Research To Distinguish Bos Grunniens,Bos Taurus And Bubalus From Meat Products Mixture Based On Mitochondrion 12S RRNA Gene

Mitochondrial DNA sequence is highly conserved in the intraspecific.Besides highly conserved,12S rRNA gene is able to endure degeneration and high temperature;it was used gradually in identification of feedingstuff,fresh meat,processed meat and traceability.This study use Bos grunniens,Bos taurus and Bubalus as materials,according to characteristic of processed food,sample was divide into two group:fresh sample and processed sample.Processed sample was processed at 100℃,120℃,140℃,160℃,180℃, to study effect for identification.Use correct way to extracting DNA.In order to evaluate the test sensitivity,four different percentages,1%,0.1%,0.01%,0.001%were prepared by dilute fresh sample DNA.Design primer to amplify a 137 bp fragment on the nuclear 18S rDNA gene from eukaryotic DNA,serving as endogenous control for PCR amplifiable DNA in the processed sample.Design universal primers that amplify a 453bp(Bos taurus is 452 bp) form Bos grunniens,Bos taurus and Bubalus,three specific sites were found out in the universal primers region of mitochondrial 12S rRNA gene,the specific sites can distinguish Bos grunniens,Bos taurus and Bubalus in fresh meat and processed meat mixture,it was identified by sequencing,establishing a PCR-RFLP way to identify Bos grunniens,Bos taurus and Bubalus.Use this way to identify sample,including two Bos grunniens breeds,Eight Bos Taurus breeds,Four Bubalus breeds,all above come from Sichuan,Gansu,Yunnan,Shanxi, Anhui,Guangdong,Hubei,Every breed collects 30 sample of muscle,420 in total.The primers can amplify correct fragment exactly.Use BspHⅠ,EcoNⅠ,HpaⅡto digestion that fragment.The result were validated by sequencings. The fragment of yak was digested to 134bp and 318bp,scalper was digested to 134bp and 318bp and buffalo was digested to 86bp and 367bp,proving its correctness by sequencings.There was no effect of routinely used additives or cooking temperature(100℃,120℃, 140℃,160℃,180℃) on the efficacy of PCR amplification,but signal was weak at 120℃.When the percentage of DNA is 0.01%,the fragment of 12S rRNA was every weak, proving the lowest percentage of DNA is 0.01%to identify Bos grunniens,Bos taurus and Bubalus.Use this way to identify 24 brands Yak dried meat which come from Yaan,Kangding, Chengdu Supermarket,the result is that:only 3 brands are the real yak dried meat,6 brands of the rest are the cattle dried meat,4 brands of the rest are the buffalo dried meat.In addition to the other meat products.Note beef jerky on the market with cattle,buffalo and even posing as other meat products Yak stem the phenomenon is very serious.This study establishment the China's first way that it is The simple,fast and cheap in identification of fresh meat and processed meat.