CN111304350A

CN111304350A - Method for detecting overwintering amount of plasmopara viticola soil through real-time fluorescent quantitative PCR

Info

Publication number: CN111304350A
Application number: CN202010009702.1A
Authority: CN
Inventors: 顾沛雯; 闫思远; 肖瑞刚; 李文学; 张游; 李嘉泓
Original assignee: Ningxia University
Current assignee: Ningxia University
Priority date: 2020-01-06
Filing date: 2020-01-06
Publication date: 2020-06-19

Abstract

The invention provides a method for detecting the overwintering amount of plasmopara viticola soil by real-time fluorescent quantitative PCR, which has important significance for accurately estimating the field conditions in the early stage, determining the disease attack place and making timely and effective prevention and treatment measures; a specific primer pair Pv5/Pv6 is designed according to the full-length ITS gene (2337bp) of the plasmopara viticola, a Real-time PCR molecular detection system of the plasmopara viticola is constructed, the source quantity of the plasmopara viticola wintering bacteria in soil is quantitatively detected, and technical support is provided for early monitoring, early warning and accurate prevention and control of the plasmopara viticola in fields.

Description

Method for detecting overwintering amount of plasmopara viticola soil through real-time fluorescent quantitative PCR

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to a method for detecting the overwintering amount of plasmopara viticola soil by real-time fluorescent quantitative PCR.

Background

Grape downy mildew caused by Plasmopara viticola (Plasmopara viticola) is one of the most serious fungal diseases that harm grapes, causing serious economic losses in grape production, throughout all grape production areas throughout the world. The strain overwinter in soil mainly in the form of oospores. In the next year, oospores germinate under proper conditions to produce blastocysts, and zoospores are produced by the blastocysts. The infection is carried out by wind and rain, invading from the stomata of the back of the leaf and carrying out primary infection. And (3) after 7-12 d of incubation period, generating sporangium peduncles and sporangium in the diseased part, and germinating the sporangium to generate zoospores for secondary infection. Therefore, establishing a rapid quantitative detection technology for the overwintering amount of the plasmopara viticola soil has important significance for guiding the early monitoring, prevention and control of the disease.

Grapevilloa (p.viticola) is a fungus which is parasitized by nutrition and specificity of living organisms, and cannot be cultured artificially at present, so that the invention in the aspects of early diagnosis, monitoring, population genetic differentiation and the like of the grapevilloa is restricted to a certain extent. The traditional method for detecting the plasmopara viticola in the soil is characterized by soil enrichment screening, microscopic morphology identification, observation and counting, and has the disadvantages of complicated procedures, long period and inaccurate quantification. The Real-time quantitative PCR (Real-time quantitative PCR) technology is simple and convenient to operate, fast, efficient and high in sensitivity, and is very suitable for detecting and quantifying microorganisms in the environment. Real-time PCR is a technique for nucleic acid quantification by detecting changes in fluorescence signals, which is introduced by Applied Biosystems (ABI) of the United states, and is mainly Applied to the invention of rapid molecular detection and quantification. In 1992, the results of Real-time PCR invention of Zheng wavelet and the like prove that the survival rate of the cotton boll germ oospore after 5-6 months of burying in soil for overwintering is still higher, and the isolated oospore can survive in the soil independently and cause the disease of cotton seedlings in the next year. In 2009, the Real-time PCR method for Kangzheimei was used for quantitative detection of tobacco black rot parasitic on tobacco, and theoretical basis is provided for early diagnosis and prevention of tobacco black rot field diseases. In 2013, Xuxin et al detected the pathogenic variety of Phaseolus vulgaris common bacterial blight bacterium Xanthomonas carpi Phaseolus vulgaris bean and brown subspecies Xanthomonas fuscus in soil and plant residue through winter by Real-time PCR. In 2014, Xuna quantitatively detects Rhizoctonia cerealis in soil by using a Real-time PCR method, and the occurrence dynamics of pathogenic bacteria is determined. Peng et al artificially simulate the bacterium-bearing soil and the diseased residue soil to perform the overwintering invention, and use Real-time PCR to verify that the watermelon acidophilic bacteria can survive the overwintering in Zheng Zhou, Xinjiang Jichang and Gansu Jinta areas in Henan. At present, no report about a quantitative method for the overwintering amount in the soil of the plasmopara viticola exists in China. According to the invention, a specific primer pair Pv5/Pv6 is designed according to the ITS gene of the plasmopara viticola, a Real-time PCR molecular detection system of the plasmopara viticola is constructed, the source quantity of the plasmopara viticola in soil is quantitatively detected, and a technical support is provided for early monitoring, early warning and accurate prevention and control of the plasmopara viticola in fields.

