Development of loop-mediated isothermal amplification (LAMP) assay for rapid and sensitive identification of ostrich meat

Abstract

Animal species identification is one of the primary duties of official food control. Since ostrich meat is difficult to be differentiated macroscopically from beef, therefore new analytical methods are needed. To enforce labeling regulations for the authentication of ostrich meat, it might be of importance to develop and evaluate a rapid and reliable assay. In the present study, a loop-mediated isothermal amplification (LAMP) assay based on the cytochrome b gene of the mitochondrial DNA of the species Struthio camelus was developed. The LAMP assay was used in combination with a real-time fluorometer. The developed system allowed the detection of 0.01% ostrich meat products. In parallel, a direct swab method without nucleic acid extraction using the HYPLEX LPTV buffer was also evaluated. This rapid processing method allowed detection of ostrich meat without major incubation steps. In summary, the LAMP assay had excellent sensitivity and specificity for detecting ostrich meat and could provide a sampling-to-result identification-time of 15 to 20 minutes.

Figure 1. The anneal curve reactions of different ostrich meat reference DNA.

The products showed a melting temperature of 86.5°C (±0.25).

Figure 2. Box plots of detection time of LAMP assay with a detection limit up to 0.01% of ostrich.

The Box plots was generated by using six replecates for each concentration.

Figure 3. Amplification signal of ostrich meat DNA using the direct swab and HYPLEX buffer test method.

The red curve is the signal of ostrich DNA isolated with DNeasy tissue Isolation Kit (detection time of 6∶18 min). The other positive curves in the oval shap are the direct swab samples with a detection time of 11∶03–12∶18 min. The pink curve is the negative control (see table 3).