CN102534024A - Real-time fluorescent polymerase chain reaction (PCR) detection method for European Lymantria dispar, and primers and probe for detection - Google Patents

Real-time fluorescent polymerase chain reaction (PCR) detection method for European Lymantria dispar, and primers and probe for detection Download PDF

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CN102534024A
CN102534024A CN 201210030281 CN201210030281A CN102534024A CN 102534024 A CN102534024 A CN 102534024A CN 201210030281 CN201210030281 CN 201210030281 CN 201210030281 A CN201210030281 A CN 201210030281A CN 102534024 A CN102534024 A CN 102534024A
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european
eu
real
gypsy moth
probe
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CN102534024B (en )
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叶军
周国梁
易建平
朱雅君
林瑶
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中华人民共和国上海出入境检验检疫局
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Abstract

The invention discloses a real-time fluorescent polymerase chain reaction (PCR) detection method for European Lymantria dispar, and primers and a probe for detection. The specific primers EU-F/R and the probe EU-TaqMan are designed according to sequences of partial genes in mtDNA of Lymantria dispar geographical populations such as Asian gypsy moth, European Lymantria dispar and North American Lymantria dispar, and the real-time fluorescent PCR detection method for the European Lymantria dispar is established, so that an aim of quickly and accurately detecting and identifying the European Lymantria dispar is fulfilled.

Description

欧洲型舞毒蛾的实时荧光PCR检测方法及检测用引物和探针 European gypsy moth type real-time PCR detection method and the detection primers and probes

技术领域 FIELD

[0001] 本发明涉及林业和植物检疫技术领域,具体涉及一种欧洲型舞毒蛾的检测方法; 尤其涉及一种欧洲型舞毒蛾的实时荧光PCR检测方法;此外,本发明还涉及检测欧洲型舞毒蛾的引物和探针。 [0001] The present invention relates to a technical field of phytosanitary and forestry, in particular, to a method for the detection of the European gypsy moth; Europe in particular to a type of real-time PCR gypsy moth detection; The present invention further relates to the detection of the European gypsy moth primers and probes.

背景技术 Background technique

[0002]舞毒蛾 Lymantria dispar (Linnaeus)属鱗翅目(Lepidoptera)、毒蛾科(Lyman triidae)、毒蛾属(Lymantria),是世界公认的全北温带地区森林和观赏性林木害虫。 [0002] gypsy moth Lymantria dispar (Linnaeus) Lepidoptera (Lepidoptera), moth Branch (Lyman triidae), is a moth (Lymantria), is recognized worldwide as the whole of north temperate regions ornamental trees and forest pests. 舞毒蛾幼虫寄主范围广,主要有杨、柳、榆、栎、落叶松、椴、云杉等树木。 Gypsy moth larvae wide host range, mainly poplar, willow, elm, oak, larch, linden, spruce and other trees. 主要分布在欧洲、北美、中国、日本、朝鲜,根据其形态特征、生物学习性和地理分布,舞毒蛾被分为亚洲亚种(L. disparasiatica Vnukovsk ii)、欧洲亚种(L. dispardispar (Linnaeus))和日本亚种(L. dispar japonica (Motschulsky))。 Mainly in Europe, North America, China, Japan, North Korea, according to their morphological characteristics, biological habits and geographical distribution, Asian gypsy moth is divided into subspecies (L. disparasiatica Vnukovsk ii), the European subspecies (L. dispardispar (Linnaeus )) and Japanese subspecies (L. dispar japonica (Motschulsky)). 亚洲亚种和日本亚种被统称为亚洲型舞毒蛾,其中亚洲亚种在我国有分布,欧洲亚种则被称为欧洲型舞毒蛾。 Asiatica and Japanese subspecies are collectively referred to as the Asian gypsy moth, which the Asian subspecies distribution in our country, the European subspecies is called the European gypsy moth. 亚洲型舞毒蛾主要分布在亚洲和部分欧洲地区,雌成虫飞行能力强,可为害约500种寄主植物。 Asian gypsy moth mainly in Asia and parts of Europe, a strong female adults the ability to fly, can damage about 500 host plants. 欧洲型舞毒蛾原产欧洲,于1869年由欧洲传入美国,雌成虫不能飞行,为害约250种寄主植物。 European gypsy moth native to Europe, in 1869 the United States imported from Europe, adult female can not fly, damage of about 250 host plants.

