Laboratory method validation of meat species-specific detection using Real-Time PCR test-system “Meat-Test”
Journal Title: Наукові горизонти. Scientific Horizons - Year 2018, Vol 71, Issue 9
Abstract
Intralaboratory validation of meat-species identification method was performed according to european standards and regulations. Validation was performed by designed test-system "Meat-test" on the basis of TagMan technology Real-Time PCR.This test-system allows to perform species-specific identification of animal tissues in meat raw material, grinded up meat raw material in the content of semi-finished meat products, finished untreated meat products, and meat products under thermal treatment. Test-system id designed as multiplex and it allows to identify three targets-chicken, pork and beef, simultaneously. Method validation was performed for such basic parameters as specificity, limit of detection, analytical sensitivity, amplification efficiency and linearity. Method specificity was 100% for all three indicators – Fam (chicken),Hex (pork), and Rox (beef). No cross reactions was detected. No false-negative and false-positive results was detected, what shows high level of applied method. Limit of detection and analytical sensitivity for Fam and Hex was 1×10-6 and 1×10-5 ng, respectively. Limit of detection was 1×10-7ng, and analytical sensitivity was 1×10-6 ng for Rox. Obtained values of slope of calibration curves (α) were within –3,58 to –3,10,what shows normal amplification efficiency. Calculated PCR efficiency values for Fam, Hex, Rox, was 103,2, 100,0 and 94,9 %, respectively, what is high enough value for diagnostic test-system. Clear dependence of analysed samples Ct values on logarithm of initial concentration of added substrate was shown (R2 ≥ 0,98). Obtained validation parameters meet the EU PCR-performing standards. It enables application of this method in laboratory practice. This approach can be recommended to test laboratories for high reliability implementation of obtained results and analytical errors (false-positive and false-negative results) elimination.
Authors and Affiliations
R. Oblap
Keywords
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- EP ID EP631672
- DOI 10.33249/2663-2144-2018-71-9-10-96-103
- Views 249
- Downloads 0
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