火鸡源性成分数字PCR通用定量检测方法的研究
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摘要
建立了一种特异、灵敏和通用的火鸡源性成分数字PCR(digital Polymerase Chain Reaction,dPCR)定量检测方法,可对肉制品中火鸡源性成分的质量百分比进行准确定量,同时适用于微滴式数字PCR(droplet digital PCR,ddPCR)和芯片式数字PCR(chip digital PCR,cdPCR)平台。ddPCR平台对火鸡源性成分拷贝数浓度定性检测限(Limit of Detection,LOD)和定量检测限(Limit of Quantitation,LOQ)分别为1.75和6.43 copies·μL~(-1),cdPCR平台对火鸡源性成分拷贝数浓度LOD和LOQ分别为1.71和3.43 copies·μL~(-1)。对混合鲜肉中火鸡源性成分的质量百分比定量检测限为5%。本方法可对食品中火鸡源性成分进行定量检测。
A digital polymerase chain reaction(dPCR) was established to be applied as a specific,sensitive and universal method to quantify detection of turkey-derived components. The dPCR method can precisely quantify the mass percentage of the turkey-derived components in meat products,which is available from a droplet digital PCR(ddPCR) and chip digital PCR(cdPCR) platforms. The limit of detection( LOD) and limit of quantitation( LOQ) of the turkey-derived components detected byddPCR platform were 1. 75 and 6. 43 copies·μL~(-1),respectively. The LOD and LOQ for turkey-derived components detected by cd PCR platform were 1. 71 and 3. 43 copies·μL~(-1),respectively. The quantitative limit for detection of the mass percentage of turkey-derived components in mixed fresh meat was 5%( w/w). This method can be used for the quantitative detection of turkey-derived ingredients in poultry based products.
A digital polymerase chain reaction(dPCR) was established to be applied as a specific,sensitive and universal method to quantify detection of turkey-derived components. The dPCR method can precisely quantify the mass percentage of the turkey-derived components in meat products,which is available from a droplet digital PCR(ddPCR) and chip digital PCR(cdPCR) platforms. The limit of detection( LOD) and limit of quantitation( LOQ) of the turkey-derived components detected byddPCR platform were 1. 75 and 6. 43 copies·μL~(-1),respectively. The LOD and LOQ for turkey-derived components detected by cd PCR platform were 1. 71 and 3. 43 copies·μL~(-1),respectively. The quantitative limit for detection of the mass percentage of turkey-derived components in mixed fresh meat was 5%( w/w). This method can be used for the quantitative detection of turkey-derived ingredients in poultry based products.
引文
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[19]REN J,DENG T,HUANG W,et al.A digital PCR method for identifying and quantifying adulteration of meat species in raw and processed food[J].PLo S One,2017,12(3):e0173567.
[20]CAI Y,HE Y,LV R,et al.Detection and quantification of beef and pork materials in meat products by duplex droplet digital PCR[J].PLo S One,2017,12(8):e0181949.
[21]苗丽,张秀平,陈静,等.微滴数字PCR法对肉制品中牛源和猪源成分的定量分析[J].食品科学,2016,37(8):187-191.
[22]苗丽,张秀平,陈静,等.肉制品中羊源性成分微滴数字PCR法定量检测方法的研究[J].食品工业科技,2016,37(4):73-76.
[23]SCOLLO F,EGEA L A,GENTILE A,et al.Absolute quantification of olive oil DNA by droplet digital-PCR(dd PCR):comparison of isolation and amplification methodologies[J].Food Chemistry,2016,213:388-394.
[2]俞霖.政府监管视野下的食品安全问题研究[D].南昌:江西财经大学,2013.
[3]WANG Q,ZHANG X,ZHANG H Y,et al.Identification of 12 animal species meat by T-RFLP on the 12S rRNA gene[J].Meat Science,2010,85(2):265-269.
[4]HAIDER N,NABULSI I,AL-SAFADI B.Identification of meat species by PCR-RFLP of the mitochondrial COI gene[J].Meat Science,2012,90(2):490-493.