Disclosure of Invention

In order to solve the problems, the invention provides a method for detecting the overwintering amount of downy mildew soil by real-time fluorescence quantitative PCR, which comprises the following steps:

step 1: collecting a soil sample and extracting soil DNA;

step 2: designing specific primers, and performing conventional PCR detection on DNA of plasmopara viticola, erysiphe necator, botrytis cinerea and grape anthracnose by using the specific primers, wherein the blank control is ddH₂O；

And step 3: preparing recombinant plasmids by the specific primers designed in the step 2;

and 4, step 4: carrying out sensitivity detection on the recombinant plasmid prepared in the step 3;

and 5: taking the recombinant plasmid diluted by 10 times of gradient as a standard substance, carrying out Real-time PCR detection by using the specific primer designed in the step 2 and constructing a Real-time PCR standard curve;

step 6: performing Real-time PCR detection on the DNA of the soil sample in the step 1 to obtain a Ct value, and quantifying according to the linear relation between the recombinant plasmid and the Ct value in the step 3;

and 7: detecting the overwintering amount of the soil according to the Real-time PCR standard curve in the step 5 and the quantitative result in the step 6;

furthermore, the soil sampling method in the step 1 adopts a grid method to set soil sampling points according to the size of the field, a GPS instrument performs point marking, and the positions of the sampling points are determined; generally, 25-30 sampling points are designed for 100 mu of land. The soil sampling method is a 5-point sampling method, each soil sampling point adopts a cross-shaped 5-point method to collect soil samples, and the soil samples are uniformly mixed to serve as 1 sampling point; the specific sampling method comprises the following steps: determining 4 points in four directions 3-5 m away from the center of the sampling point, wherein after removing large blocks of dry soil with the surface of 0-5 cm from 5 points including the center point, each point is 5-10 cm away from the ground, and taking a soil sample with the thickness of 2 cm; and (3) uniformly mixing the soil samples with the 5 points, sieving the mixture by using a 20-mesh sieve, taking 3-5 parts of about 20g of soil samples, repeating the steps for 3-5 times, respectively filling the soil samples into plastic packaging bags, sealing the plastic packaging bags, putting the plastic packaging bags into an ice box, and carrying back the plastic packaging bags to the ice box for storage at-80 ℃ for later use. (ii) a

Further, the specific primer design in the step 2 specifically comprises:

designing a specific primer pair PV5/PV6 with a 105bp fragment through an ITS full-length gene of the plasmopara viticola; wherein PV5 is 5'-CGCATATTGCACTTTCGGGTT-3'; PV6 is 5'-TGGCTTTACTTCCACCGACT-3';

further, the preparation of the recombinant plasmid in the step 3 specifically comprises:

purifying an amplification product of a plasmopara viticola specific primer Pv5/Pv 6;

and

connecting Simple Cloning Vector at 25 ℃, transferring to Trans1-T1 competent cells, culturing overnight at 37 ℃, picking white monoclonal with sterilized toothpick, transferring to LB liquid culture medium containing Amp, and performing shaking culture at 37 ℃ and 200r/min for 12 h;

extracting recombinant plasmids by using a plasmid DNA small-scale kit, and detecting whether the target fragments are correctly inserted or not by using a PCR (polymerase chain reaction) verification and sequencing method;

the ultramicro spectrophotometer determines the concentration and purity of plasmid DNA, and calculates the DNA copy number concentration contained in unit volume of plasmid according to Moore's law;

further, the method for detecting the sensitivity in step 4 specifically comprises: the genome DNA is diluted by 10 times after being measured by an ultramicro spectrophotometer, the detection is carried out by adopting the amplification method of conventional PCR and Real-time PCR, and the sensitivity of the primer is judged according to the dilution concentration and the fluorescence value of the DNA;

further, the Real-time PCR detection and Real-time PCR standard curve construction in the step 5 specifically comprises the following steps: performing Real-time PCR detection by using a primer Pv5/Pv6, selecting a concentration range with a stable fluorescence value and good linear proportion, and constructing a Real-time PCR standard curve by taking a copy number logarithmic value as an x axis and a C t value as a y axis; verifying the validity according to the correlation coefficient, the slope, the PCR amplification efficiency and the like of the Real-time PCR standard curve; wherein the correlation coefficient R2 is more than 0.98; the slope of the curve is-3.0 to-3.5; the PCR amplification efficiency E is 0.9-1.2; the standard deviation is less than 0.2; real-time PCR amplification efficiency E is 10^(-1/slope)-1；

