Validation of a Commercial Enzyme-Linked Immunosorbent Assay for Screening Tetracycline Residues in Foods of Animal Origin from the Perspective of Bangladesh

et al. 2019

Preprint

The study carried out a primary validation of Charm II tests for the detection of antimicrobial residues in aquaculture fish. The Validation was performed according to European Commission Decision 2002/657/ EC and the parameters determined included: detection capability, repeatability, reproducibility, specificity and robustness for the detection of antimicrobial residues in fish. Fish materials from different species including cat fish, trout, salmon, sea bass, tilapia, lingue and pangasius, were spiked with varying concentrations of selected antibiotics including sulfonamides, beta-lactams, macrolides, tetracyclines and aminoglycosides to determine the detection capabilities and other validation parameters of the Charm II tests. Results of the validation showed that the detection capabilities for the tetracyclines ranged from 25 µg/kg to 100 µg/kg, while the sulfonamides and aminoglycosides were detected at 25 µg/kg for all species under study. The detection capabilities for the beta-lactams ranged from 25 µg/kg to 300 µg/kg and were 100 µg/kg for the tested macrolides. Results of the study showed that there was no significant difference between counts for samples read immediately after addition of the scintillation fluid and those read fourteen hours after addition of the scintillation fluid, provided that there was good vortexing before analysis. There was also no significant difference between counts for the same samples analyzed in different runs under repeatability and reproducibility conditions at the same spiking concentrations for the different fish species analyzed. The relative standard deviation for both repeatability and reproducibility ranging from 0.02 to 0.1 was recorded.The Charm II tests were found to be 100% group specific, as none of antimicrobials kits, gave false positive results when testing non-target antimicrobial drugs. The results of this study prove the robustness, specificity, reliability and accuracy of the Charm II tests in the detection of various antimicrobial residuals in fish. Results also confirm the suitability of the Charm II technique as a rapid screening tool for detection of antimicrobial residues in a variety of fish species; and its applicability for the rapid evaluation of the quality of aquaculture fish for safety and trade purposes.

Section: Detection Capability For the Different Antimicrobials In Selmentioning

confidence: 55%

Primary validation of Charm II tests for the detection of antimicrobial residues in a range of aquaculture fish

Mukota

et al. 2019

Preprint

“…1 The detection capabilities and maximum residue limits for the different antimicrobials antimicrobials involved in the study were within the 5% requirement of the EU decision 2002/657, and therefore the validation results are satisfactory. The Charm II technique exhibited better CCβ for tetracyclines at 25 ppb (0.25 MRL) compared to other rapid screening techniques such as the ELISA kit of R-Biopharm for screening tetracycline antibiotic residues in the muscle of chicken, beef, and shrimp, which detected the same at 100 ppb (MRL) [27]. In another study, results of the revolutionary Biochip Array Technology showed better detectability for tylosin A and oxytetracycline at 0.10 and 0.5 of the respective MRL in samples [28].…”

Section: Detection Capability For the Different Antimicrobials In Selmentioning

confidence: 96%

“…In the case Fm < T the false positive rate is below 5%. If more than 5% of the spiked samples at the screening target concentration gave a response greater than the cut-off level (deemed false negative), the concentration chosen for the spiking is considered too low for validation and a higher concentration is tested [26,27]. From the results presented in Table 3, the Fm values obtained using the EU guideline and the respective calculated CP according to the Charm II protocol are comparable.…”

Section: Evaluation Of the Control Points For The Different Drug Resimentioning

confidence: 99%

Primary validation of Charm II tests for the detection of antimicrobial residues in a range of aquaculture fish

