肝水解肽样品中牛源及猪源性成分的PCR检测

目的: 建立肝水解肽各工艺步骤段中样品的PCR检测方法,用于检测生产原料的动物来源是牛源性或猪源性。 方法: 分别对猪源性及牛源性肝水解肽各工艺步骤段中样品进行了DNA提取,并加入猪、牛特异性引物进行扩增。对扩增产物进行酶切及测序验证。 结果: 工艺段为超滤液四之前的样品均可提取并扩增出相应的DNA片段,牛源性PCR产物与Genbank中牛序列的同源性为100%,猪源性PCR产物与猪序列的同源性为99.4%,PCR扩增的检测限为0.5 mg·mL^-1的肝细胞酶解液。 结论: 可用本方法检测肝水解肽各工艺步骤段中超滤液四之前的样品。

Objective: To develop a PCR method for identification bovine or porcine derived materials in liver hydrolysate samples. Methods: DNA were extracted from liver hydrolysate samples which prepared by different steps of the preparation process,and then they were amplified with specific primers.PCR products were digested by enzyme and sequenced to verify the sequence identity. Results: Characteristic DNA fragments were extracted and amplified in the samples prepared in the process steps before the fourth ultrafiltrate.It was found that PCR products amplified from bovine-derived samples and porcine-derived samples have 100% and 99.4% homologies to that of gene in the Genbank respectively.The method was confirmed with a minimum detection level of 0.5 mg·mL^-1 liver cell hydrolysate. Conclusion: The method can be applied to detect the DNA fragments in the samples prepared in the process before the fourth ultrafiltrate.

1 CAO Ji-juan(曹际娟),LU Xing-an(卢行安),WANG Gang(王刚),et al.Method on PCR detection of bovine materials in animal feedstuffs(PCR技术检测肉骨粉中牛源性成分的方法).J Hyg Res (卫生研究),2003,32,(1):30
2 LUO Jia-qin(罗家琴),WANG Jia-qi(王加启),BU Deng-pan(卜登攀),et al.Development and application of a PCR detection of bovine,sheep,pig and chichen derived materials in feedstuff(饲料中牛、羊、猪、鸡源性成分的PCR检测方法及其应用).Scien Agric Sin(中国农业科学),2008,41(7):2112
3 National standard from Regional one of Chemical Pharmaceutics by State Drug Administration,Vol.9(国家药品标准化学药品地标升国标)WS-10001-(HD-0808)-2002.2002.74
4 Identification of bovine ,sheep,and goat derived materials in import animal derived feedstuff –PCR method(进口动物源性饲料中牛羊源性成分检测方法 PCR方法)SN/T1119-2002
5 Identification of procine derived materials in animal-originated feedstuffs–PCR method(动物源性饲料中猪源性成分定性检测方法 PCR方法)GB/T 21101-2007
6 GAO Dan-dan(高丹丹),CAO Yu-sheng(曹郁生),WANG Ying-hua(王迎华).Application of PCR technique to detection of the animal origin of food(PCR技术在食品动物源性检测中的应用).Food Res Dev(食品研究与开发),2007,28(01):141
7 GAO Lin(高琳),XU Xing-lian(徐幸莲),ZHOU Guang-hong(周光宏),et al.Identification of porcine- and bovine-derived materials in meat products by PCR-PFLP assay(应用PCR-RFLP法鉴别肉制品中的猪和牛源性成分).J Nanjing Agric Univ(南京农业大学学报),2008,31(2):135
8 Tartaglia M,Saulle E,Pestalozza S,et al.Detection of bovine mitochondrial DNA in ruminant feeds:a molecular approach to test for the presence of bovine derived materials.J Food Prot,1998,61:513
9 Tartaglia M,Saulle E.Rapid communication:Nucleotide sequence of procine and ovine tRNA^lys and ATPase mitochondrial genes. J Anim Sci,1998,76:1
10 Lahiff S ,Glennon M ,O'Brien L,et al.Species-specific PCR for the identification of ovine,procine and chicken species in meat and bone meal(MBM).Mol Cell Probes,2001,15:27
11 John J.Dooley,Kelly E.Paine,Stephen D.Garrett,et al.Detection of meat species using TaqMan real-time PCR assays.Meat science ,2004,68:413
12 LI Lin(李林),CHEN Ji-ming(陈继明),WANG Zhi-liang(王志亮).Real-time PCR and conventional PCR detection of bovine materials in meat and bone meal(Real-time PCR和常规PCR方法快速鉴定肉骨粉中的牛源性成份).Chin J Prev Vet Med(中国预防兽医学报),2005,27(1):50