Double-Tube Multiplex TaqMan Real-Time PCR for the Detection of Eight Animal-Derived Dairy Ingredients

• Published in: Journal of agricultural and food chemistry Vol. 72; no. 20; pp. 11640 - 11651
• Main Authors: Ma, Xinyu, Xia, Huili, Pan, Yingqiu, Huang, Yafang, Xu, Ting, Guan, Feng
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 22.05.2024
• Subjects: Animals; Buffaloes - genetics; Camelus - genetics; Cattle - genetics; Dairy Products - analysis; DNA Primers - genetics; Equidae - genetics; Food and Beverage Chemistry/Biochemistry; Food Contamination - analysis; Goats - genetics; Horses - genetics; Milk - chemistry; Multiplex Polymerase Chain Reaction - methods; Real-Time Polymerase Chain Reaction - methods; Sheep - genetics; double-tube and duplex real-time PCR; TaqMan probe; simultaneous identification; milk and dairy products; adulteration
• ISSN: 0021-8561; 1520-5118
• DOI: 10.1021/acs.jafc.4c01294

Milk and dairy products represent important sources of nutrition in our daily lives. The identification of species within dairy products holds importance for monitoring food adulteration and ensuring traceability. This study presented a method that integrated double-tube and duplex real-time polymerase chain reaction (PCR) with multiplex TaqMan probes to enable the high-throughput detection of animal-derived ingredients in milk and dairy products. The detection system utilized one pair of universal primers, two pairs of specific primers, and eight animal-derived specific probes for cow, buffalo, goat, sheep, camel, yak, horse, and donkey. These components were optimized within a double-tube and four-probe PCR multiplex system. The developed double-tube detection system could simultaneously identify the above eight targets with a detection limit of 10–0.1 pg/μL. Validation using simulated adulterated milk samples demonstrated a detection limit of 0.1%. The primary advantage of this method lies in the simplification of the multiplex quantitative real-time PCR (qPCR) system through the use of universal primers. This method provides an efficient approach for detecting ingredients in dairy products, providing powerful technical support for market supervision.