Real-Time Fluorescence PCR Method For Identification Of Ruminant Animals-derived Materials In Feedstuff

The feedstuff containing ruminant animals-derived materials is the main approach ofspreading of BSE and Scrapic. Many studies have been made on the method for identificationof ruminant animals-derived materials with real-time fluorescence PCR. However, report onusing SYBR Green I dye detection format has not been found. This paper aims to develop anew reliable, simple and easy method for identification of ruminant animals-derived materialsin feedstuff by using SYBR Green I detection format and a species-specific PCR primer toavoid the imported animals-derived feedstuff and prevent China from the BSE.The common conserved sequence was carried out according to the BLAST result of fourruminat mitochondrial Cytb gene sequence. According to this sequence a set of commonlyused species-specific primer was designed. By using SYBR Green I detection format underthe optimized PCR system and condition the real-time fluorescence PCR was utilized toidentify the presence of ruminant animals-derived materials in feedstuff. The probability ofdistinguishing the species of four ruminants by their melting temperature was examined. Teston the specificity, repeatability, detection limit and primer competition was also carried out.Real-time PCR results present that all four ruminants DNA are amplified and their Tmvalue are all different. There is only amplification from four ruminants DNA, but noamplification from other animals. Excellent repeatability of this method was proved. The Tmvalue of bovine, goat, sheep and deer is failed in the range of 82.40±0.026℃, 79.95±0.056℃,81.13±0.063℃and 79.50±0.063 individually and no cross is found. The detection limit of thismethod can reach 0.001％at least. Primer competition test shows main component dominatesthe PCR reaction and in ruminants mixture with similar proportion all components can beidentified except deer because deer and goat's melting curve overlapped.This paper designed a commonly used ruminants-specific primer. The method foridentification of ruminant animals-derived materials by SYBR Green I detection format wasdeveloped. The species of four ruminants can be distinguished by Tm value. It is easy, rapid,sensitive method with good specificity and repeatability and can be used to identify ruminatproducts. It will play an key role in preventing China from BSE, keeping national economicsafety and peple healthy and promoting the development of food and feedstuff industry.