[0003] 欧洲型舞毒蛾在欧洲为害严重,分布广泛,1948年Cointat就描述了舞毒蛾在法国普罗旺斯地区的大爆发,1962年Kailidis报道了舞毒蛾在希腊的危害,1972年Keremidchiev报道了舞毒蛾在保加利亚的危害等。 [0003] European gypsy moth infestation in Europe is serious, widespread distribution, 1948 Cointat to describe the gypsy moth outbreak in France, Provence region, in 1962 Kailidis reported Gypsy Moth harm in Greece, 1972 Keremidchiev reported gypsy moth in Bulgaria and other hazards. 舞毒蛾在亚洲和欧洲的危害历史久远, 近年普遍受到关注的原因是欧洲型和亚洲型舞毒蛾先后被引入了北美,美加持续关注。 Gypsy Moth harm in Asia and Europe has a long history, because in recent years, widespread concern that the European and Asian gypsy moth type has been introduced in North America, United States and Canada continue to focus. 舞毒蛾最先被引入北美是由一次人为意外。 The first North American gypsy moth was introduced by a man-made accident. 在1869年由E. Leopold Trouvelot将舞毒蛾做为产丝昆虫从欧洲引入美国的波士顿附近,由于在实验室饲养时部分成虫逃逸,10年后舞毒蛾在Trouvelot家的周围大爆发。 In 1869 by the E. Leopold Trouvelot the gypsy moth insect introduced as a production Sikun near Boston, United States from Europe, as some laboratory-reared adults to escape when, 10 years after the outbreak of gypsy moth around Trouvelot home. 1890年美国政府和州政府开始了采取喷药进行根除舞毒蛾的行动,但是,由于无法根除彻底而导致行动失败。 In 1890 the US government and state governments began to take action to eradicate gypsy moth spraying carried out, however, due to the action can not be eradicated completely and lead to failure. 从那以后,舞毒蛾以每年6〜9km的速度向美国的西南方向蔓延。 Since then, gypsy moth at an annual rate of 6~9km spread to the southwest of the United States. 现在,欧洲型舞毒蛾在北美的分布包括了美国的东北方以及东南和中西部的部分地区,受危害的林木面积达200万hm2,还有加拿大东部的部分地区。 Now, the distribution of European gypsy moth in North America, including the northeastern United States and parts of the Southeast and Midwest, compromised forest area of ​​200 million hm2, as well as parts of eastern Canada. 由于舞毒蛾的检疫重要性,因此,寻求快速、准确的欧洲型舞毒蛾的检测鉴定方法是现阶段检疫技术亟待解决的问题和发展的方向。 Because of gypsy moth quarantine importance, therefore, to seek rapid and accurate detection and identification methods of the European type of gypsy moth quarantine technology is the direction of the problem at this stage to be solved and development.

发明内容 SUMMARY

[0004] 本发明要解决的技术问题是提供一种欧洲型舞毒蛾的实时荧光PCR检测方法;为此,本发明还提供用于上述方法的检测引物和探针。 [0004] The present invention is to solve the technical problem of providing a European type Lymantria dispar real-time PCR detection method; for this reason, the present invention also provides primers and probes for the detection method described above. 利用欧洲型舞毒蛾检测引物及探针可以快速、准确地对欧洲型舞毒蛾进行检测鉴定。 European gypsy moth type using primers and probes can be detected quickly and accurately Lymantria dispar European type detection and identification.

[0005] 本发明根据亚洲型舞毒蛾、欧洲型舞毒蛾、北美型舞毒蛾这几个地理种群的mtDNA 中部分基因的序列设计特异性引物和探针,建立了欧洲型舞毒蛾的实时荧光PCR检测方法,以达到快速准确检测鉴定欧洲型舞毒蛾的目的。 [0005] According to the present invention is designed mtDNA sequences Asian gypsy moth, European gypsy moth, North America geographical type gypsy moth populations in several parts of the gene-specific primers and probes, the establishment of a European type of real-time PCR gypsy moth detection methods, in order to achieve the purpose of European type fast and accurate detection and identification of the gypsy moth. [0006] 在本发明的一方面,提供一种欧洲型舞毒蛾的实时荧光PCR检测方法,包括如下步骤: [0006] In one aspect of the present invention, there is provided a European type Lymantria dispar real-time PCR detection method, comprising the steps of:

