Development of ISO Standard for Real-Time Polymerase Chain Reaction Detection of Turkey-Derived Material

Abstract

In this study, the specific fragment of turkey chromosome Z-DNA sequence was selected as the target sequence for turkey-derived material detection. A real-time polymerase chain reaction (real-time PCR) method was established, which had good interspecies specificity and interspecies consistency. The target sequence was cloned into the plasmid pUC57, and after being diluted to different concentrations, the plasmid was tested by real-time PCR. The absolute detection limit of this method was 5 copies/PCR reaction. An international collaborative validation trial was conducted to validate this method. The results showed that the false positive and negative rates of the proposed method were both 0%, and the absolute detection limit was 5 copies/PCR reaction. The results of qualitative analysis of the diluted plasmid showed that the inter-laboratory standard deviation was 0.30, less than the maximum permitted value of 1; at a detection probability of 95%, the absolute detection limit was 3.2 copies/PCR reaction, less than the maximum allowable value of 20 copies/PCR reaction. Based on voting results and reviewers’ comments, this method was approved by the International Organization for Standardization (ISO) as an international standard method (ISO/TS 20224-8:2022).

Keywords

• turkey; real-time polymerase chain reaction; detection; international organization for standardization