CN111206115B - PCR (polymerase chain reaction) detection primer group for Valeriana officinalis kurz and application of PCR detection primer group - Google Patents

PCR (polymerase chain reaction) detection primer group for Valeriana officinalis kurz and application of PCR detection primer group Download PDF

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CN111206115B
CN111206115B CN202010171604.8A CN202010171604A CN111206115B CN 111206115 B CN111206115 B CN 111206115B CN 202010171604 A CN202010171604 A CN 202010171604A CN 111206115 B CN111206115 B CN 111206115B
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primer
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primer group
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CN111206115A (en
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李志勇
白辉
王璐
王永芳
董志平
马继芳
刘磊
全建章
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Grain Research Institute of Hebei Academy of Agriculture and Forestry Sciences
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Abstract

The invention discloses a PCR detection primer group for Valeriana officinalis L.and application thereof, belonging to the technical field of biology. The invention designs a pair of PCR specific amplification primers according to the ITS gene sequence of the fusarium wilt pathogen: an upstream primer Gw-15 '-GAAAAACTCCAACCCCTGTGA-3'; the downstream primer Gw-25 '-TGCGTCCAAAGATTCGATGA-3'. A specific 225bp band can be amplified in the blast valley disease bacteria and plant tissues infected by the blast valley disease bacteria through PCR amplification. The specific detection primer and the detection method can be applied to the rapid detection of pathogenic bacteria in plants infected by the Valsa pestilence in millet production, and can provide early diagnosis for the occurrence of the Valsa pestilence in a field, so that corresponding disease early warning is formulated, and the specific detection primer and the detection method have a very good application prospect.

