Study On The Identification Method Of Deer Blood By PCR-RFLP

Deer blood is a kind of traditional Chinese medicine that is the blood of sika deer(Cervus Nippon)or red deer(Cervus elaphus).Deer blood has been used as medicinal for more than one thousand years in our country.As people’s demand for health continues to increase,the value of deer blood’s is increasingly at home and abroad.Deer blood is developed to commercial products such as liquor,powder,pills,etc.,and its potential market is recognized as huge.To gain extra profit,there are fake deer blood or its products which mixed with blood from animals other than sika deer or red deer in the markets.The common source of blood are pig,ox,sheep,duck,etc.,and it is difficult to distinguish them from genuine deer blood with traditional methods,such as appearance identification,microscopic identification,and physical and chemical identification.To establish a rapid and convenient method to identify deer blood,we initiated this research.Objective:1.To establish a polymerase chain reaction coupled with restriction fragment length polymorphism(PCR-RFLP)method which can identify deer blood.2.To search for a restriction enzyme which can distinguish the blood of deer or non-deer and determine the detection limit.3.To search for a restriction enzyme which can distinguish the blood of sika deer or red deer and determine the detection limit.Methods:1.Select an optimal genomic DNA extraction kit in three differentsuppliers,with the best genomic DNA concentration and purity.2.Select a pair of universal primers within the primers of COI or cytb,which are both widely used as DNA barcoding markers in animals.Optimize the condition of PCR using this pair of primers.3.Purify the PCR product of this pair of primers,and sub-clone into a pMD20-T vector to sequence this PCR product.4.Search for the appropriate restriction enzymes according to the sequencing results using a software(http://nc2.neb.com/NEBcutter2/).5.Determine the minimum detected proportion added to blood of deer with other non-deer sample.And of sika deer with red deer.Results:1.Blood & Cell Culture DNA Mini Kit(25)(QIAGEN,13323)can generate genomic DNA with high concentration and purity;Blood & Cell Culture DNA Mini Kit(25)with less genomic DNA degradation.2.The PCR product of a pair of primers of cytb gene is one clear and specific band.In the contrast,the PCR product of a pair of primers of coI gene has multiple bands even with smear.3.The sequencing results of cytb gene fragments showed that it can strongly differentiate each sample.4.Two DNA restriction enzymes of EcoRV and MseI were selected to distinguish deer from other species and to sika deer from red deer,respectively.EcoRV digestion made two fragments of 287 bp and 185 bp in deer,but no digestion in other species;MesI digestion made two fragments of 281 bp and191bp in sika deer,but three fragments of 281 bp,126bp and 65 bp in red deer.The 191 bp fragment is a characteristic marker of sika deer,and the 126 bp red deer.5.The minimum detected proportion added to blood of deer with other sample was 3%,6% of sika deer with red deer.Conclusion:1.A PCR-RFLP method according to the sequences of cytb gene was established.This method can identify the blood of sika deer rapidly andconveniently.2.The method we established in this study has a potential to use as a powerful quality control method of deer blood and its products.