Further, the Real-time PCR detection in step 6 specifically obtains a Ct value as follows: real-time PCR amplification System-20. mu.L:

green qPCR SuperMix 10. mu.L, forward and reverse primers 0.4. mu.L, template 1. mu. L, ddH2O 9.2.2. mu.L; the reaction condition is that the pre-denaturation is carried out for 30s at 94 ℃; denaturation at 94 ℃ for 5s, annealing at 59 ℃ for 15s, extension at 72 ℃ for 30s, and then collecting fluorescence signals for 40 cycles; increasing the temperature from 60 ℃ to 95 ℃ according to 5 ℃ s-1, collecting fluorescence signals for 1 time every 0.5 ℃ during temperature rising, and establishing a melting curve;

further, the step 7 of detecting the soil overwintering amount specifically comprises the following steps: constructing a Real-time PCR standard curve by fluorescence quantitative software; carrying out curve simulation through statistical software; detecting the amount of plasmopara viticola DNA in the soil;

further, the method for extracting the soil DNA in the step 1 specifically comprises the following steps:

(1) weighing 0.5g of soil sample, placing into a centrifugal oscillation tube, adding 725 mu L of SLX-Mlus Buffer, fully centrifuging to dissolve the sample, removing a small amount of supernatant, adding 72 mu L of DS Buffer, mixing uniformly, placing in 70 ℃ water bath for 10min, and shaking gently once every 5 min;

(2) centrifuging to obtain supernatant, adding ice-bath 135 μ L P2 Buffer, mixing, standing at low temperature for 3min, centrifuging to obtain supernatant, adding 200 μ L cHTR Buffer, mixing, and standing at room temperature for 2 min;

(3) centrifuging and transferring the supernatant to HiBind DNA Mini Colum, adding an equivalent amount of XP1Buffer, and mixing uniformly; centrifuging to remove the filtrate, and adding 500 mu L of HBC Buffer;

(4) centrifuging and discarding the filtrate, and transferring the HiBind DNAmini Colum into a new 2mL collecting pipe; adding 700 mu LDNAWash Buffer, centrifuging for 1min, discarding the filtrate, and repeating for 1 time;

(5) centrifuging the DNA Mini Colum, transferring the DNA Mini Colum into a 1.5ml centrifuge tube, adding 50-100 mu L of Elution Buffer at 70 ℃ in a water bath, and standing for 1min at room temperature; centrifuging, eluting the filtrate with HiBind DNAMini Colum for 1 time, and standing at room temperature for 1 min; after centrifugation, the eluted DNA is stored at-20 ℃; measuring the DNA concentration and purity of the sample by using an ultramicro spectrophotometer;

further, the conventional PCR detection in step 2 specifically comprises: conventional PCR amplification System-25. mu.L:

PCR SuperMix12.5. mu.L, positive and negative primer 0.5. mu. L, DNA template 1. mu. L, ddH2O 10.5. mu.L; the reaction condition is that the pre-denaturation is carried out for 10min at 94 ℃; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 1min, and 35 cycles; extending for 10min at 72 ℃, and storing at 4 ℃; detecting a target band of the PCR product through 1.5% agarose gel electrophoresis;

the invention has the following beneficial effects:

1. the method for monitoring and quantifying the plasmopara viticola in the soil is established, and has important significance for early and accurate estimation of field conditions, determination of disease attack places and formulation of timely and effective prevention and treatment measures;

2. a specific primer pair Pv5/Pv6 is designed according to the ITS gene of the plasmopara viticola, a Real-time PCR molecular detection system of the plasmopara viticola is constructed, the source quantity of the plasmopara viticola in soil is quantitatively detected, and technical support is provided for early monitoring, early warning and accurate prevention and control of the plasmopara viticola in fields.