Description

PCR (polymerase chain reaction) detection primer group for Valeriana officinalis kurz and application of PCR detection primer group
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a PCR detection primer group for rice blast germs and application thereof.
Background
The millet originates from China, is rich in nutrition, drought-resistant and barren-resistant, is a high-quality drought-resistant crop in vast arid areas in the north of China, and is a preferred grain and grass dual-purpose crop for developing livestock breeding under the condition of short supply of cultivated land, and the millet is also a high-quality forage grass for cattle and sheep. The blast disease is an epidemic fungal disease of millet outbreak caused by Magnaporthe oryzae (Magnaporthe oryzae), which can be caused in the whole growth period, especially the final stage blast has a large influence on the yield, and the serious outbreak can cause the failure of production. In recent years, with the change of large-scale planting and farming conditions, the blast disease has become one of important diseases affecting the millet production in China, which poses a serious threat to the healthy development of the millet industry in China. Because of the lack of disease-resistant varieties in the current production and the frequent variation of physiological races of blast bacteria, the disease is very difficult to control. Chemical pesticides are most commonly used for preventing and treating blast disease in production, but the disease has short incubation period and high epidemic speed, and is difficult to prevent and treat after the disease is found in the field, so the prevention and treatment effect is poor.
In addition, many leaf spot diseases exist in the field, some leaf spot diseases are similar to the plague spots of paddy, many technicians cannot accurately identify the pathogeny of the leaf spot diseases, the leaf spot diseases also need to be isolated and cultured in a laboratory, the identification needs a long time, and the best prevention and control time is often missed. Therefore, early diagnosis of blast disease is urgently needed in production. With the development of molecular biology technology in recent years, PCR technology is widely applied to the detection of plant pathogenic fungi. For example, a nested PCR detection system of the rust fungi on the millet is established by the royal nanmu and the like, and the rust fungi in the millet can be specifically detected. Wangzhirong et al rapidly detected chlorotic virus in tomato by RT-PCR technique. Therefore, the detection of the blast disease of the paddy rice by utilizing the PCR technology can be applied to the accurate identification of the blast disease of the paddy rice and the early discovery of diseases, can save precious time for the prevention and control of the diseases and is greatly helpful for improving the prevention and control effect of chemical prevention and control.
Disclosure of Invention
In order to solve the technical problems, the invention provides a PCR detection primer group for Valeriana officinalis L.and application thereof.
At present, the conventional separation and microscopic morphology identification of the blast disease bacteria have long period, delay the optimal disease control period, and simultaneously require technical personnel with rich separation and identification experience of plant pathogenic bacteria. The PCR detection technology is used for accurately identifying the blast disease pathogen, and the early detection of the blast disease bacteria in the millet leaves can be realized.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a primer group for PCR detection of Valeriana officinalis L.var.oryzae, which comprises an upstream primer with a nucleotide sequence shown as SEQ ID No.4 and a downstream primer with a nucleotide sequence shown as SEQ ID No.5, wherein the method for obtaining the specific PCR primer group comprises the following steps:
(1) extracting the genomic DNA of the blast disease bacteria;
(2) PCR amplification is carried out by using fungus ITS universal primers ITS 15 ' -TCCGTAGGTGAACCTGCGG-3 (namely SEQ ID No.1) and ITS 45 ' -TCCTCCGCTTATTGATATGC-3 ' (namely SEQ ID No.2) and taking the genomic DNA of the blast bacterium as a template, so as to obtain a specific fragment of 543 bp;
(3) connecting the 543bp specific fragment recovered and purified in the step (2) into a PMD19-T vector;
(4) transferring the ligation product into escherichia coli JM109 competent cells by a calcium chloride mediated transformation method, carrying out colony PCR by using universal primers ITS1 and ITS4, detecting positive clones, sending the positive clones to Beijing Zhongkoschilin company for sequencing, and obtaining a nucleotide base sequence of a specific fragment of the blast bacterium oryzae ITS after removing a vector sequence by sequencing analysis, wherein the nucleotide base sequence is shown as SEQ ID No.3 in a sequence table;
(5) designing a PCR primer for the specificity of the blast disease bacteria: according to the nucleotide sequence result of the nucleotide polymorphism (ITS) of the blast disease bacterium, the BioXM2.6 software is used for designing a specific primer sequence, and specifically comprises an upstream primer Gw-15 ' -GAAAAACTCCAACCCCTGTGA-3 ' (namely SEQ ID No.4) and a downstream primer Gw-25 ' -TGCGTCCAAAGATTCGA TGA-3 (namely SEQ ID No. 5).
Compared with the prior art, the invention has the following beneficial effects:
