面粉中毒麦成分检测方法研究
摘要
面粉作为人类的主要食物来源有着举足轻重的地位。作为加工面粉的原料小麦,经常能截获到有毒植物种子——毒麦。资料显示,我国小麦产区以及出口小麦的国家如：美国、澳大利亚、德国、法国、阿根廷及东北亚、中亚和独联体国家均是毒麦的主要分布区,也有多起国人因误食含毒麦碱的面粉而引起中毒事件发生的报道。
毒麦种子含有毒麦碱(Temuline-C7H12N2O),能麻痹动物中枢神经。人吃了含4%毒麦成分的面粉就会引起中毒反应,家禽食入体重0.7%剂量的毒麦时就会中毒。据不完全统计,1983年我国因食用毒麦或含毒麦成分的面粉制品而中毒的人数多达1200人次。
目前我国对于面粉中毒麦成分含量的检测未见有相关报道,因此,建立有效、准确、实用、快速的分子生物学检测鉴定方法非常重要。
本研究应用单核苷酸多态性(SNP)技术和双重实时荧光定量PCR技术对面粉中的毒麦成份进行快速检测。采用SNP技术和TaqMan-MGB探针对面粉中的毒麦成分的单碱基进行检测分析。结果表明：探针Du-57可以特异性鉴定出面粉中的毒麦成分。使用的TaqMan-MGB实时荧光PCR技术稳定性和特异性高,且其淬灭基团与报告基团比其他探针在空间位置上更加接近,实验结果更精确。
在上述两种检测方法中：单核苷酸多态性(SNP)技术在近似种鉴定上具有稳定性高,读数方便,同时可检测上百个数据,具有基因芯片检测技术等高通量特点,适用于大规模样品的筛查,缩短了检疫鉴定时间；TaqMan-MGB实时荧光定量PCR技术具有特异性强,敏感度高,重复性好,并有较强安全性的特点。
Flour has a very important status as human's staple food. Frequently, we can intercept one kind of poisonous plant seed from wheat which as the raw material for processing bread flour-Lolium temulentum. From many data we can tell that the wheat's main production area of our country and the export country are all the main distribution area of Lolium temulentum, the export country include US, Australia, Germany, France, Argentina and Northeast Asia, Central Asia and Commonwealth of Independent States. Meanwhile, many reports of poisoning accidents all because of the people eat the flour which contains the loline-type alkaloids.
Lolium temulentum's seed contains the loline-type alkaloids which could cause Animal's central nervous system affected with paralysis. Human could result in toxic reaction if eat the flour which contain four percent Lolium temulentum, poultry could result in toxic reaction if eat the Lolium temulentum's dosage is 0.7 percent of it's body weight. According to incomplete statistics, more than 1200 person result in toxic reaction because of eat Lolium temulentum or flour products which contains Lolium temulentum.
Currently, there is no report about the detection of Lolium temulentum content in flour, therefore, it is crucial to establish a effective、accurate、practical and rapid method involved in the molecular biology.
In this study we use the Single Nucleotide Polymorphisms (SNP) and duplex Real-time Fluorescent Quantitative PCR detection technology to quick detect the Lolium temulentum content in flour. We select the SNP TaqMan-MGB probe (Du-57) to detect and analyze the single-base of of Lolium temulentum content in flour. The results indicated that Du-57 probe can identified the Lolium temulentum content in flour specificity. The degree of TaqMan-MGB Real-time PCR detection technology's stability and specificity were higher, the probe's quencher and reporter closer in space than other probes, so the result of this study will more precision.
Through the two methods described:Single nucleotide polymorphism (SNP) technology has a high stability, easy reading, while test hundreds of data in the identification of approximately, with high flux features of gene chip technology, the technology is applicable to the large-scale sample screening, shorten the identification of the quarantine time. TaqMan-MGB Real-time Fluorescent Quantitative PCR's degree of specifity、repeatability、security were higher.
