A novel multiplex xMAP assay for generic detection of avian, fish, and ruminant DNA in feed and feedstuffs.

The identification of animal species in feed and feedstuffs is important for detecting contamination and fraudulent replacement of animal components that might cause health and economic problems. A novel multiplex assay, based on xMAP technology and the generic detection of closely related species, was developed for the simultaneous differential detection of avian, fish, and ruminant DNA in products. Universal primers and probes specific to avian, fish, or ruminant species were designed to target a conserved mitochondrial DNA sequence in the 12S ribosomal RNA gene (rRNA). The assay specificity was validated using samples of 27 target and 10 nontarget animal species. The limits of detection of the purified DNA were determined to be 0.2 pg/µL-0.1 ng/µL by testing the meat samples of six species and four feedstuffs. The detection sensitivity of the experimental mixtures was demonstrated to be 0.01% (weight percentage). The assay's suitability for practical application was evaluated by testing feed samples; unlabeled animal ingredients were detected in 32% of the 56 samples. The assay differentially detected the three targeted categories of animal species in less than 2 h, reflecting improvements in speed and efficiency. Based on these results, this novel multiplex xMAP assay provides a reliable and highly efficient technology for the routine detection of animal species in feed and other products for which this information is needed.

[Faster detection of Vibrio parahaemolyticus in foods by FQ-PCR technique].

OBJECTIVE: To establish a rapid and accurate qualitative and quantitative method to detect Vibrio parahaemolyticus in food. METHODS: Primers and Taqman probe were designed according to the sequence of gyrase gene of Vibrio parahaemolyticus. The PCR fragment was cloned into PTM vector and was used as positive template for establishing the criterion curve. The simulated samples, made from negative food samples with Vibrio parahaemolyticus positive strain, were used to evaluate the sensitivity of the PCR reaction. 8 Vibrio parahaemolyticus standard strains and other 24 negative strains (8 strains were Vibrionaceae other than Vibrio parahaemolyticus, the rest 16 strains were none-Vibrionaceae) were treated in the same way to evaluate the specificity. All samples were detected with PE7000 sequence detection system and DA620 fluorescene detection system. RESULTS: FQ-PCR method had good specificity and high sensitivity. All 8 strains of Vibrio parahaemolyticus tested showed positive results while all other 24 strains were negative (8 strains were Vibrionaceae, 16 strain were from different). The correlation was 0.9871 between the concentration of positive template and the quantitative curve circular threshold. The threshold for detecting Vibrio parahaemolyticus in pure culture is 10 cfu/ml, and the threshold is as low as 2 cfu/ml with 16 simulative samples after these samples were incubated for a period of time. By direct quantitative detection for uncultured 16 food samples, the threshold is 100 cfu/g. CONCLUSION: FQ-PCR method has high sensitivity and specificity, and it is a handy and rapid detection method. Comparing with regular PCR method, it is not easily contaminated in operation, and can achieve high sensitivity and specificity with domestic instruments. FQ-PCR method has potential and applied value for detection of pathogenic bacteria in food.