Drawings

FIG. 1 is a diagram showing the results of conventional PCR and Real-time PCR amplification of the test strains Plasmopara viticola, Blastomyces viticola, Botrytis cinerea and Colletotrichum viticola by using the primers Pv5/Pv6 of the present invention;

FIG. 2 is a graph showing that the unique absorption peak of the lysis curve of only P.viticola by Real-time PCR amplification in the present invention;

FIG. 3 is a graph comparing the results of conventional PCR detection of inserted fragments and sequencing according to the present invention;

FIG. 4 is a diagram showing primer sensitivity detection of 10-fold dilution of recombinant plasmid standard by conventional PCR in the present invention;

FIG. 5 is a diagram of primer sensitivity detection of real-time PCR of recombinant plasmid standard in 10-fold dilution according to the present invention;

FIG. 6 is a real-time PCR assay of standards prepared by 10-fold gradient dilution of the recombinant plasmid with primers Pv5/Pv6 according to the present invention;

FIG. 7 is a graph of a standard curve constructed by using the logarithm of copy number of plasmids with different dilution gradients as the x-axis and the Ct value as the y-axis in the present invention;

FIG. 8 is a graph showing the detection of the DNA content of Plasmopara viticola in soil according to the present invention;

FIG. 9 shows 2.81X 10 of the present invention⁴copies·μL^-1The DNA content of Plasmopara viticola in the soil of (1).

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. On the contrary, the invention is intended to cover alternatives, modifications, equivalents and alternatives which may be included within the spirit and scope of the invention as defined by the appended claims. Furthermore, in the following detailed description of the present invention, certain specific details are set forth in order to provide a better understanding of the present invention. It will be apparent to one skilled in the art that the present invention may be practiced without these specific details.

The invention is further described with reference to the following figures and specific examples, which are not intended to be limiting. The following are preferred examples of the present invention:

as shown in FIGS. 1-9, the invention provides a real-time fluorescent quantitative PCR method for detecting the overwintering amount of downy mildew soil, which comprises the following steps:

step 1: collecting a soil sample and extracting soil DNA;

step 2: designing a specific primer, and carrying out conventional PCR detection on the DNA of the grape downy mildew bacterium plasmid, the grape powdery mildew bacterium plasmid, the grape gray mold bacterium plasmid and the grape anthracnose by using the specific primer, wherein the blank control is ddH₂O；

and 7: and (4) detecting the overwintering amount of the soil according to the Real-time PCR standard curve in the step 5 and the quantitative result in the step 6.

Example 1:

1. materials and methods

1.1 soil sample sources

The soil samples were sampled for 2018 for 4-7 months, and were from a cattail wine garden, a zhihuiyuan stone wine garden, a zhangyu wine garden, and a yuanhuang wine garden in the bronze gorge city in the eastern foot producing area of the Ningxia herland (table 1). The whole field is subjected to a grid method, each soil sample is collected by a cross-shaped 5-point method, and the soil samples are uniformly mixed to serve as 1 sample. The sampling method comprises the following specific steps: and determining 4 points in four directions 3-5 m (determined according to the size of the field) from the center of the sampling point, wherein after 5 points including the center point remove large dry soil on the surface by about 0-5 cm, each point is 5-10 cm away from the ground, and taking a soil sample with the thickness of 2 cm. And (3) uniformly mixing the soil samples at 5 points, sieving the soil samples by a 20-mesh sieve, taking about 20g of soil samples, sealing the soil samples by a plastic packaging bag, putting the soil samples into an ice box, and carrying back the soil samples to be stored at-80 ℃ for later use.

TABLE 1 soil sample sources

Note: in the table, the soil sample numbers are from random soil sample points of different vineyards

1.2 test strains

P. viticola, Uncinula necator, Botrytis cinerea, Colletotrichum gloeosporioides are obtained from single-spot purification in plant pathology laboratory of college of agriculture of Ningxia university.

1.3 test reagents and instruments

Extraction kit for total DNA of soil microorganism (A)

Soil DNA Kit, Shanghai assist saint Biotech Co., Ltd.); gel Purification Kit (Universal DNA Purification Kit, TIANGEN, Germany); plasmid DNA minikit (

Plasma Miniprep Kit, CORNING, usa);

PCRSuperMix (+ dye) (total gold);

TipGreen qPCR SuperMix (total gold). qTOWER 2.2 fluorescent quantitative PCR instrument (Jena, germany); azure c200 gel imaging system (Azurebiosystems, Inc.)United states); simpli Nano ultramicro spectrophotometer (GE corporation, USA).