[0007] (I)设计引物和探针; [0007] (I) design primers and probes;

[0008] 该引物的序列为: [0008] The sequence of the primer was:

[0009]上游引物 EU-F :5' -AACTTCAGGATGTCCGAAAAATCA-3' (SEQ ID NO. I); [0009] The upstream primer EU-F: 5 '-AACTTCAGGATGTCCGAAAAATCA-3' (SEQ ID NO I.);

[0010]下游引物 EU-R :5,-ACAGCTTTCCTTCTACITTTATCTTTACCT-3,(SEQ ID NO. 2); [0010] The downstream primer EU-R: 5, -ACAGCTTTCCTTCTACITTTATCTTTACCT-3, (SEQ ID NO 2.);

[0011] 该探针的序列为: [0011] The sequence of the probe was:

[0012]欧洲型舞毒蛾探针 EU-MGB :FTGGATCTCCTCCTCCTP (SEQ ID NO. 3)或其互补链; [0012] European gypsy moth probe EU-MGB: FTGGATCTCCTCCTCCTP (. SEQ ID NO 3) or its complementary strand;

[0013] (2)提取欧洲型舞毒蛾DNA ; [0013] (2) extracting the DNA European gypsy moth;

[0014] (3)采用步骤⑴设计的引物和探针进行实时荧光PCR检测。 [0014] (3) using the procedure ⑴ design primers and probes for real-time PCR assay.

[0015] 步骤⑵具体为:取欧洲型舞毒蛾单头虫样放入研钵,加液氮磨成粉末状,用试剂盒提取样品DNA。 [0015] Step ⑵ specifically: taking European gypsy moth single head type worm-like into a mortar, adding liquid nitrogen, ground into powder, extracted sample DNA kit.

[0016] 步骤(3)中,所述实时荧光PCR检测的反应体系为25 iiL,包括2 X Premix Ex Taq [0016] Step (3) in the reaction system for the real time PCR detection 25 iiL, comprising 2 X Premix Ex Taq

12. 5 u L, 50 X ROX Reference Dye0.5iiL,步骤(I)设计的上下游引物EU-F/R 各I. 0 y L, EU-MGB探针l.OuL, DNA模板2. Ou L,超纯水补至25 u L。 12. 5 u L, 50 X ROX Reference Dye0.5iiL, step (I) of the upstream and downstream primers designed EU-F ​​/ R each I. 0 y L, EU-MGB probe l.OuL, DNA template 2. Ou L ultrapure water up to 25 u L.

[0017] 步骤(3)中,所述实时荧光PCR检测的反应程序为:预变性95°C 30seC ;然后95°C 5sec,65°C 34sec 循环40 次。 [0017] Step (3), the reaction sequence is detected by real-time PCR: denaturation at 95 ° C 30seC; then 95 ° C 5sec, 65 ° C 34sec 40 cycles.

[0018] 在本发明的另一方面,提供一种用于检测欧洲型舞毒蛾的引物,其序列为: [0018] In another aspect of the present invention, there is provided a primer for detecting a European-type Lymantria dispar, having the sequence:

[0019]上游引物 EU-F :5' -AACTTCAGGATGTCCGAAAAATCA-3' (SEQ ID NO. I); [0019] The upstream primer EU-F: 5 '-AACTTCAGGATGTCCGAAAAATCA-3' (SEQ ID NO I.);

[0020]下游引物 EU-R :5,-ACAGCTTTCCTTCTACITTTATCTTTACCT-3,(SEQ ID NO.2)。 [0020] The downstream primer EU-R: 5, -ACAGCTTTCCTTCTACITTTATCTTTACCT-3, (SEQ ID NO.2).

[0021] 在本发明的另一方面,提供一种用于检测欧洲型舞毒蛾的探针,其序列为: EU-MGB :FTGGATCTCCTCCTCCTP(SEQ ID NO. 3)或其互补链。 [0021] In another aspect of the present invention, there is provided a method for detecting a European-type probes for the gypsy moth, having the sequence: EU-MGB: FTGGATCTCCTCCTCCTP (. SEQ ID NO 3) or its complementary strand.