毒麦种子含有毒麦碱(Temuline-C7H12N2O),能麻痹动物中枢神经。人吃了含4%毒麦成分的面粉就会引起中毒反应,家禽食入体重0.7%剂量的毒麦时就会中毒。据不完全统计,1983年我国因食用毒麦或含毒麦成分的面粉制品而中毒的人数多达1200人次。
目前我国对于面粉中毒麦成分含量的检测未见有相关报道,因此,建立有效、准确、实用、快速的分子生物学检测鉴定方法非常重要。
本研究应用单核苷酸多态性(SNP)技术和双重实时荧光定量PCR技术对面粉中的毒麦成份进行快速检测。采用SNP技术和TaqMan-MGB探针对面粉中的毒麦成分的单碱基进行检测分析。结果表明：探针Du-57可以特异性鉴定出面粉中的毒麦成分。使用的TaqMan-MGB实时荧光PCR技术稳定性和特异性高,且其淬灭基团与报告基团比其他探针在空间位置上更加接近,实验结果更精确。
在上述两种检测方法中：单核苷酸多态性(SNP)技术在近似种鉴定上具有稳定性高,读数方便,同时可检测上百个数据,具有基因芯片检测技术等高通量特点,适用于大规模样品的筛查,缩短了检疫鉴定时间；TaqMan-MGB实时荧光定量PCR技术具有特异性强,敏感度高,重复性好,并有较强安全性的特点。
Flour has a very important status as human's staple food. Frequently, we can intercept one kind of poisonous plant seed from wheat which as the raw material for processing bread flour-Lolium temulentum. From many data we can tell that the wheat's main production area of our country and the export country are all the main distribution area of Lolium temulentum, the export country include US, Australia, Germany, France, Argentina and Northeast Asia, Central Asia and Commonwealth of Independent States. Meanwhile, many reports of poisoning accidents all because of the people eat the flour which contains the loline-type alkaloids.
Lolium temulentum's seed contains the loline-type alkaloids which could cause Animal's central nervous system affected with paralysis. Human could result in toxic reaction if eat the flour which contain four percent Lolium temulentum, poultry could result in toxic reaction if eat the Lolium temulentum's dosage is 0.7 percent of it's body weight. According to incomplete statistics, more than 1200 person result in toxic reaction because of eat Lolium temulentum or flour products which contains Lolium temulentum.
Currently, there is no report about the detection of Lolium temulentum content in flour, therefore, it is crucial to establish a effective、accurate、practical and rapid method involved in the molecular biology.
In this study we use the Single Nucleotide Polymorphisms (SNP) and duplex Real-time Fluorescent Quantitative PCR detection technology to quick detect the Lolium temulentum content in flour. We select the SNP TaqMan-MGB probe (Du-57) to detect and analyze the single-base of of Lolium temulentum content in flour. The results indicated that Du-57 probe can identified the Lolium temulentum content in flour specificity. The degree of TaqMan-MGB Real-time PCR detection technology's stability and specificity were higher, the probe's quencher and reporter closer in space than other probes, so the result of this study will more precision.
Through the two methods described:Single nucleotide polymorphism (SNP) technology has a high stability, easy reading, while test hundreds of data in the identification of approximately, with high flux features of gene chip technology, the technology is applicable to the large-scale sample screening, shorten the identification of the quarantine time. TaqMan-MGB Real-time Fluorescent Quantitative PCR's degree of specifity、repeatability、security were higher.
引文
[1]中华面粉网http://www.cnflour.com中国面粉粉师协会.
[2]中国园林植保网http://www.lawnchina.com/
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[2]中国园林植保网http://www.lawnchina.com/
[3]贺光,邱广蓉,孙开来,等.DNA测序中的一些常见问题和对策[J].中国医科大学学报,2002,31(5)：366-367.
[4]Smith L.M, Sanders J.Z,Kaiser R.J.,et al. Fluorescence detection in automated DNA sequenceanalysis. Nature,1986,321(6071):674-679
[5]郭晓强.拉斯克基础医学奖简介(2).生物学通报,2003,38(10)：59-60.