the PCR technology can be used for rapidly detecting the Valley blast bacterium, and the primer has good specificity and is not influenced by other common plant pathogenic fungi on the millet. Although the number of germs in leaves is very small after 1 day after the inoculation of the blast disease, the method can detect the germs and provide early warning for the occurrence of the blast disease germs. The primer has the advantages of less drug consumption after early prevention and treatment, obvious prevention and treatment effects, and good application prospect in prediction and forecast of blast disease.
Drawings
FIG. 1 is an electrophoretogram of PCR amplification products in example 1.
FIG. 2 is an electrophoretogram of PCR amplification products in example 2.
FIG. 3 is an electrophoretogram of PCR amplification products in example 3.
Detailed Description
Example 1
In this example, the specificity of the PCR primer sets shown in SEQ ID No.4 and SEQ ID No.5 is detected by the following steps:
(1) the extraction of common fungus DNA on millet comprises the following steps of extracting common fungus DNA on the millet and other 6 kinds of common plant pathogenic fungi on the millet, namely the millet blast (Magnaporthe oryzae), the millet white hair (Sclerospora graminicola), the millet sheath blight (Rhizoctonia solani), the maize black ear fungus (Utilia crameri), the maize macrophoma (Bipolaris setaria), the Curvularia lunata and the rust (Uromyces setaria-italica), wherein the detailed steps are as follows:
a. after activation of the fusarium wilt germs, the millet sheath blight germs, the millet northern leaf blight germs and the curvularia vulgare germs by solid PDA, selecting a bacterial disc to inoculate into a liquid PDA culture medium, collecting hyphae after seven days of culture, and pressing the hyphae dry by using sterilized filter paper to extract genome DNA. The rice rust and smut bacteria directly use spore powder to extract DNA, and the white hair bacteria use the oospore to extract DNA.
b. And extracting genome DNA from pathogenic bacteria on the millet by adopting CTAB. The method comprises the following specific steps: putting 200mg of mycelium or spore powder into a mortar, grinding the mycelium or spore powder into powder by using liquid nitrogen, and putting the ground sample into a 1.5ml centrifuge tube precooled in the liquid nitrogen, wherein each tube is about 0.1 g; ② adding 400 μ L lysis buffer (100mM Tris-HCl,20mM EDTA,1.4M NaCl, 2% CTAB), water bath at 65 ℃ for 40min, reversing and mixing once every 10 min; ③ cooling at room temperature, adding 400 mu L of phenol/chloroform/isoamyl alcohol (volume ratio is 25:24:1), reversing, mixing evenly, and centrifuging at 12000rpm for 10min at 4 ℃; fourthly, the supernatant is absorbed and transferred into a new centrifuge tube, CTAB-NaCl with the volume of 1/10 is added and mixed gently, equal volume of phenol/chloroform/isoamylol is taken and mixed gently, and centrifugation is carried out for 10min at 12000rpm at the temperature of 4 ℃. Fifthly, absorbing the supernatant, transferring the supernatant into a new centrifuge tube, adding equal volume of frozen isopropanol to precipitate DNA, gently mixing the DNA, placing the mixture at the temperature of minus 20 ℃ for 15 to 30min, and then separating the mixture at the temperature of 4 ℃ and 12000rpm for 10 min. Sixthly, pouring off the supernatant, adding 1ml of 70% ethanol, washing and precipitating for 2 times, adding 50-100 mu L of TE after the precipitate is dried, standing at 37 ℃ overnight, taking out and storing at-20 ℃.
(2) Carrying out PCR amplification by taking 7 kinds of genome DNA in the step (1) as templates and the nucleotide sequences shown in SEQ ID No.4 and SEQ ID No.5 as primers respectively, wherein the reaction system is 25 mu L, 2.0 mu L of the DNA template, 10.5 mu L of the upstream primer, 20.5 mu L of the downstream primer, 12.5 mu L of 2 XEs Taq MasterMix, ddH...2...O9.5. mu.L. The PCR amplification procedure was: pre-denaturation at 95 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles, and extension at 72 ℃ for 10 min. The results after electrophoresis of the PCR amplification products are shown in FIG. 1, wherein M: DNA marker 2000; lanes 1-2: bacterial blight (Magnaporthe oryzae); lane 3: white germ (Sclerospora graminicola); lane 4: rhizoctonia solani (Rhizoctonia solani); lane 5: smut bacteria (Ustilago crameri); lane 6: northern leaf blight (Bipolaris setaria); lane 7: curvularia lunata (Curvularia lunata); lane 8: rust disease (Uromyces setariae-italica). The pestivirus cerealis amplified a single band 225bp in length (lanes 1-2), whereas none of the other test strains amplified a specific band (lanes 3-8). The detection results show that the primer designed by the invention has good specificity and can be used for specificity detection of the fusarium wilt.
Example 2
This example analyzes the sensitivity of the PCR primer sets shown in SEQ ID No.4 and SEQ ID No.5 by the following steps:
dilution of DNA concentration
The genomic DNA concentration of the extracted blast disease strain was determined using a NanoDrop 1000 spectrophotometer, the initial concentration of DNA was adjusted to 10. mu.g/. mu.L, and the samples were diluted 10-fold to 7 concentration gradients of 10. mu.g/. mu.L, 1. mu.g/. mu.L, 100 ng/. mu.L, 10 ng/. mu.L, 1 ng/. mu.L, 100 pg/. mu.L, and 10 pg/. mu.L.
PCR amplification sensitivity detection
(1) The PCR amplification system is 25 mu L, wherein the DNA template is 2.0 mu L, the upstream primer is Gw-10.5 mu L, the downstream primer is Gw-20.5 mu L, and 2 XEs Taq MasterMix 12.5μL，ddH...2...O9.5. mu.L. The PCR amplification procedure was: pre-denaturation at 95 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s, wherein the total time is 35 cycles, and extension at 72 ℃ for 10 min.