[0022] 上述实时荧光PCR检测方法采用MGB (全称为Minor Groove Binder)探针。 [0022] The real-time PCR assay using MGB (all known Minor Groove Binder) probe. MGB 探针一是在探针的3'端标记了自身不发光的淬灭荧光分子,以取代常规可发光的TAMRA荧光标记;二是探针的3'端另结合了Minor groove binder结合物,使探针的Tm值提高,大大增加了探针的杂交稳定性。 First, the probe of the MGB probe 3 'end of itself does not quench the fluorescence emission of the molecule to replace a conventional fluorescent label capable of emitting light TAMRA; Second probe 3' binding of the other end of the Minor groove binder combination, that the Tm value of the probe is increased, greatly increases the stability of the hybridization probe. 该方法具有如下优点:1、更容易设计;2、探针更短:3、提高配对与司E配对模板间的Tm值差异;4、实验结果更精确、分辩率更高;5、杂交的稳定性提高; This method has the following advantages: 1, are easier to design; 2, a shorter probe: 3, increase the value of Tm differences between the template pair and the pair E Division; 4, results more accurate, higher resolution; 5, hybridized improved stability;

6、低背景;7、重复性更强。 6, low background; 7, more reproducible. 该方法适合病原体的检测、SNP和突变体检测,既可以进行基因定量分析,又可以进行基因突变分析。 This method is suitable for detection of pathogens, the SNP and mutation detection, quantitative analysis of gene may be performed, and gene mutation analysis may be performed.

[0023] 与现有技术相比,本发明的有益效果在于: [0023] Compared with the prior art, the beneficial effects of the present invention:

[0024] I、检测时间大大缩短,可以在六个小时内完成检测,传统的常规方法需10-20天; [0024] I, the detection time is shortened, detection can be completed within six hours, the conventional method requires the conventional 10-20 days;

[0025] 2、对欧洲型舞毒蛾的鉴定准确率增大,传统的常规方法还需要其他详细产地等信息支持才能鉴定。 [0025] 2, identify the exact type of European gypsy moth increases, the traditional conventional methods require additional detailed information such support in order to identify the origin.

附图说明 BRIEF DESCRIPTION

[0026] 图I是本发明的引物EU-F/EU-R和探针EU-MGB在欧洲型舞毒蛾上的位置示意图; [0026] FIG. I is a schematic view of the position of the primers of the present invention, EU-F ​​/ EU-R EU-MGB probe, and the European type Lymantria dispar;

[0027] 图2是亚洲型、欧洲型、北美型舞毒蛾序列在探针位置的比对示意图; [0027] FIG. 2 is a type in Asia, Europe type, North American gypsy moth type sequence alignment schematic view of the probe position;

[0028] 图3是不同来源的5个舞毒蛾样品实时荧光PCR扩增的结果示意图; [0028] FIG. 3 is a schematic view of five different sources gypsy moth sample time PCR amplification result;

[0029] 图4是不同来源的20个舞毒蛾样品实时荧光PCR扩增的结果示意图;[0030] 图5是不同稀释浓度的欧洲型舞毒蛾样品实时荧光PCR扩增的结果示意图。 [0029] FIG. 4 is a schematic diagram of the results of different sources 20 gypsy moth time PCR amplification samples; [0030] FIG. 5 is a schematic diagram of the results of various dilutions of the sample gypsy moth European type real-time PCR amplification. 具体实施方式 detailed description

[0031] 下面结合附图和实施例对本发明作进一步详细的说明。 Drawings and embodiments of the present invention will be further described in detail [0031] below in conjunction. 应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。 It should be understood that these embodiments are illustrative only and the present invention is not intended to limit the scope of the invention. 下列实施例中未注明具体条件的实验方法,通常按常规条件,例如Sambrook等人,分子克隆:实验室手册(New York : Co Id Spring Harbor Laboratory Press, 1989)中所述条件,或按制造厂商所建议的条件。 Experimental methods without specific conditions in the examples below, generally by conventional conditions, for example, Sambrook et al, Molecular Cloning: A Laboratory Manual (New York: Co Id Spring Harbor Laboratory Press, 1989) in the conditions according to the manufacturer, or conditions recommended by the manufacturer.