1.4 methods

1.4.1 primer design and Synthesis

In the invention, when designing a primer, the following four pairs of primers are designed according to the full-length ITS sequence (2337bp) of Plasmopara viticola:

1)：pv-1:GCTTACGCTTGCGTTTGTCT

pv-2:CACACGGAGACCCAAATCCA

2)：PV-3:GGACGTCAAGGTCAGTTCGT

PV-4:GCGACTACTCGTCCACCAAG

3)：pv-5：CGCATATTGCACTTTCGGGTT

pv-6：TGGCTTTACTTCCACCGACT

4)：pv-7：CGCATATTGCACTTTCGGGT

pv-8：TCTGGCTTTACTTCCACCGAC

after specific detection, selecting the primer with the best specificity:

PV5 (5'-CGCATATTGCACTTTCGGGTT-3')/PV 6 (5'-TGGCTTTACTTCCACCGACT-3'), the designed fragment was 105bp, and primer synthesis was performed by Biotechnology engineering (Shanghai) GmbH.

ITS sequence of Plasmopara viticola (Plasmopara viticola)

Plasmopara viticola internal transcribed spacer 1,5.8S ribosomal RNAgene,and internal transcribed spacer 2,complete sequence GenBank:DQ665668.1

CCACACCTAAAAACTTTCCACGTGAACCGTTTCAACCAAATAGT TGGGGATGAAATAGGCAGCGACTACTGACTTTATTGTTGGCGGT TGCAGCTAATGGATTCCTATCATAGTGAAATAGTTTGGAATTTAT TCCGAGCTAGTAGCTATTTTTAAACCATTACTAAATACTGATTATA CTGTGAGGACGAAAGTCTTCGCTTTTACTAGATACAACTTTCAG CAGTGGATGTCTAGGCTCGCACATCGATGAAGAACGCTGCGAA CTGCGATACGTAATGCGAATTGCAGGATTCAGTGAGTCATCGAA ATTTTGAACGCATATTGCACTTTCGGGTTAGTCCTGGAAGTATGC CTGTATCAGTGTCCGTACAATAAACTTGGCATTTCATCCTTCCGT GTAGTCGGTGGAAGTAAAGCCAGATGTGAAGTGTCTTGCGCTT AATTTTAAATTGATTGCGAGTCCTTTTAAATGTACTCACTGTACT TCTCTTTGCTCAAAAAGCATAGCGATTTTGGTTGTTAGACTTTGT GATTAGTAACAAAACTGCTCCCGGTTTGTTTGTCGAGGCAATAA TGAAAGAGTATTTAATTTGCGGAAGCTGGCCTCGGCTAAGCTAT ACGCTTATATAGTATGCTTTCTGGCATGACATTTACAGGTGGGTC GTAGTTACGACGTTGCTTTGTCTTTTAACCGGTTTTGCTGTTATA AAAGACTTTCATCTGTAGCCAATCGGCGATCAATTTCTTCTTGCT AAAGCATTTAAGAAAATTTGTTATAAATATGAACTATATCAGCTT TTGCTTGATACTGTGCTTATAAAACATTTTTCTTGCTGCGGCAGA ATAACTTGGTGAACCGTAGTTATATTTGATACTTTGGTCTTCTAAT CGGCATTATTGCTGCGAAAGGTTTTGCTTGTACTTGTCGGCGAC CGATTTATTCTTCTTAAGCACTAAGAAAATTAGTATGAAAAGCG CTAATTAAAGCTCTGCTTTAATAATGCACTTTTTGAACATTTTTTTCTGCTATAGATAATAATAAAATGGTGAACCGTAGTTATGAGCTTA AATCTTTTAACGTGTAATGTTGTGTGAAGGCTGTCGTTTGTAGC AAGTCGGCGATCATTGTTTTTGCTGAAGCACTTAAAGAAAATTT GTTATATATTCGTACAATATCAGCTTTGGCTTGTTAATGTGCTTAT TAAACATTTTTTTTGCTGCGGCAGAATACTTTGGTGAACCGTAG TTATATTCGATACTATAGTCTTCTAATCGGCATTATTGTTGCGAAA GGTTTTGCTTGTACTTGTCGGCGACCAATTTATTCTTCTTAAGCA CTAAGAAAATTGGTATGAAAAGCGCTATTTAAAGCTCTGCTTTA ATAATGCACTTTTTGAACTTTTTTTCTGCTATAGATAATAATAAAA TGGTGAACCGTAGTTATGAGCTTTAAGTCTTTTGATGTTTAATGT TGTGTGAAGGCTGTCGTTTGTAGCAAGTCGGAGATCATTTTTTT TGCTGAAGCACTTAAAAAAAATTTGTTATATATTTGTACAATATC AGCTTTGGCTTGTTAATGTGCTTATTAAACATTTTTTTTGCTGCG GCAGAATACTTTGGTGAACCGTAGTTATATTCGATACTATAGTCT TCTAATCGGCATTATTGTTGCGAAAGGTTTTGCTTGTACTTGTCG GCGACCGATTTAATTTCTTAAGCACTAAGAAAATTGGTATGAAA AGCGCTATTTAAAGCTCTGCTTTAATAATGCACTTTTTGAACTTT TTTTCTGCTATAGATTAATTATAGAGTGGTGAACCGTAGTTATGA GCTTAAAGTCTTTTAATCTATAATGCTGTGTGAAGGCTGTCGTTT TTGACCGGTCGGCGATCACTTTGTTTATACTTAAAGCATAAGAA AAAAATATTTTAGTATGTGTTTTGATAGCTTCGGCTTGAAAATGT GCTAATAAATTCTTTTCTTATTGCAGTATAATAACTTGATGAACC GTAGTCATTAGTGACAAAGTCTTTTATTAAGCTATACCAGTTTGA AGTTTGTCTATTGTGGCCAGTCGGCGACCAATTTATTTGCTGTTG CATTAAAGATTTTTTTTTGATTTGCGGTATGGTTGGCTTCGGCTA AACAATGCGTTTATTAAATTAATTTCTGTGATGGCTTAATGAATC GGTGAACCGTAGCTATATGTGACTATGCTTTCAATCAGTTTTGCT ATTGCGAAGTAGAGTGGCAGCTTTGGCTGCCGAGGGTCGATCC ATTTGGGAAATAATGTGTATTTCGGTATGCATCTCAGTTGGACCT GATATCAGGCAA。

