重组酶介导等温扩增技术快速检测牛肉及牛肉制品中的牛源性成分
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摘要
目的建立重组酶介导等温扩增技术快速检测牛肉及牛肉制品中牛源性成分的方法。方法应用重组酶聚合酶等温扩增技术(recombinase polymerase amplification,RPA),根据牛源性线粒体细胞色素b(Cytb)基因序列设计筛选了一对可用于扩增的引物,建立基于重组酶介导的等温扩增技术检测方法,并对该方法进行扩增温度、特异性和灵敏性验证。结果 40℃等温扩增条件下,所设计的引物的特异性为100%,该检测方法的灵敏性可达0.1 ng/μL。结论本研究成功建立了牛源性肉制品的等温扩增方法,该方法快速简便,具有较高的特异性和灵敏性,适用于市售生鲜肉制品及加工肉制品中牛源性成分的鉴定。
Objective To establish a method for rapid detection of bovine ingredient in beef and its derivates by recombinase polymerase amplification assay(RPA). Methods According to the Cytb gene sequence of bovine, a pair of RPA primers was designed and a novel event-specific detection method for beef and its derivates based on RPA was established. The specificity and sensitivity of this method were respectively performed for validation. Results Primers for RPA had good specificity under the condition of 40 ℃. This method had a high sensitivity of 0.1 ng/?L. Conclusion The RPA method for detection of bovine in meat and meat products is successfully established. This method is rapid, efficient and suitable for the identification of meat and its derivates from the market.
Objective To establish a method for rapid detection of bovine ingredient in beef and its derivates by recombinase polymerase amplification assay(RPA). Methods According to the Cytb gene sequence of bovine, a pair of RPA primers was designed and a novel event-specific detection method for beef and its derivates based on RPA was established. The specificity and sensitivity of this method were respectively performed for validation. Results Primers for RPA had good specificity under the condition of 40 ℃. This method had a high sensitivity of 0.1 ng/?L. Conclusion The RPA method for detection of bovine in meat and meat products is successfully established. This method is rapid, efficient and suitable for the identification of meat and its derivates from the market.
引文
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[13]高威芳,朱鹏,黄海龙.重组酶聚合酶扩增技术:一种新的核酸扩增策略[J].中国生物化学与分子生物学报,2016,32(6):627-634.Gao WF,Zhu P,Huang HL.Recombinase polymerase amplification:Anew DNA/RNA amplification strategy[J].Chin J Biochem Mol Biol.2016,32(6):627-634.
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[15]Clarke CM,Connor LO,Carreskinner H,et al.Development and performance evaluation of a recombinase polymerase amplification assay for the rapid detection of group B streptococcus[J].BMC Microbiol,2016,16(1):221.
[16]Eid C,Santiago JG.Assay for Listeria monocytogenes cells in whole blood using isotachophoresis and recombinase polymerase amplification[J].Analyst,2017,142(1):48-54.
[2]李宗梦,赵良娟,赵宏,等.肉及肉制品动物源性成分鉴别技术研究进展[J].食品研究与开发,2014,35(18):122-127.Li ZM,Zhao LJ,Zhao H,et al.Research progress in identification techniques of animal ingredient in meat and meat products[J].Food Res Dev,2014,35(18):122-127.
[3]Macedo-Silva A,Barbosa S,Alkmin M,et al.Hamburger meat identification by dot-ELISA[J].Meat Sci,2000,56(2):189-192.
[4]Cozzolino D,Murray I.Identification of animal meat muscles by visible and near infrared reflectance spectroscopy[J].LWT-Food Sci Technol,2004,37(4):447-452.
[5]Sch?nherr J.Analysis of products of animal origin in feeds by determination of carnosine and related dipeptides by high-performance liquid chromatography[J].J Agric Food Chem,2002,50(7):1945-1950.
[6]Nurjuliana M,Man YC,Hashim DM,et al.Rapid identification of pork for halal authentication using the electronic nose and gas chromatography mass spectrometer with headspace analyzer[J].Meat Sci,2011,88(4):638-644.
[7]Pfeiffer I,Burger J,Brenig B.Diagnostic polymorphisms in the mitochondrial cytochrome b gene allow discrimination between cattle,sheep,goat,roe buck and deer by PCR-RFLP[J].BMC Genetics,2004,5(1):30.
[8]Montiel-Sosa J,Ruiz-Pesini E,Montoya J,et al.Direct and highly species-specific detection of pork meat and fat in meat products by PCRamplification of mitochondrial DNA[J].J Agric Food Chem,2000,48(7):2829-2832.
[9]孙晶莹,孙丽君,王晓红,等.肉制品中牛源性成分多重实时荧光PCR检测方法的建立及初步应用[J].食品安全质量检测学报,2016,7(9):3756-3760.SUN JY,SUN LJ,WANG XH,et al.Establishment and application of multiple real-time fluorescent PCR to detect the beef ingredient in meat products[J].J Food Saf Qual,2016,7(9):3756-3760.
[10]沙才华,杨素,廖秀云,等.应用三重PCR方法鉴别肉制品中猪和牛源性成分[J].畜禽业,2011,(12):52-53.Sha CH,Yang S,Liao XY,et al.Applies triple PCR Simultaneously to Examine the porcine and Bovine Derived Materials[J].Livest Poultry Ind,2011,(12):52-53.
[11]Piepenburg O,Williams CH,Stemple DL,et al.DNA detection using recombination proteins[J].PLo S Biol,2006,4(7):1115-1121.
[12]Crannell ZA,Rohrman B,Richards-Kortum R.Equipment-free incubation of recombinase polymerase amplification reactions using body heat[J].PLo S One,2014,9(11):e112146.
[13]高威芳,朱鹏,黄海龙.重组酶聚合酶扩增技术:一种新的核酸扩增策略[J].中国生物化学与分子生物学报,2016,32(6):627-634.Gao WF,Zhu P,Huang HL.Recombinase polymerase amplification:Anew DNA/RNA amplification strategy[J].Chin J Biochem Mol Biol.2016,32(6):627-634.
[14]Santiagofelipe S,Tortajadagenaro LA,Puchades R,et al.Recombinase polymerase and enzyme-linked immunosorbent assay as a DNAamplification-detection strategy for food analysis[J].Anal Chim Acta,2014,811(81-87).
[15]Clarke CM,Connor LO,Carreskinner H,et al.Development and performance evaluation of a recombinase polymerase amplification assay for the rapid detection of group B streptococcus[J].BMC Microbiol,2016,16(1):221.
[16]Eid C,Santiago JG.Assay for Listeria monocytogenes cells in whole blood using isotachophoresis and recombinase polymerase amplification[J].Analyst,2017,142(1):48-54.