[0032] 实施例I [0032] Example I

[0033] I.设计引物和探针 [0033] I. Design of primers and probes

[0034] 根据舞毒蛾这几个地理种群的mtDNA (线粒体DNA)中部分基因的序列设计如下特异性引物EU-F/R和探针EU-MGB (引物和探针由上海基康公司合成): [0034] The specific primers EU-F ​​/ R and the probe EU-MGB (primers and probes were synthesized by the company GeneCore) mtDNA sequence was designed according to these geographic gypsy moth populations (mitochondrial DNA) as part of gene :

[0035] 欧洲型舞毒蛾引物: [0035] European gypsy moth primer:

[0036]上游引物 EU-F :5' -AACTTCAGGATGTCCGAAAAATCA-3' (SEQ ID NO. I); [0036] The upstream primer EU-F: 5 '-AACTTCAGGATGTCCGAAAAATCA-3' (SEQ ID NO I.);

[0037]下游引物 EU-R :5,-ACAGCTTTCCTTCTACITTTATCTTTACCT-3,(SEQ ID NO. 2); [0037] The downstream primer EU-R: 5, -ACAGCTTTCCTTCTACITTTATCTTTACCT-3, (SEQ ID NO 2.);

[0038]欧洲型舞毒蛾探针 EU-MGB :FTGGATCTCCTCCTCCTP (SEQ ID NO. 3)或其互补链。 [0038] European type probe Lymantria dispar EU-MGB: FTGGATCTCCTCCTCCTP (. SEQ ID NO 3) or its complementary strand.

[0039] 上述引物及探针的检测原理如图I所示,3种不同类型舞毒蛾序列在探针位置的比对见图2。 [0039] The above-described detection principle of primers and probes is shown in Figure I, 3 different types of gypsy moth probe location in the sequence of Figure 2.

[0040] 2、提取舞毒蛾DNA [0040] 2, gypsy moth extracted DNA

[0041] 取舞毒蛾单头虫样(样品来源见表I)放入研钵,加液氮磨成粉末状,用DNeasy® Blood&Tissue Kitt (Qiagen 公司)提取样品DNA。 [0041] Lymantria dispar taken single-headed worm-like (Sample Source see Table I) into a mortar, adding liquid nitrogen, ground into powder, extracted sample DNA with DNeasy® Blood & Tissue Kitt (Qiagen Company).

[0042] 表I舞毒蛾样品信息 [0042] Table I sample information Lymantria dispar

[0043] [0043]

Figure CN102534024AD00051

[0044] [0044]

Figure CN102534024AD00061

LL05 吉林松花江 2010 釆集LL07 黑龙江牡丹江 2011 釆集LL08 吉林长春 2010 釆集LLlO 内蒙古克旗阿斯哈图石林 2011 釆集LL60 河南 2010 赠送LL61 辽宁瓦房店 2010 赠送LL62 河南新乡辉县关山 2011 赠送LL65 黑龙江哈尔滨 2010 釆集JXLS 江西庐山 2010 釆集 LL05 Jilin Songhua 2010 Bian Bian 2011 set LL07 Mudanjiang in Heilongjiang, Jilin, Changchun set LL08 2010 preclude the collection LLlO Inner Mongolia Keqi Siha Stone Forest Bian 2011 collection presented LL61 LL60 Henan 2010 2010 Wafangdian, Liaoning, Henan Xinxiang Huixian gift LL62 gift Guanshan 2011 LL65 Harbin 2010 Bian Bian 2010 Lushan Mountain in Jiangxi set JXLS set