(2) And (3) detection results: 10 μ L of the PCR amplification product was detected by electrophoresis in 1.2% agarose gel, and the results of the electrophoresis were analyzed by imaging with a gel imaging system, as shown in FIG. 2, where M: DNA marker 2000; lanes 1-7: the concentrations of the template DNAs were 10. mu.g/. mu.L, 1. mu.g/. mu.L, 100 ng/. mu.L, 10 ng/. mu.L, 1 ng/. mu.L, 100 pg/. mu.L, and 10 pg/. mu.L, respectively. At DNA concentrations of 10. mu.g/. mu.L, 1. mu.g/. mu.L, 100 ng/. mu.L, 10 ng/. mu.L, and 1 ng/. mu.L, a 225bp specific band (lanes 1-5) can be amplified by using the specific primers SEQ ID No.4 and SEQ ID No.5 designed by the present invention, while at DNA concentrations of 100 pg/. mu.L and 10 pg/. mu.L, the target band (lanes 6-7) cannot be amplified, indicating that the minimum detection concentration of the PCR reaction is 1 ng/. mu.L.
Example 3
Detection of Valley fever in inoculated plants
(1) Sample treatment: taking healthy millet leaves with consistent growth period, inoculating fusarium wilt pathogen conidia (1 × 10) by adopting a spray inoculation method5One per ml), inoculating, placing into a constant temperature incubator at 26 ℃ for moisturizing and dark culture for 1 day. After the moisture preservation culture, the millet plants are placed in an incubator for normal illumination culture, and are sprayed in real time to keep the humidity. Sampling was carried out 1 day, 2 days and 3 days after the culture, leaves were also collected from a control in which the millet was additionally inoculated with clear water, 5 leaves were collected from each sample, and genomic DNA was extracted after mixing.
(2) And (3) extracting DNA of millet leaves: extracting genomic DNA of a millet leaf sample by adopting an improved CTAB method, which comprises the following specific steps:
grinding 0.2g of millet leaves into powder by using liquid nitrogen, transferring the powder into a 1.5mL centrifuge tube, adding 650 mu L of CTAB extraction buffer solution preheated at 65 ℃, and shaking to uniformly disperse the powder in the extracting solution;
② water bath is carried out for 40min at 65 ℃, and the samples are reversed and mixed evenly for 3-4 times;
cooling to room temperature, adding equal volume of phenol: chloroform: isoamyl alcohol (volume ratio is 25:24:1), mixing gently, centrifuging at 4 ℃ and 12000rpm for 15 min;
taking the supernatant, transferring the supernatant into a new tube, and adding chloroform with the same volume: isoamyl alcohol (volume ratio is 24:1), mixing gently, centrifuging at 4 ℃ and 12000rpm for 15 min;
fifthly, taking the supernatant, adding 650 mu L of isopropanol with the same volume, and standing at-20 ℃ for 30-60 min;
sixthly, centrifuging for 15min at 4 ℃ and 12000 rpm;
seventhly, pouring off the supernatant, washing for 1-2 times by using 75% ethanol, and air-drying the DNA;
the DNA is dissolved by 100 mul TE buffer solution, and PCR amplification is carried out on the millet leaves inoculated for 1 day, 2 days and 3 days and clear water control DNA. The PCR amplification system is 25 mu L, wherein the DNA template is 2.0 mu L, the upstream primer is Gw-10.5 mu L, the downstream primer is Gw-20.5 mu L,2 XEs Taq MasterMix is 12.5 mu L, ddH...2...O9.5. mu.L. The PCR amplification procedure was: pre-denaturation at 95 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles, and extension at 72 ℃ for 10 min. 10 μ L of PCR amplification product was detected by electrophoresis on 1.2% agarose gel.
(3) The result of the detection
The detection results are shown in fig. 3, in which: m: DNA marker 2000; lanes 1-4: inoculating for 1 day; lanes 5-8: inoculating for 2 days; lanes 9-12: inoculating for 3 days; lanes 13-16: in sterile water, when the inoculation time is 1d, 2d and 3d, 225bp specific bands (lanes 1-12) can be amplified in the millet leaves, and the amplified bands become brighter with the increase of the inoculation time, which indicates that the pathogen content in the pathogenic tissues is higher and higher. Therefore, the specific primer can be used for identifying whether the millet plants are infected with the fusarium oxysporum.
The above-described embodiments are only intended to illustrate the preferred embodiments of the present invention, and not to limit the scope of the present invention, and various modifications and improvements made to the technical solution of the present invention by those skilled in the art without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Sequence listing
<110> millet institute of academy of agriculture, forestry and science of Hebei province
<120> PCR detection primer group for rice blast germs and application thereof
<130> 2020.2.12
<141> 2020-02-27
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213> Artificial sequence ()
<400> 1
tccgtaggtg aacctgcgg 19
<210> 2
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 2
tcctccgctt attgatatgc 20
<210> 3
<211> 543
<212> DNA
<213> Valley blast bacterium ()
<400> 3
tccgtaggtg aacctgcgga gggatcatta ctgagttgaa aaactccaac ccctgtgaac 60
ataacctctg tcgttgcttc ggcgggcacg cccgccggag gttcaaaact cttatttttt 120
tcagtatctc tgagcctaaa agacaaataa tcaaaacttt caacaacgga tctcttggtt 180
ctggcatcga tgaagaacgc agcgaaatgc gataagtaat gtgaattgca aaattcagtg 240
aatcatcgaa tctttggacg cacattgcgc ccgccggtat tccggcgggc atgcctgttc 300
gagcgtcatt tcaaccctca agcctcggct tggtgttggg gcgcccgggc cctccgcggc 360
ccggggcccc caagttcatc ggcgggctcg tcggtacact gagcgcagta aaacgcggta 420
aaacgcgaac ctcgttcgga tcgtcccggc gtgctccagc cgctaaaccc ccaatttttt 480
aaaggttgac ctcggatcag gtaggaatac ccgctgaact taagcatatc aataagcgga 540
gga 543
<210> 4
<211> 21
<212> DNA
<213> Artificial sequence ()
<400> 4
gaaaaactcc aacccctgtg a 21
<210> 5
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 5
tgcgtccaaa gattcgatga 20