[5]GIRISH P S,ANJANEYULU A S R,VISWAS K N,et al.Polymerase chain reaction-restriction fragment length polymorphism of mitochondrial 12S rRNA gene:a simple method for identification of poultry meat species[J].Veterinary Research Communications,2007,31(4):447-455.
[6]张舒亚,谌鸿超,宋青,等.食品和饲料中火鸡源性成分的实时荧光PCR检测方法[J].食品与生物技术学报,2013,32(2):207-211.
[7]LPEZ-ANDREO M,LUGO L,GARRIDO-PERTIERRA A,et al.Identification and quantitation of species in complex DNA mixtures by real-time polymerase chain reaction[J].Analytical Biochemistry,2005,339(1):73-82.
[8]LOPEZ-OCEJA A,NUEZ C,BAETA M,et al.Species identification in meat products:a new screening method based on high resolution melting analysis of cyt b gene[J].Food Chemistry,2017,237:701-706.
[9]HINDSON B J,NESS K D,MASQUELIER D A,et al.High-throughput droplet digital PCR system for absolute quantitation of DNA copy number.[J].Analytical Chemistry,2011,83(22):8604-8610.
[10]余桂容,张维,杜文平,等.抗草甘膦转基因玉米外源基因dd PCR拷贝数分析[J].西南农业学报,2017,30(8):1707-1712.
[11]MORISSET D,TEBIH D,MILAVEC M,et al.Quantitative analysis of food and feed samples with droplet digital PCR[J].PLo S One,2013,8(5):e62583.
[12]CAI Y,LI X,LV R,et al.Quantitative analysis of pork and chicken products by droplet digital PCR[J].Bio Med Research International,2014,2014:810209.
[13]陈晨,张岩,李永波,等.微滴式数字PCR对肉制品中羊肉和猪肉定量分析[J].现代食品科技,2018,1:221-226.
[14]王珊,李志娟,苗丽.微滴式数字PCR与实时荧光PCR检测羊肉制品中羊源和猪源性成分方法的比较[J].肉类工业,2015,7:38-41.
[15]ROTHROCK M J,HIETT K L,KIEPPER B H,et al.Quantification of zoonotic bacterial pathogens within commercial poultry processing water samples using droplet digital PCR[J].Advances in Microbiology,2013,3(5):403-411.
[16]LAST A,MOLINA-GONZALEZ S,CASSAMA E,et al.Development and evaluation of a next-generation digital PCR diagnostic assay for ocular Chlamydia trachomatis infections[J].Journal of Clinical Microbiology,2013,51(7):2195-2203.
[17]ZHAO H,WILKINS K,DAMON I K,et al.Specific q PCR assays for the detection of orf virus,pseudocowpox virus and bovine papular stomatitis virus[J].Journal of Virological Methods,2013,194(1-2):229-234.
[18]任君安,邓婷婷,黄文胜,等.微滴式数字聚合酶链式反应精准定量检测羊肉中掺杂猪肉[J].食品科学,2017,38(2):311-316.
[19]REN J,DENG T,HUANG W,et al.A digital PCR method for identifying and quantifying adulteration of meat species in raw and processed food[J].PLo S One,2017,12(3):e0173567.
[20]CAI Y,HE Y,LV R,et al.Detection and quantification of beef and pork materials in meat products by duplex droplet digital PCR[J].PLo S One,2017,12(8):e0181949.
[21]苗丽,张秀平,陈静,等.微滴数字PCR法对肉制品中牛源和猪源成分的定量分析[J].食品科学,2016,37(8):187-191.
[22]苗丽,张秀平,陈静,等.肉制品中羊源性成分微滴数字PCR法定量检测方法的研究[J].食品工业科技,2016,37(4):73-76.
[23]SCOLLO F,EGEA L A,GENTILE A,et al.Absolute quantification of olive oil DNA by droplet digital-PCR(dd PCR):comparison of isolation and amplification methodologies[J].Food Chemistry,2016,213:388-394.