关键词: 枸杞子；大肠杆菌；质粒标准品；快速检测

Abstract: The virulence genes escV, stx2 and hlyA of Escherichia coli were selected as target sequences to construct plasmid standards for the rapid detection of E. coli. The recombinant plasmids were verified by colony PCR and sequencing, and after passaging for 15 generations, sequencing was carried out to verify the stability of the plasmid standards. The escV, stx2, and hlyA plasmid standards were prepared, and they were evaluated by limit of detection (LOD), homogeneity, stability and qualitative tests. These plasmid standards were applied to detect goji berries contaminated with E. coli. The results of colony PCR and sequencing showed successful construction of the escV, stx2 and hlyA plasmid standards. The detection sensitivity of the plasmid standards was at the pg level by the PCR system. The PCR results showed that the plasmid standards were specific, and they remained stable for 15 generations. The LODs of the escV, stx2 and hlyA plasmid standards were 3.93 × 106, 2.41 × 105 and 2.14 × 105 copies/mL, respectively, and they had good stability at 25, 4 and ?20 ℃. When these plasmid standards were used to detect E. coli in 34 batches of goji berries, escV and stx2 were undetectable while hlyA was detected in 3 batches with a detection rate of 8.82%. Using the method specified in the national standard GB 4789.6?2016 Food Hygiene Microbiology Test Standard combined with 16S rRNA gene sequencing, E. coli was not detected in the 3 batches of goji berry samples indicating that the detection sensitivity of the plasmid standards was high, and the detection rate was greatly improved as compared with traditional microbial separation and identification.

Key words: Lycium barbarum; Escherichia coli; plasmid standard; rapid detection