1.4.2 specific assays

Primers Pv5/Pv6 were used to treat Plasmopara viticola (P.viticola) plasmid, Staphylotrichum viticola (U.necator), Botrytis cinerea (B.cinerea) and grape anthracnose (C.gloeo. disease)spiroides) by ddH₂O is blank control, and the primer specificity is detected by conventional PCR.

1.4.3 amplification systems and reaction conditions

Conventional PCR amplification System (25. mu.L):

PCR Supermix (+ dye) 12.5. mu.L, reciprocal primer 0.5. mu. L, DNA template 1. mu. L, ddH₂O10.5. mu.L. The reaction condition is that the pre-denaturation is carried out for 10min at 94 ℃; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 1min, and 35 cycles; extending for 10min at 72 ℃, and storing at 4 ℃. The PCR product was subjected to electrophoresis on a 1.5% agarose gel to detect the band of interest.

Real-time PCR amplification System (20. mu.L):

green qPCR Supermix 10. mu.L, forward and reverse primers 0.4. mu.L, template 1. mu. L, ddH₂O9.2. mu.L. The reaction condition is that the pre-denaturation is carried out for 30s at 94 ℃; denaturation at 94 ℃ for 5s, annealing at 59 ℃ for 15s, extension at 72 ℃ for 30s (fluorescence signal collection), 40 cycles. From 60 ℃ and then according to 5 ℃ s^-1The temperature is increased to 95 ℃, and fluorescence signals are collected for 1 time every 0.5 ℃ when the temperature is increased, so that a melting curve is established.

1.4.4 preparation of recombinant plasmids

The amplification product of the Plasmopara viticola specific primer Pv5/Pv6 was purified by gel purification Kit (Universal DNAPurification Kit), and

after ligation at 25 ℃ with Simple Cloning Vector, the cells were transferred to Trans1-T1 competent cells, cultured overnight at 37 ℃, white single clones were picked with sterilized toothpicks, transferred to LB liquid medium containing Amp, and cultured with shaking at 37 ℃ and 200r/min for 12 hours. Recombinant plasmids were extracted using a plasmid DNA minikit, and whether the target fragment was correctly inserted was determined using PCR verification and sequencing (Shanghai Bioengineering Co., Ltd.). The plasmid DNA concentration and purity are determined by an ultramicro-spectrophotometer, and the DNA copy number concentration per unit volume of plasmid is calculated according to Moore's law.

