实时荧光PCR法鉴定食用淀粉植物来源
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摘要
以红薯g3pdh基因、藕ITS1-5.8S-ITS2基因、山药rbcL基因和葛根CHI基因等设计红薯、藕、山药以及葛根特异性引物探针;结合文献报道的木薯、大米、小麦、玉米及马铃薯特异性引物探针,建立红薯、木薯、马铃薯、藕、山药、葛根、大米、玉米及小麦等9种常见食用淀粉的实时荧光PCR检测方法。该方法特异性强,绝对灵敏度0.01 ng/μL,相对灵敏度1%(质量分数)。对50份市售淀粉样品的检测表明,在红薯淀粉、马铃薯淀粉、藕粉及葛根粉等中均检出非标识的马铃薯、木薯或玉米等成分,标签错误标识率达32%。
The specific primers and probes for sweet potato, lotus root, Chinese yam, and kudzu were designed based on the sequence differences of g3 pdh, ITS1-5.8 S-ITS2, rbcL and CHI gene. Combined with cassava-specific,rice-specific, wheat-specific, maize-specific, and potato-specific primers and probes which had been reported, nine real-time PCR systems with the characteristic of high specificity were developed for detecting sweet potato, cassava, potato, lotus root, Chinese yam, kudzu, rice, wheat, and maize component in edible starch. The absolute and relative sensitivity of these methods was 0.01 ng/μL DNA, 1%, respectively. Fifty commercial starches were tested by all the realtime PCR methods, and unlabeled components such as potato, cassava and/or maize were found in sweet potato starch,potato starch, lotus root powder, and kudzu starch. The mislabeled rate was 32%.
The specific primers and probes for sweet potato, lotus root, Chinese yam, and kudzu were designed based on the sequence differences of g3 pdh, ITS1-5.8 S-ITS2, rbcL and CHI gene. Combined with cassava-specific,rice-specific, wheat-specific, maize-specific, and potato-specific primers and probes which had been reported, nine real-time PCR systems with the characteristic of high specificity were developed for detecting sweet potato, cassava, potato, lotus root, Chinese yam, kudzu, rice, wheat, and maize component in edible starch. The absolute and relative sensitivity of these methods was 0.01 ng/μL DNA, 1%, respectively. Fifty commercial starches were tested by all the realtime PCR methods, and unlabeled components such as potato, cassava and/or maize were found in sweet potato starch,potato starch, lotus root powder, and kudzu starch. The mislabeled rate was 32%.
引文
[1] CASTILLO-VALDIVIA M E, L魷PEZ-MONTES A,ESPEJO T, et al. Identification of starch and deter mination of its botanical source in ancient manuscripts by MEKC-DAD and LDA[J]. Microchemical Journal,2014, 112:75-81.
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[3]康维民,肖念新.甘薯淀粉掺假的快速检测研究[J].食品科技, 2003(1):78-79.
[4] DHITAL S, SHRESTHA A K, GIDLEY M J. Relationship between granule size and in vitro digestibil ity of maize and potato starches[J]. Carbohydrate Polymers, 2010, 82(2):480-488.
[5] CHO D H, HAN J A, LIM S T. Identification of botanical origin of starch by using peptide mass fingerprinting of granule-bound starch synthase[J]. Journal of Cereal Science, 2013, 3(57):264-266
[6] YOON J W, JUNG J Y, CHUNG H J, et al. Identification of botanical origin of starches by SDSPAGE analysis of starch granule-associated proteins[J]. Journal of Cereal Science, 2010, 52(2):321-326.
[7] HAN J X, WU Y J, HUANG W S, et al. PCR and DHPLC methods used to detect juice ingredient from 7 fruits[J]. Food Control, 2012, 25(2):696-703.
[8] GUO L L, WU Y J, LIU M C, et al. Authentication of Edible Bird’s nests by TaqMan-based realtime PCR[J]. Food Control, 2014, 44:220-226.
[9] SN/T 3729.2-2013出口食品及饮料中常见水果品种的鉴定方法第2部分:杏成分检测实时荧光PCR法[S].
[10]韩建勋,吴亚君,王斌,等.实时荧光PCR法快速检测食用淀粉中的木薯成分[J].检验检疫学刊,2015(6):4-8.
[11] SN/T 1198-2013转基因成分检测马铃薯检测方法[S].
[12] SN/T 2584-2010水稻及其产品中转基因成分实时荧光PCR检测方法[S].
[13] MARTA H, MARIE-NO魮LLE D, GEORGES B, et al. Development and comparison of four real-time polymerase chain reaction systems for specific detection and quantification of Zea mays L[J]. J Agric Food Chem, 2004, 52(15):4632-4637.
[14] SN/T 1943-2007小麦中转基因成分PCR和实时荧光PCR定性检测方法[S].
