 10 N/A 3 34.66 11 N/A 3 N/A 11 N/A 4 35.72 12 37.20 4 N/A 12 N/A 5 N/A 13 N/A 5 N/A 13 N/A 6 N/A 14 N/A 6 N/A 14 N/A 7 N/A 15 N/A 7 N/A 15 N/A 8 N/A 16 36.22 8 N/A 16 N/A Tables show Cq result for each sa
Slide 15: Rapid Test Method Summary
Rapid Test Method Summary Rapid Test Method ✓ A rapid test method that detects microorganisms from 100 CFU in around 4 hours ✓ A clean method with relatively little background noise → Results affected somewhat by sample type and the testing environment Likely suitable for real-world applications whe
Slide 16: Contents
Contents 1 Background 2 BactFinder /FungiFinder 3 Rapid Test Method 4 High Sensitivity Test Method 5 Conclusion Copyrights © Shimadzu Diagnostics Corporation. All rights reserved. 15
Slide 17: Background to High Sensitivity Test Method
Background to High Sensitivity Test Method Rapid Test Method ✓ Result in around 4 hours → Great! ✓ 100 CFU sensitivity → Would like higher sensitivities ✓ Occasional background noise → Would like no false positive results We developed a high sensitivity test method with a higher sensitivity and lowe
Slide 18: Features of the High Sensitivity Test Method
Features of the High Sensitivity Test Method Rapid Test Method High Sensitivity Test Method Expected Use Basic sample screening Product release testing, in-process testing — Preculture Test Method Steps Preparation and nucleic acid extraction Real-time PCR Detection Sensitivity 100 CFU 1 CFU Testing
Slide 19: High Sensitivity Test Method
High Sensitivity Test Method Preculture Preparation and nucleic acid extraction PCR Aerobic Anaerobic Zero-hour group Sample Judgment criterion Preparation and Culture group Culture Cq - 0h Cq = ΔCq nucleic acid extraction Culture Culture for fixed duration Negative/Positive result based on size of
Slide 20: Tested Attributes
Tested Attributes We evaluated the performance of the high sensitivity test method by testing the following attributes. Test items Definition Limit of Detection Lowest concentration of microorganisms that can be detected Equivalence Degree of closeness between tests results of rapid and compendial m
Slide 21: Method
Method N = 3 of each concentration Preparation → Nucleic acid 30 CFU 3 CFU 0.3 CFU extraction → PCR No turbidity Aerobic Anaerobic Aerobic Anaerobic Aerobic Anaerobic in culture Culture Cq - 0h Cq = ΔCq Judgment criterion ΔCq ≤ -6 Positive ΔCq > -6 Negative Note: All samples contain 1.0 × 106 Jurkat
Slide 22: Limit of Detection
Limit of Detection Results after 48-hour culture of 30 to 0.3 CFU samples 30 CFU 3 CFU 0.3 CFU Microorganism strain Turbidity PCR Positive Turbidity PCR Positive Turbidity PCR Positive Aspergillus brasiliensis ATCC 16404 3/3 3/3 0/3 Candida albicans ATCC 10231 3/3 3/3 2/3 Bacillus subtilis subsp. sp
Slide 23: Equivalence
Equivalence 30 CFU 3 CFU 0.3 CFU High Sensitivity compendial High Sensitivity compendial High Sensitivity compendial Microorganism strain Test Method method Test Method method Test Method method Aspergillus brasiliensis ATCC 16404 3/3 + 3/3 + 0/3 ー Candida albicans ATCC 10231 3/3 + 3/3 + 2/3 + Bacil
Slide 24: Range of Species
Range of Species The acne bacterium is a high contamination risk microorganism and detection by the compendial method is time-consuming. We attempted to detect acne bacteria using the high sensitivity test method with a 48-hour and 72-hour preculture. 48h 72h 29 CFU 5 CFU 0.3 CFU 29 CFU 5 CFU 0.3 CF
Slide 25: Range of Species
Range of Species Microorganism strain Stenotrophomonas maltophilia Kocuria rhizophila Bacillus licheniformis Micrococcus luteus Brevibacterium casei Staphylococcus epidermidis Yersinia enterocolitica Paenibacillus glucanolyticus Enterococcus hirae Corynebacterium jeikeium Pseudomonas fluoresens Cory
Slide 26: Specificity
Specificity Test conditions : Aerobic medium / Anaerobic medium + 1.0 × 106 Jurkat cells Culture time : 48 h Samples : N = 12 BactFinder FungiFinder Sample No. Turbidity PCR（ΔCq） Turbidity PCR（ΔCq） 1 ー ー ー ー 2 ー ー ー ー 3 ー ー ー ー 4 ー ー ー ー 5 ー ー ー ー 6 ー ー ー ー 7 ー ー ー ー 8 ー ー ー ー 9 ー ー ー ー 10 ー ー ー ー 1
Slide 27: High Sensitivity Test Method Summary
High Sensitivity Test Method Summary High Sensitivity Test Method ✓ High sensitivity test method that detects living microorganisms from 1 CFU within 48 hours 72 hours required for some microorganisms ✓ No background noise and low risk of false positives Currently in initial phases of validation C
Slide 28: Contents
Contents 1 Background 2 BactFinder /FungiFinder 3 Rapid Test Method 4 High Sensitivity Test Method 5 Conclusion Copyrights © Shimadzu Diagnostics Corporation. All rights reserved. 27
Slide 29: Conclusion
Conclusion BactFinder /FungiFinder Rapid Test Method ⚫ Determines if microbial nucleic acids are present in around 4 hours High Sensitivity ⚫ Detects living microorganisms within 2-3 days from 1 CFU Test Method Use one PCR kit and choose between two test methods according to sterility test objective