Plasmid copy number ═ plasmid concentration (ng. mu.L)^-1) Plasmid volume (. mu.L). times.6.02X 10²³]/[ (vector length bp + fragment length bp) × 660g · mol^-1]

1.4.5 sensitivity detection

The genome DNA is diluted by 10 times after being measured by an ultramicro spectrophotometer, the detection is carried out by adopting the amplification method of conventional PCR and Real-time PCR, and the sensitivity of the primer is judged according to the dilution concentration and the fluorescence value of the DNA.

1.4.6Real-time PCR standard curve drawing and method validity test

The recombinant plasmid was diluted at a concentration of 50ng in a 10-fold gradient to prepare a standard. The primer Pv5/Pv6 is used for Real-time PCR detection, a concentration range with stable fluorescence value and good linear proportion is selected, the logarithm of the copy number is taken as an x axis, the C t value is taken as a y axis, and a Real-time PCR standard curve (generated by software Real-time PCR soft 3.2) is constructed. And verifying the validity of the method according to the correlation coefficient, the slope, the PCR amplification efficiency and the like of the Real-time PCR standard curve.

Wherein the correlation coefficient R2 is more than 0.98; the slope of the curve is-3.0 to-3.5; the PCR amplification efficiency (E) is 0.9-1.2; the standard deviation is less than 0.2; real-time PCR amplification efficiency E is 10^(-1/slope)-1。

1.4.7 quantitative detection of soil samples

1.4.7.1 soil DNA extraction

Extraction of DNA in soil DNA is extracted according to a soil microorganism total DNA extraction kit. The method comprises the following steps: (1) weighing 0.5g of soil sample, placing into a centrifugal oscillation tube, adding 725 mu L of SLX-Mlus Buffer, fully centrifuging to dissolve the sample, removing a small amount of supernatant, adding 72 mu L of DS Buffer, mixing uniformly, placing in 70 ℃ water bath for 10min, and shaking gently once every 5 min; (2) centrifuging to obtain supernatant, adding ice-bath 135 μ LP2 Buffer, mixing, standing at low temperature for 3min, centrifuging to obtain supernatant, adding 200 μ L cHTR Buffer, mixing, and standing at room temperature for 2 min; (3) centrifuging to transfer the supernatant to HiBind DNA Mini Colum, adding an equivalent amount of XP1Buffer, and mixing uniformly. The filtrate was centrifuged and 500. mu.L of HBC Buffer (100% isopropanol dilution) was added; (4) centrifuging to remove filtrate, and transferring Hibind DNAMini Colum into new product2mL collection tube. Adding 700 μ L DNA Wash Buffer (diluted by 100% ethanol), centrifuging for 1min, discarding the filtrate, and repeating for 1 time; (5) centrifuging the DNAMini Colum, transferring the DNAMini Colum into a 1.5ml centrifuge tube, adding 50-100 mu L of Elution Buffer at 70 ℃ in a water bath, and standing for 1min at room temperature. Centrifuging, collecting filtrate, eluting HiBindDNAMini Colum for 1 time, and standing at room temperature for 1 min. After centrifugation, the eluted DNA was stored at-20 ℃. Determination of DNA concentration and purity of sample by ultramicro-spectrophotometer (A)₂₆₀/A₂₈₀)。

1.4.7.2 Real-time PCR detection of soil samples

Taking 7 soil samples of DNA, and adopting a Real-time PCR method in 1.4.3 to detect. And (3) obtaining a Ct value, and quantifying according to the linear relation between the DNA of the downy mildew grapevine and the Ct value established by 1.4.6.

1.5 data processing

Constructing a Real-time PCR standard curve by using fluorescent quantitative software Real-time PCR soft 3.2; statistical software the Regression-current Estimation program of IBM SPSS Statistics 19.0 performs Curve simulation.