[15] WANG W, HAN J X, WU Y J, et al. Simultaneous detection of eight food allergens using optical thin-film biosensor chips[J]. Journal of Agricultural and Food Chemistry, 2011, 59(13):6889-6894.
[16] PAULI E D, BARBIERI F, GARCIA P S, et al.Detection of ground roasted coffee adulteration with roasted soybean and wheat[J]. Food Research International, 2014, 61(7):112-119.
[17] CASAZZA A P, MORCIA C, PONZONI E, et al.A reliable assay for the detection of soft wheat adulteration in Italian pasta is based on the use of new DNA molecular markers capable of discriminating between Triticum aestivum and Triticum durum[J]. Journal of Cereal Science, 2012, 56(3):733-740.
[18] XU L, SHI P T, YE Z H, et al. Rapid analysis of adulterations in Chinese lotus root powder(LRP)by near-infrared(NIR)spectroscopy coupled with chemometric class modeling techniques[J]. Food Chemistry, 2013, 141(3):2434-2439.
[19]王绍清,武士奎,穆同娜,等.扫描电镜和稳定碳同位素比质谱法鉴别马铃薯淀粉中的掺假玉米淀粉[J].食品科学, 2010, 31(22):332-335.
[2] LIU J, LI J C, CHEN J, et al. Discrimination of kudzu starch adulteration by fourier transform infrared spectroscopy[J]. Food Science, 2011, 8(32):226-230.
[3]康维民,肖念新.甘薯淀粉掺假的快速检测研究[J].食品科技, 2003(1):78-79.
[4] DHITAL S, SHRESTHA A K, GIDLEY M J. Relationship between granule size and in vitro digestibil ity of maize and potato starches[J]. Carbohydrate Polymers, 2010, 82(2):480-488.
[5] CHO D H, HAN J A, LIM S T. Identification of botanical origin of starch by using peptide mass fingerprinting of granule-bound starch synthase[J]. Journal of Cereal Science, 2013, 3(57):264-266
[6] YOON J W, JUNG J Y, CHUNG H J, et al. Identification of botanical origin of starches by SDSPAGE analysis of starch granule-associated proteins[J]. Journal of Cereal Science, 2010, 52(2):321-326.
[7] HAN J X, WU Y J, HUANG W S, et al. PCR and DHPLC methods used to detect juice ingredient from 7 fruits[J]. Food Control, 2012, 25(2):696-703.
[8] GUO L L, WU Y J, LIU M C, et al. Authentication of Edible Bird’s nests by TaqMan-based realtime PCR[J]. Food Control, 2014, 44:220-226.
[9] SN/T 3729.2-2013出口食品及饮料中常见水果品种的鉴定方法第2部分:杏成分检测实时荧光PCR法[S].
[10]韩建勋,吴亚君,王斌,等.实时荧光PCR法快速检测食用淀粉中的木薯成分[J].检验检疫学刊,2015(6):4-8.
[11] SN/T 1198-2013转基因成分检测马铃薯检测方法[S].
[12] SN/T 2584-2010水稻及其产品中转基因成分实时荧光PCR检测方法[S].
[13] MARTA H, MARIE-NO魮LLE D, GEORGES B, et al. Development and comparison of four real-time polymerase chain reaction systems for specific detection and quantification of Zea mays L[J]. J Agric Food Chem, 2004, 52(15):4632-4637.
[14] SN/T 1943-2007小麦中转基因成分PCR和实时荧光PCR定性检测方法[S].
[15] WANG W, HAN J X, WU Y J, et al. Simultaneous detection of eight food allergens using optical thin-film biosensor chips[J]. Journal of Agricultural and Food Chemistry, 2011, 59(13):6889-6894.
[16] PAULI E D, BARBIERI F, GARCIA P S, et al.Detection of ground roasted coffee adulteration with roasted soybean and wheat[J]. Food Research International, 2014, 61(7):112-119.
[17] CASAZZA A P, MORCIA C, PONZONI E, et al.A reliable assay for the detection of soft wheat adulteration in Italian pasta is based on the use of new DNA molecular markers capable of discriminating between Triticum aestivum and Triticum durum[J]. Journal of Cereal Science, 2012, 56(3):733-740.
[18] XU L, SHI P T, YE Z H, et al. Rapid analysis of adulterations in Chinese lotus root powder(LRP)by near-infrared(NIR)spectroscopy coupled with chemometric class modeling techniques[J]. Food Chemistry, 2013, 141(3):2434-2439.
[19]王绍清,武士奎,穆同娜,等.扫描电镜和稳定碳同位素比质谱法鉴别马铃薯淀粉中的掺假玉米淀粉[J].食品科学, 2010, 31(22):332-335.