[0045] 3、实时荧光PCR检测 [0045] 3, real-time PCR Detection

[0046] 实时荧光PCR扩增体系25 ii L (大连宝生物公司TaKaRa Premix Ex Taq™): 2 XPremix Ex Taql2. 5 UL, 50 X ROX Reference DyeO. 5 ii L,上下游引物EU_F/R(5umol/L) 各I. 0 ii L,EU-MGB 探针(IOumoI/L) I. OuL, DNA 模板2. 0 u L,超纯水补至25 u L。 [0046] The real-time PCR amplification system 25 ii L (Takara TaKaRa Premix Ex Taq ™):. 2 XPremix Ex Taql2 5 UL, 50 X ROX Reference DyeO 5 ii L, upstream and downstream primers EU_F / R (5umol. / L) of each I. 0 ii L, EU-MGB probe (IOumoI / L) I. OuL, DNA template 2. 0 u L, ultra-pure water up to 25 u L. 实时荧光PCR 扩增在7500Real-Time PCR System 定量PCR 扩增仪上进行。 Real-time PCR amplification was carried out on 7500Real-Time PCR System quantitative PCR amplification. 打开“7500Fast System Software”,设置PCR反应条件,两步法扩增反应程序:预变性95°C 30sec ;然后95°C 5sec, 65°C 34sec循环40次。 Open "7500Fast System Software", setting PCR conditions, two-step reaction sequence amplification: denaturation at 95 ° C 30sec; then 95 ° C 5sec, 65 ° C 34sec 40 cycles. 点击运行,进行PCR反应,Ih左右反应结束,保存文件,打开分析软件。 Click Run, PCR reaction, the reaction is completed in about Ih, save the file, open the analysis software.

[0047] 检测结果如图3、4、5所示,在图3中,有6条曲线(数字1_6曲线),数字1_6曲线表示的样品详细信息如表I所示。 [0047] 3,4,5 detection results shown in FIG. In FIG. 3, there are six curves (curve digital 1_6), sample numbers 1_6 curves represent details shown in Table I. 在图3中,1-3分别表示样品编号为BE、TC1、TC2的欧洲型舞毒蛾样品,4表不样品编号为PA的北美型舞毒蛾样品,5表不样品编号为LL65的亚洲型舞毒蛾样品,6表示没有模板DNA的空白对照。 In Figure 3, respectively, represent the sample numbers 1-3 to BE, TC1, TC2 European gypsy moth samples, the sample No. 4 table is not for the North American gypsy moth samples type PA, the table does not sample No. 5 LL65 Asian gypsy moth sample 6 represents the control without template DNA. 在图4中,有21条曲线(数字1-21曲线),数字1-21曲线表示的样品详细信息如表I所示。 In FIG. 4, there are 21 curves (curve numbers 1-21), sample numbers 1-21 curves represent details shown in Table I. 在图4中,I表示样品编号为BE的欧洲型舞毒蛾样品,2-20分别表示样品编号为PA、MA、ME、WV、NJ、M、J13、頂、R4、LL01、LL05、 LL07、LL08、LLlO、LL60、JXLS、LL62、LL65、LL61的欧洲型舞毒蛾和亚洲型舞毒蛾样品,21表示没有模板DNA的空白对照。 In Figure 4, I indicates that the sample No. BE Lymantria dispar European type samples, respectively, represent the sample numbers 2-20 as PA, MA, ME, WV, NJ, M, J13, top, R4, LL01, LL05, LL07, LL08, LLlO, LL60, JXLS, LL62, LL65, LL61 type of European gypsy moth and Asian gypsy moth sample, 21 means no blank template DNA. 由图3、4表明此次实时荧光PCR检测到的是欧洲型舞毒蛾, 表明引物EU-F/R和探针EU-MGB可以特异性检测欧洲型舞毒蛾。 Figures 3 and 4 show the real time PCR detection of the European type of the gypsy moth, show primers EU-F ​​/ R, and EU-MGB probes that can specifically detect European gypsy moth.

[0048] 在图5中,有8条曲线(数字1-8曲线),数字1-7曲线表示欧洲型舞毒蛾DNA 浓度分别为252 ug/ml,25. 2 ug/ml,2. 52 ug/ml,2. 52X KT1 yg/ml、2. 52X 1(T2 yg/ml、 [0048] In FIG. 5, eight curves (curve number 1-8), 1-7 curve represents the European type digital Lymantria dispar DNA concentrations were 252 ug / ml, 25. 2 ug / ml, 2. 52 ug / ml, 2. 52X KT1 yg / ml, 2. 52X 1 (T2 yg / ml,

2. 52X10、g/ml、2. 52X10、g/ml的实时荧光PCR扩增曲线,8表示没有模板DNA的空白对照。 2. 52X10, g / ml, 2. 52X10, g / ml of time PCR amplification curve 8 represents the control without template DNA.

