两种PCR方法检食品中花生过敏原Arah1成分.pdf

食品研究与开及
2011年9月
基础研究
Food Research And Development
第32卷第9期
两种和PCR方法检測食品中花生过原Arah1成分
陈家杰,王海燕,梁秋妮,吉坤美,刘志刚
(呼吸疾病国家重点实验室深圳大学变态反应分室,广东深圳518060)
摘要:采用套式PCR和荧光实时定量PCR两种方法检测食品中花生主受过敏原 Ara hi基因成分,从而推断食品中是
否含有花生过敏原成分。根据 Genbank提供的花生主要过敏原基因 Ara hi DNA序列(登录号为AF432231)的中一段序
列分别设计2对内外特异性引物,扩增目的基因片段来建立套式PCR方法;再用上述内引物扩增目的片段,建立SYBR
Green I荧光实时定量PCR方法,绘出拷贝数-CT标准曲线;并通过这2种PCR方法检测8种食品中含有花生主要过敏
原 Ara hi基因成分。套式PCR方法具有较高的持异性和灵敏度, SYBR GreenⅠ荧光实时定量PCR方法的标准曲线在3x
10至3x10° copley范閩内线性关系良好,R2值为0.9935,尬测低展设定为3x10 copies。2种方法检涮8种食品样品,结果
均与食物过敏原标注内容相符。本研究建立的2种方法具有快遠、灵敏等优点,可用于食品中花生主要过敏原基因
Arah1成分的检测。
关键词:套式RPCR;荧光实时定量PCR;花生主要过敏原; Ara hi
Development of Two PCR Assays to Detect Peanut Allergen Ara hi DNA Residue in Food Products
CHEN Jia-jie, WANG Hai-yan, Liang Qiu-ni, JI Kun-mei, LIU Zhi-gang
基金项目:深港创新圈计划项目(2007)
作者简介:陈家杰(1985-),男(汉),硕土研究生,研究方向:食品中花生过敏原检测方法的研究。
通信作者
2.5稳定性的检测
标准偏差为1.36%<5%,说明本实验研究方法的准确
取苦丁茶样品溶液3份,按上述测定方法重复测度较高。
定3次,各样品的相对标准偏差在0.0-0.4%之间,说明
此方法稳定性很好。
3结论
表3精密度的检测记录
采用超声波萃取法提取苦丁茶中黄酮类化合物,
Table 3 Precision test
用三波长分光光度法测定苦丁茶中黄酮的含量,方法
编号
平均值
RSD/@o
简便可靠。正交实验表明,在乙醇浓度为70%,料液比
△A
0.077
0.078
0.078
0.078
0.4%
为1:20(gmL),超声时间为15min,苦丁茶中黄酮的含
2.6加标回收
量达到1.81%,实验的重现性好、准确度高。
取苦丁茶黄酮提取液5mL于50mL容量瓶中,加
人0.1mL0.2000gL的芦丁标准溶液,用前述方法测定参考文献
吸光度。其结果见表4。
]张建,蔡保国,杜福彦.山绿茶中黄酮的超声波提取研究们食品
寝4加标回收致据表
工业,2006(1:49-50
Table 4 Determination results of recovery
]郭孝武超声技术在中草药成分提取中的应用中草药,1993,24
标题样品mgL)加标量(ロgL)实测量んmL)回收率院平均回收率%RSD%
(10):548-549
2.88
[3]回瑞华,候冬岩,关崇新,等三波长分光光度法测定红茶及其饮
101
料中黄的含量「〕食品科技,2004(5):67-69
100.20
4李明静,张卫,赵东保,等.三波长分光光度法测定黑沙蒿中的
10
总黄酮]分析实验窒,2007(3):99-10
0.40
10
加标回收试验中标样的回收率为10020,其相对
收稿日期:2010-12-24
万方数据
陈家杰,等:两种PCR方法检测食品中花生过歙原ArhI成分
基础研究
(State Key Laboratory of Respiratory Disease for Allergy at Shengzhen University, She
ity, She
enzhen
518060, Guangdong, China)
Abstract To develop new methods for detection of the peanut major allergen component Ara hi DNA in foods
by using Nested PCR and SYBR Green I Real-time quantitative PCR technology. A DNA fragment of the peanut
major allergen Ara hl gene was amplified through the design of two pair specific primers according to data of
Genbank No. AF432231. The two pairs of primers were used in Nested PCR. The inner pair of primers for
amplification of short DNA fragment was used in SYBR Green I Real-time PCR, in which the standard curve is
made according to relationship between the copies and Ct value. These two methods could be used to detect
peanut allergen Ara hi DNA residue in foods. These two methods were very specific and sensitive, and in SYBR
Green I Real-time PCR the linear correlation ranged from 310 o 3x10copies with 0. 935 of R2 value and 3 x
10copies of detection limit. Through these two PCR assays, the results of eight foods were consistent with the
peanut allergen labels from their manufactures. The two PCR methods were suitable for detection of the peanut
major allergen Ara hi DNA residue in foods with good sensitivity and accuracy
Key words: Nested PCR; Real-time quantitative PCR; peanut major allergen; Ara h1
花生是一种营养丰富、深受人们喜爱的常见食1.12主要试剂
物,但也是常见的一种主要的食物过敏原。花生食物
定量PCR试剂( SY