CN105543363A - 一种Cry3Bb1实时荧光PCR检测方法及其试剂盒 - Google Patents

一种Cry3Bb1实时荧光PCR检测方法及其试剂盒 Download PDF

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CN105543363A
CN105543363A CN201610009420.5A CN201610009420A CN105543363A CN 105543363 A CN105543363 A CN 105543363A CN 201610009420 A CN201610009420 A CN 201610009420A CN 105543363 A CN105543363 A CN 105543363A
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cry3bb1
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王仲敏
赵璟源
姜一
方焱
张裕君
贺艳
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Tianjin Entry Exit Inspection and Quarantine Bureau of Animals Plants and Food Inspection Center
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Abstract

本发明“一种Cry3Bb1实时荧光PCR检测方法及其试剂盒”涉及转基因检测领域。通过设计针对Cry3Bb1基因特异序列的特异引物，应用实时荧光PCR扩增技术，实现了Cry3Bb1基因的快速检测。该方法特异性强，灵敏度高和稳定性好，对于提高Cry3Bb1基因检测的准确性、缩短检测时间具有重要的作用。

Description

一种Cry3Bb1实时荧光PCR检测方法及其试剂盒
技术领域
本发明涉及转基因植物极其产品检测领域，提供了一种利用实时荧光PCR扩增技术快速检测抗虫基因Cry3Bb1的方法及其试剂盒，适用于口岸检验检疫实验室应用。
背景技术
Cry3Bb1基因编码表达一种从苏云金芽孢杆菌中来的抗鞘翅类昆虫的蛋白质，目前该基因已被美国孟山都公司用于转基因抗虫玉米培育研发中，并将MON863和MON88017两个含有Cry3Bb1基因的转基因玉米品系投入市场，来控制玉米根蠕虫危害。欧盟和我国也先后批准了这两个转基因玉米品系进口，我国目前进口转基因玉米用于饲料等加工工业，未批准其用于人类食品，也不允许国内商业化种植。
目前在我国转基因生物安全检测标准中尚无实时荧光PCR检测方法。由于我国玉米及其制品进口量逐年上升，检测精度、速度要求越来越高，传统的定性PCR检测方法已不能满足商品快速通关的需要。
本研究通过巧妙地设计针对外源基因Cry3Bb1特异引物序列及Taqman探针，应用实时荧光PCR扩增技术，实现了Cry3Bb1的高效快速检测。该方法特异性强，灵敏度高和稳定性好，对于提高Cry3Bb1检测的准确性、缩短检测时间具有重要的作用，适合口岸检验检疫部门运用和推广。
发明内容
本发明需要解决的技术问题是提供用于检测Cry3Bb1的特异性引物及Taqman探针。
本发明需要解决的另一问题是提供包含上述引物和Taqman探针的PCR检测试剂盒。
本发明用于检测Cry3Bb1的PCR引物及Taqman探针序列为：
上游引物Cry3Bb1-F：5’—GAGCTCCAGACCAACCACAAC—3’
下游引物Cry3Bb1-R：5’—AACTCCTTGTAGTTCAGCTCTTCCA—3’
Taqman探针Cry3Bb1-P：5’FAM—AGTACCCGCTGGCCGACAACCC—BHQ13’
本发明的Cry3Bb1检测试剂盒，包括以下成分：
（1）Cry3Bb1DNA模板和阴性对照DNA模板
（2）Cry3Bb1上下游引物及Taqman探针：
Cry3Bb1-F：5’—GAGCTCCAGACCAACCACAAC—3’
Cry3Bb1-R：5’—AACTCCTTGTAGTTCAGCTCTTCCA—3’
Cry3Bb1-P：5’FAM—AGTACCCGCTGGCCGACAACCC—BHQ13’
（3）10×ExTaqPCRBuffer,DDH2O,2.5mMdNTP,5U/μlExTaqHS酶
（4）判定结果：Ct值小于35，说明样品中含有Cry3Bb1；Ct值大于等于40，则认为样品中不含Cry3Bb1成分；Ct值介于35和40之间，需要对样品进行复验，复验Ct值小于40，则认为样品含有Cry3Bb1。
与现有技术相比，本发明的进步在于：检测速度快，节省时间，特异性强，灵敏度高，稳定性好，适合口岸检验检疫实验室快速鉴定Cry3Bb1。
附图说明
图1为Cry3Bb1试剂盒特异性测试结果，1-8号样品为8个转基因玉米品系，分别为MON89034、Bt11、Bt176、TC1507、MIR604、MON88017、MIR162和MON863，9号样品为非转基因玉米。从图1可以看出，含有Cry3Bb1的6号和8号样品有明显的扩增曲线产生，其他对照样品均无荧光信号，用本试剂盒能准确地检测出Cry3Bb1。
具体实施方式
实时荧光PCR引物、探针设计:
根据Cry3Bb1序列，利用PrimerPremier5.0引物设计软件，设计一组引物及TaqMan探针，引物探针位置如上所示，引物及TaqMan探针由Invitrogen（上海英俊生物技术有限公司）公司合成：
上游引物Cry3Bb1-F：5’—GAGCTCCAGACCAACCACAAC—3’
下游引物Cry3Bb1-R：5’—AACTCCTTGTAGTTCAGCTCTTCCA—3’
Taqman探针Cry3Bb1-P：5’FAM—AGTACCCGCTGGCCGACAACCC—BHQ13’
标本来源
本实验测试了9个玉米样品，其中包括8个转基因玉米品系样品，1个非转基因玉米样品，见下表：
实施例1、Cry3Bb1的检测
提取：按照Promega组织基因组DNA提取试剂盒说明书进行操作，提取9份样品玉米基因组DNA溶于100uLTE中，-20℃保存备用。
扩增：以无菌水作为空白对照，分别以MON89034、Bt11、Bt176、TC1507、MIR604、MON88017、MIR162、MON863和非转基因玉米基因组DNA作为模板，进行实时荧光PCR扩增。
25μlPCR反应体系：
10×ExTaqBuffer2.5μl
2.5mmol/LdNTP2μl
20μmol/LCry3Bb1-F0.5μl
20μmol/LCry3Bb1-R0.5μl
20μmol/LCry3Bb1-P0.5μl
模板DNA2μl
5U/μLTaq酶0.125μl
ddH2O16.875μl
PCR反应条件：95℃预变性2min，95℃变性10s，60℃退火30s，40个反应循环，4℃降温。
结果：
从图1结果可以看出，6号和8号样品为Cry3Bb1，荧光信号积累出现典型扩增曲线，其他各种转基因玉米和9号非转基因玉米都没有荧光信号积累升高。结果表明，本发明中的Cry3Bb1特异引物只能从样品中扩增出Cry3Bb1基因，其他样品及阴性对照都没有出现扩增产物，显示了该引物良好的特异性。
序列表
SEQUENCELISTING
<110>天津出入境检验检疫局动植物与食品检测中心
<120>一种Cry3Bb1实时荧光PCR检测方法及其试剂盒
<141>2016－1－6
<160>3
<210>1
<211>21
<212>DNA
<213>人工序列
<400>1
gagctccagaccaaccacaac21
<210>2
<211>25
<212>DNA
<213>人工序列
<400>2
aactccttgtagttcagctcttcca25
<210>3
<211>22
<212>DNA
<213>人工序列
<400>3
agtacccgctggccgacaaccc22