Claims (6)

  1. 1. 一种欧洲型舞毒蛾的实时荧光PCR检测方法,其特征在于,包括如下步骤:(1)设计引物和探针;该引物的序列为:上游引物EU-F :5' -AACTTCAGGATGTCCGAAAAATCA-3' (SEQ ID NO. I);下游引物EU-R :5,-ACAGCTTTCCTTCTACITTTATCTTTACCT-3,(SEQ ID NO. 2);该探针的序列为:欧洲型舞毒蛾探针EU-MGB :FTGGATCTCCTCCTCCTP (SEQ ID NO. 3)或其互补链;(2)提取欧洲型舞毒蛾DNA ;(3)采用步骤(I)设计的引物和探针进行实时荧光PCR检测。 Real-time PCR detection method for Lymantria dispar the European type, the method comprising the steps of: (1) Design of primers and probes; sequence of the primer was: upstream primer EU-F: 5 '-AACTTCAGGATGTCCGAAAAATCA-3 '(. SEQ ID NO I); downstream primer EU-R: 5, -ACAGCTTTCCTTCTACITTTATCTTTACCT-3, (. SEQ ID NO 2); the sequence of the probe was: the European gypsy moth type probe EU-MGB: FTGGATCTCCTCCTCCTP (SEQ . ID NO 3) or its complementary strand; (2) extracting the European gypsy moth DNA; (3) employed in step (I) design primers and probes for real-time PCR assay.
  2. 2.如权利要求I所述的欧洲型舞毒蛾的实时荧光PCR检测方法,其特征在于,步骤⑵ 具体为:取欧洲型舞毒蛾单头虫样放入研钵,加液氮磨成粉末状,用试剂盒提取样品DNA。 2. The real-time PCR detection method of the European type I gypsy moth claim, wherein the step of specifically ⑵: taking European gypsy moth single head type worm-like into a mortar, adding liquid nitrogen, ground into powder , extracted sample DNA kit.
  3. 3.如权利要求I所述的欧洲型舞毒蛾的实时荧光PCR检测方法,其特征在于,步骤⑶ 中,所述实时荧光PCR检测的反应体系为25 ii L,包括2 X PremixEx Taq 12. 5 UL, 50 X ROX Reference DyeO. L,步骤(I)设计的上下游引物EU-F/R 各I. 0 y L,EU-MGB 探针l.OuL, DNA模板2. 0 ii L,超纯水补至25 u L。 3. The real-time PCR detection method of the European type I gypsy moth claim, wherein the step ⑶, the real time PCR detection reaction system is 25 ii L, comprising 2 X PremixEx Taq 12. 5 UL, 50 X ROX Reference DyeO. L, step (I) of the upstream and downstream primers designed EU-F ​​/ R each I. 0 y L, EU-MGB probe l.OuL, DNA template 2. 0 ii L, ultrapure water up to 25 u L.
  4. 4.如权利要求I或3所述的欧洲型舞毒蛾的实时荧光PCR检测方法,其特征在于, 步骤(3)中,所述实时荧光PCR检测的反应程序为:预变性95°C 30seC;然后95°C 5sec, 65 °C 34sec 循环40 次。 4. I or real-time PCR detection method of the European gypsy moth claimed in claim 3, wherein, in step (3), the reaction procedure of the real time PCR detection was: denaturation at 95 ° C 30seC; then 95 ° C 5sec, 65 ° C 34sec 40 cycles.
  5. 5. 一种用于检测欧洲型舞毒蛾的引物,其特征在于,其序列为:上游引物EU-F :5' -AACTTCAGGATGTCCGAAAAATCA-3' (SEQ ID NO. I);下游引物EU-R :5,-ACAGCTTTCCTTCTACITTTATCTTTACCT-3,(SEQ ID NO.2)。 5. A method for detecting a European-type Lymantria dispar primers, wherein the sequence of which is: upstream primer EU-F: 5 '-AACTTCAGGATGTCCGAAAAATCA-3' (. SEQ ID NO I); downstream primer EU-R: 5 , -ACAGCTTTCCTTCTACITTTATCTTTACCT-3, (SEQ ID NO.2).
  6. 6. 一种用于检测欧洲型舞毒蛾的探针,其特征在于,其序列为:EU-MGB :FTGGATCTCCTCCTCCTP (SEQ ID NO. 3)或其互补链。 A probe for detecting a European-type Lymantria dispar, wherein the sequence is: EU-MGB: FTGGATCTCCTCCTCCTP (. SEQ ID NO 3) or its complementary strand.
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