The real-time PCR test detects DNA of fish according to directive (EC) 1169/2011 qualitatively and / or quantitatively. Each reaction contains an internal amplification control (IAC).
For the quantitative determination the use of the laboratory reference material SureFood® QUANTARD Allergen 40 (S3301) containing 40 mg fish / kg food sample and a detection system including a standard dilution is required.
For DNA preparation, the use of SureFood® PREP Advanced (S1053), protocol 1, is recommended.
The real-time PCR assay can be performed with commonly used real-time PCR instruments, equipped for detection of two fluorescence emissions at the channels FAM and VIC/HEX at the same time. The technical validation of instruments was performed on Roche LightCycler® 480 II, Roche cobas® z 480 Analyzer, Qiagen Rotor-Gene Q, Applied Biosystems 7500, Bio-Rad CFX96, Bio Molecular Systems MIC, AriaDx and Agilent Mx3005P.
The method is validated for quantitative determination of 1 mg to 400 mg allergenic substance / kg food sample using the reference material SureFood® QUANTARD Allergen 40 containing 40 mg allergenic substance / kg food sample.
|Test format||100 reactions|
|LOD (Detection limit)||≤ 1 mg / kg|
|LOQ (Limit of quantification)|
4 mg / kg using SureFood® PREP Advanced, protocol 1
Cross reactivity was observed with DNA extracts from muscovy duck (Cairina moschata).
|Evaluation||Real-time PCR instrument with two detection channels (510 nm and 580 nm)|