Claims (3)

1.一种用于Cry3Bb1检测的特异性引物、探针，其特征在于，序列为：
Cry3Bb1-F：5’—GAGCTCCAGACCAACCACAAC—3’
Cry3Bb1-R：5’—AACTCCTTGTAGTTCAGCTCTTCCA—3’
Cry3Bb1-P：5’FAM—AGTACCCGCTGGCCGACAACCC—BHQ13’。
2.一种用于检测Cry3Bb1的试剂盒，其特征在于，包括如下成分：
（1）阳性对照DNA模板和阴性对照玉米DNA模板
（2）Cry3Bb1上下游引物
Cry3Bb1-F：5’—GAGCTCCAGACCAACCACAAC—3’
Cry3Bb1-R：5’—AACTCCTTGTAGTTCAGCTCTTCCA—3’
（3）Cry3Bb1TaqMan探针
Cry3Bb1-P：5’FAM—AGTACCCGCTGGCCGACAACCC—BHQ13’
（4）10×ExTaqPCRBuffer,ddH2O,2.5mMdNTP,5U/μlTaq酶。
3.一种Cry3Bb1的快速检测方法，包括如下步骤：
（1）25μlPCR反应体系：
10×ExTaqBuffer2.5μl
2.5mmol/LdNTP2μl
20μmol/LCry3Bb1-F0.5μl
20μmol/LCry3Bb1-R0.5μl
20μmol/LCry3Bb1-P0.5μl
模板DNA2μl
5U/μLTaq酶0.125μl
ddH2O16.875μl
（2）PCR反应条件：95℃预变性2min，95℃变性10s，60℃退火30s，40个反应循环，4℃降温
（3）判定结果：Ct值小于35，说明样品中含有Cry3Bb1；Ct值大于等于40，则认为样品中不含Cry3Bb1成分；Ct值介于35和40之间，需要对样品进行复验，复验Ct值小于40，则认为样品含有Cry3Bb1。
CN201610009420.5A 2016-01-07 2016-01-07 一种Cry3Bb1实时荧光PCR检测方法及其试剂盒 Pending CN105543363A (zh)

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Citations (3)

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KR20060101658A (ko) * 2005-03-21 2006-09-26 대한민국(국립농산물품질관리원장) 유전자 변형체의 검정을 위한 프라이머 세트，검정방법 및이에 사용되는 표준 플라스미드
EP1950311A1 (en) * 2007-01-29 2008-07-30 Scientific Institute of Public Health (IPH) Transgenic plant event detection
CN104212880A (zh) * 2013-07-24 2014-12-17 北京福德安科技有限公司 基于lamp的转基因玉米mon863基因快检试剂盒及检测方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20060101658A (ko) * 2005-03-21 2006-09-26 대한민국(국립농산물품질관리원장) 유전자 변형체의 검정을 위한 프라이머 세트，검정방법 및이에 사용되는 표준 플라스미드
EP1950311A1 (en) * 2007-01-29 2008-07-30 Scientific Institute of Public Health (IPH) Transgenic plant event detection
CN104212880A (zh) * 2013-07-24 2014-12-17 北京福德安科技有限公司 基于lamp的转基因玉米mon863基因快检试剂盒及检测方法

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YANG L等: "Event Specific Qualitative and Quantitative Polymerase Chain Reaction Detection of Genetically Modified MON863 Maize Based on the 5¢-Transgene Integration Sequence", 《J AGRIC FOOD CHEM》 *
王鸣等: "《甲型流感》", 31 March 2010, 中国中医药出版社 *

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