[6]郭晓强.自动测序仪的发明者.生物学通报,2005,40(6)：58-69.
[7]Wattana A, Aanasda NA, Sumlee D, et al Highly variable sequences in the env V3 region of HIV type 1 distributing among Thai carriers from 1995 to 1997 [J]. A D S Res Hum Retro viruses, 2000,16:283-289.
[8]张姮,康林.毒麦属6个种的rDNA ITS序列测定及分析..湖南农业大学学报,2009,35(1)：21-23.
[9]陈萍,温硕洋,曾玲.PCR-RFLP用于五种斑潜蝇的诊断和鉴别.植物检疫,2009,23(5)：22-24.
[10]张姮,康林.毒麦及其近似种rDNA ITS-RFLP初步分析,植物检疫,2008,22(2)：70-72.
[11]王师,包振民,张玲玲,等.应用PCR-RFLP技术鉴别区分中国沿海四种主要养殖扇贝.食品科学,2006,27(7)：210-213.
[12]宋敏,胡建民,何孔旺.PCR定量方法概述[J].上海畜牧兽医通讯,2005,(2)：-18-19.
[13]蔡霞.定量PCR技术及其应用现状[J].现代诊断与治疗,2005,16(2)：112-115.
[14]Vet JA, Majithia AR, Marras SA,et al. Multiplex detection of four pathogenic retroviruses using molecular beacons[J].Proc Natl Acad Sci USA,1999,96; 6394-6399.
[15]胡骑,程安春,汪铭书.荧光定量PCR技术的研究进展及应用.黑龙江畜牧兽医,2006,11：32-34.
[16]章桂明,程颖慧,王颖,等.应用Taq Man MGB探针检测小麦印度腥黑穗病菌和黑麦草腥黑穗病菌.植物病理学报,2006,36(2)：142-151.
[17]罗光华.Real-time PCR监测巨细胞病毒在肾移植中的应用[J].Clinical Chemistry, 2003,49 (2):325-327.
[18]Francis S C, Lisa D B, Aravinda C. A DNA Polymorphis Discovery Resource for Research on Human Genetic Variation. Genome Res,1998,8:1229-1231.
[19]李艳杰,龙火生,李影,等.单核苷酸多态性(SNP)的研究进展和应用.畜牧兽医杂志,2003,22(4)：16-18.
[20]杜玮南,孙红霞,方福德.单核苷酸多态性的研究进展[S].中国医学科学院学报,2000,22(4)：294-392.
[21]胡培丽,岳秉飞.单核苷酸多态性的研究进展.实验动物科学与管理,2006,23(1)：36-38.
[22]Shelly G. Yates; Richard J. Petroski; Richard G. Powell. Analysis of loline alkaloids in endophyte-infected tall fescue by capillary gas chromatography J. Agric. Food Chem.; 1990; 38(1) pp 182-185;
[23]Mark R. TePaske; Richard G. Powell; Richard J. Petroski; Melanie D. Samford; John A. Paterson. Quantitative analyses of bovine urine and blood plasma for loline alkaloids J. Agric. Food Chem.; 1993; 41 (2); 231-234.
[24]Joe D. Robbins; James G. Sweeny; Stanley R. Wilkinson; Donald Burdick. Volatile alkaloids of Kentucky 31 tall fescue seed (Festuca arundinancea) J. Agric. Food Chem.; 1972; 20 (5); 1040-1043.
[25]R. J. Petroski; S. G. Yates; D. Weisleder; R. G. Powell. Isolation, Semi-Synthesis, and Nmr Spectral Studies of Loline Alkaloids J. Nat. Prod.; 1989; 52 (4); 810-817.
[26]Mark R. TePaske; Richard G. Powell; Stephen L. Clement. Analyses of selected endophyte-infected grasses for the presence of loline-type and ergot-type alkaloids J. Agric. Food Chem.; 1993; 41 (12); 2299-2303.
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