Mukota¹,

et al. 2020

BMC Chemistry

The study carried out a primary validation of Charm II tests for the detection of antimicrobial residues in aquaculture fish. The validation was performed according to European Commission Decision 2002/657/EC and the parameters determined included: detection capability, repeatability, reproducibility, specificity and robustness for the detection of antimicrobial residues in fish. Fish materials from different species including cat fish, trout, salmon, sea bass, tilapia, lingue and pangasius, were spiked with varying concentrations of selected antimicrobials including sulfonamides, β-lactams, macrolides, tetracyclines and aminoglycosides to determine the detection capabilities and other validation parameters of the Charm II tests. Results of the validation showed that the detection capabilities for the tetracyclines ranged from 25 to 100 µg/kg, while the sulfonamides and aminoglycosides were detected at 25 µg/kg for all species under study. The detection capabilities for the beta-lactams ranged from 25 to 300 µg/kg; and was 100 µg/kg for the tested macrolides. Results of the study showed that there was no significant difference between counts for samples read immediately after addition of the scintillation liquid and those read 14 h after addition of the scintillation liquid, provided that there was good vortexing before analysis. There was also no significant difference between counts for the same samples analyzed in different runs under repeatability and reproducibility conditions at the same spiking concentrations for the different fish species analyzed. The relative standard deviation for both repeatability and reproducibility ranged from 1.2 to 15.1%. The Charm II tests were found to be 100% group specific, as none of the antimicrobials kits, gave false positive results when testing non-target antimicrobial drugs. Results of this study demonstrate the suitability of the Charm II technique as a rapid screening tool for detection of antimicrobial residues in a variety of fish species at maximum residue limits (MRL) established in the EU guidelines, with the exception of tilmicosin which was detected at 2 MRL. The results also prove the robustness, specificity, reliability and precision of the Charm II assay in the detection of various antimicrobial residuals in fish and its applicability for the rapid evaluation of the quality of aquaculture fish for safety and trade purposes.

“…Moreover, the incidences of false negative results observed for all antibiotics involved in the study were within the 5% requirement of the EU decision 2002/657, and therefore the validation results are satisfactory. The Charm II technique exhibited better detection capability for tetracyclines at 25 ppb (0.25 MRL) compared to other rapid screening techniques such as the ELISA kit of R-Biopharm for screening tetracycline antibiotic residues in the muscle of chicken, beef, and shrimp, which detected the same at 100 ppb (MRL) [27]. In another study, results of the revolutionary Biochip Array Technology showed better detectability for tylosin and oxytetracycline at 0.10 and 0.5 of the respective MRL in samples [28].…”

Section: Detection Capability For the Different Antimicrobials In Selmentioning

confidence: 97%

“…In the case Fm < T the false positive rate is below 5%. If more than 5% of the spiked samples at the screening target concentration gave a response greater than the cutoff level (deemed false negative), the concentration chosen for the spiking is considered too low for validation and a higher concentration is tested [26,27].…”

Section: Evaluation Of the Control Points For The Different Drug Resimentioning

confidence: 99%

Primary validation of Charm II tests for the detection of antimicrobial residues in a range of aquaculture fish

Mukota

et al. 2020

Preprint

The study carried out a primary validation of Charm II tests for the detection of antimicrobial residues in aquaculture fish. The Validation was performed according to European Commission Decision 2002/657/EC and the parameters determined included: detection capability, repeatability, reproducibility, specificity and robustness for the detection of antimicrobial residues in fish. Fish materials from different species including cat fish, trout, salmon, sea bass, tilapia, lingue and pangasius, were spiked with varying concentrations of selected antibiotics including sulfonamides, beta-lactams, macrolides, tetracyclines and aminoglycosides to determine the detection capabilities and other validation parameters of the Charm II tests. Results of the validation showed that the detection capabilities for the tetracyclines ranged from 25 µg/kg to 100 µg/kg, while the sulfonamides and aminoglycosides were detected at 25 µg/kg for all species under study. The detection capabilities for the beta-lactams ranged from 25 µg/kg to 300 µg/kg; and was 100 µg/kg for the tested macrolides. Results of the study showed that there was no significant difference between counts for samples read immediately after addition of the scintillation fluid and those read fourteen hours after addition of the scintillation fluid, provided that there was good vortexing before analysis. There was also no significant difference between counts for the same samples analyzed in different runs under repeatability and reproducibility conditions at the same spiking concentrations for the different fish species analyzed. The relative standard deviation for both repeatability and reproducibility ranged from 1.2 to 15.1%.The Charm II tests were found to be 100% group specific, as none of the antimicrobials kits, gave false positive results when testing non-target antimicrobial drugs. Results of this study demonstrate the suitability of the Charm II technique as a rapid screening tool for detection of antimicrobial residues in a variety of fish species at Maximum Residue Limits (MRL) established in the EU guidelines, with the exception of tilmicosin which was detected at 2MRL. The results also prove the robustness, specificity, reliability and precision of the Charm II assay in the detection of various antimicrobial residuals in fish and its applicability for the rapid evaluation of the quality of aquaculture fish for safety and trade purposes.