Identification of eleven meat species in foodstuff by a hexaplex real-time PCR with melting curve analysis

Meat product adulteration caused by temptation of economic benefit among different meats has always been a serious global problem. It is necessary to carry out a reliable and convenient method to identify meat species in foodstuff. In this paper, we developed a hexaplex real-time PCR (RT-PCR) assay using melting curve analysis to identify eleven meat species simultaneously. A total of six pairs of specific primers were designed based on mitochondrial genes, including CO I, CO II, ND4, 12S rRNA and 16S rRNA. The amplicons uniquely corresponded to six distinct melting peaks at 71.69 +/- 0.60 degrees C (Sheep/Goat), 74.47 +/- 0.34 degrees C (Horse), 78.87 +/- 0.33 degrees C (Cattle), 80.75 +/- 0.15 degrees C (Pig), 83.36 +/- 0.08 degrees C (Donkey) and 85.10-87.16 degrees C (Poultry: chicken, duck, goose, wild goose and quail), respectively. This study presented a validation including specificity, detection limit and practicability. The results showed that this method has good specificity of primers and the detection limits were 0.01-0.1 ng DNA of absolute detection limit and 0.1-1% meat mixture of relative detection limit. Validated by amplification ability and commercial products, this method can simultaneously and accurately identify eleven common meat species, indicating a good application foreground.

Automated summary

The study developed a hexaplex real-time PCR assay using melting curve analysis to identify eleven meat species simultaneously, showing good specificity of primers and detection limits of 0.01-0.1 ng DNA and 0.1-1% meat mixture. The method can accurately identify different meat species, indicating great application potential.