Claims (9)

1. A primer group for PCR detection of Gluconobacter graminearum is characterized by comprising an upstream primer with a nucleotide sequence shown as SEQ ID No.4 and a downstream primer with a nucleotide sequence shown as SEQ ID No. 5.
2. The application of the primer group for PCR detection of Valeriana fauriei according to claim 1 in detection of Valeriana fauriei.
3. Use according to claim 2, characterized in that it comprises the following steps:
(1) extracting sample DNA;
(2) carrying out PCR by taking the DNA extracted in the step (1) as a template and the nucleotide sequences shown in SEQ ID No.4 and SEQ ID No.5 as a primer group;
(3) and detecting the PCR amplification product.
4. The use according to claim 3, wherein the sample genomic DNA is extracted by modified CTAB method in the step (1).
5. The use of claim 3, wherein the PCR reaction system used in step (2) is 25 μ L, wherein the DNA template is 2.0 μ L, the upstream primer is 0.5 μ L, the downstream primer is 0.5 μ L,2 XEs Taq MasterMix 12.5 μ L, ddH2O 9.5μL。
6. The use of claim 5, wherein the PCR amplification procedure in step (2) is as follows: pre-denaturation at 95 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s, wherein the total time is 35 cycles, and extension at 72 ℃ for 10 min.
7. The use according to any one of claims 3-6, wherein the detecting step in step (3) is: 8-12 μ L of PCR amplification product was detected by 0.8-1.2% agarose gel electrophoresis.
8. The use according to claim 3, wherein the PCR in step (3) amplifies a 225bp specific band indicating that the sample has been infected by pestis cerealis.
9. The use according to claim 7, wherein the PCR in step (3) amplifies a 225bp specific band indicating that the sample has been infected by pestis cerealis.
CN202010171604.8A 2020-03-12 2020-03-12 PCR (polymerase chain reaction) detection primer group for Valeriana officinalis kurz and application of PCR detection primer group Expired - Fee Related CN111206115B (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003068988A1 (en) * 2002-02-11 2003-08-21 Secretary, Department Of Atomic Energy A method of pcr based detection and identification of rice blast fungus magnaporthe grisea
CN105925710A (en) * 2016-06-27 2016-09-07 山西大学 Rapid detection method for millet smut fungi
CN108018371A (en) * 2017-12-21 2018-05-11 辽宁省盐碱地利用研究所 Identify molecular labeling, identification method and the application of Rice Resistance To Rice Blast character
CN108707687A (en) * 2018-05-30 2018-10-26 河北省农林科学院谷子研究所 A kind of PCR Testing and appraisal methods that can distinguish millet blast bacterium and Pyricularia oryzae

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003068988A1 (en) * 2002-02-11 2003-08-21 Secretary, Department Of Atomic Energy A method of pcr based detection and identification of rice blast fungus magnaporthe grisea
CN105925710A (en) * 2016-06-27 2016-09-07 山西大学 Rapid detection method for millet smut fungi
CN108018371A (en) * 2017-12-21 2018-05-11 辽宁省盐碱地利用研究所 Identify molecular labeling, identification method and the application of Rice Resistance To Rice Blast character
CN108707687A (en) * 2018-05-30 2018-10-26 河北省农林科学院谷子研究所 A kind of PCR Testing and appraisal methods that can distinguish millet blast bacterium and Pyricularia oryzae

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