2. Results and analysis

2.1 specificity of the primers for Plasmopara viticola

Routine PCR and Real-time PCR amplification of the test strains Plasmopara viticola (P.viticola), Erysiphe viticola (U.necator), Botrytis cinerea (B.cinerea) and grape anthracnose (C.gloeosporioides) were performed using primers Pv5/Pv 6. As shown in FIG. 1, the results showed that the target band of 105bp was amplified by P.viticola, the DNA of P.viticola (U.necator) and P.viticola (B.cinerea), the DNA of P.viticola (C.gloeosporioides) and the control of clear water did not amplify the band. Real-time PCR amplification only has a single absorption peak in a P.viticola (P.viticola) lysis curve. a: m:100bp marker; 1-3 of grape powdery mildew (U.necator); 4 and 5: botrytis cinerea (b.cinerea); 6 and 7: grape anthracnose (c. gloeosporioides); 8 and 9: CK (clear water control); 10, P.viticola (P.viticola), FIG. 2 is a dissolution profile of the P.viticola (P.viticola) SYBR Green IReal-time PCR amplification reaction.

2.2 preparation of recombinant plasmids

Successfully aim atThe fragment was inserted into pEASY-T1 Simple cloning vector, and the size of the inserted fragment was about 100bp as detected by conventional PCR, as shown in FIG. 3, which is consistent with the result of 105bp as a result of sequencing. The extracted recombinant plasmid has high concentration and purity (A)₂₆₀/A₂₈₀1.858), meets the experimental requirements. In the figure, M is 100bp marker; 1 and 2, general PCR; 3 and 4, white monoclonal detection; 5 and 6, detecting recombinant plasmid DNA; 7 to 10 CK (clear water control)

2.3 primer sensitivity test and Standard Curve establishment method validity test

As can be seen from FIGS. 4 and 5, the log-curve rule of amplification of primers Pv5/Pv6 was better when the 10-fold diluted recombinant plasmid standard was subjected to conventional PCR and Real-time PCR for primer sensitivity detection, and the lower detection line of conventional PCR was 1.62X 10⁵copies·μL^-1As shown in FIG. 4, the detection result of recombinant plasmid DNA and Real-time PCR was 1.62X 10³copies·μL^-1The recombinant plasmid DNA, as shown in FIG. 5, was 100 times that of the conventional PCR. In fig. 4: m is 100bp marker; 1 to 9, in the order of 1.62X 10¹⁰～1.62×10²copies·μL^-1(ii) a CK (clear water control); in fig. 5: 1 to 9, in the order of 1.62X 10¹⁰～1.62×10²copies·μL^-1(ii) a NTC CK (clear water contrast)

2.4Real-time PCR standard curve drawing and method validity test

The primer Pv5/Pv6 recombinant plasmid 10-fold gradient dilution is used for preparing a standard substance for real-time PCR detection, as shown in FIG. 6 and FIG. 7. The amplification logarithmic curve rule of Pv5/Pv6 is better as shown in FIG. 6; and establishing a standard curve by taking the logarithm of the copy number of the plasmid with different dilution gradients as an x axis and the Ct value as a y axis. Constructing a Real-time PCR detection system, wherein a standard curve y is 45.9247-3.3621x (r)₂0.9915), the amplification efficiency was 98.35%, as shown in fig. 7. The linear range reaches 7 orders of magnitude and is 1.62 multiplied by 10³～1.62×10⁹copies·μL^-1A good linear relationship is shown.

2.5 quantitative determination of the overwintering amount of Plasmopara viticola in soil

The amount of DNA of Plasmopara viticola in the soil was detected and was low, as shown in FIG. 82.81X 10 can be detected⁴copies·μL^-1The amount of DNA of Plasmopara viticola in the soil of (1) is shown in FIG. 9 and Table 2. 1-7 in FIG. 9: soil samples numbered PS-16, ZY-3, YH-7, YH-2PS-11, PS-12 and ZH-5 in that order (Table 2)

TABLE 2 quantitative determination of the amount of downy mildew wintering in soil

3. Discussion and conclusions

The wine production area of eastern foot of Ningxia Helan orchid suffers from grape downy mildew for a long time, and rain and heat in the same season are important conditions for the attack of grape downy mildew. The plasmopara viticola overwintering in soil in the form of oospores is a main source of primary infection of the plasmopara viticola, formed spore stalks and sporangiums are continuously infected again by wind, rain and airflow transmission, and the damage is enlarged to cause epidemic. Grape downy mildew should be prevented in advance, and the prevention and treatment with the agent should be carried out in time at the early stage of the disease. Therefore, the method for monitoring and quantifying the plasmopara viticola in the soil is important for accurately estimating the field conditions in the early stage, determining the disease attack place and making timely and effective prevention and treatment measures.

At present, the invention of a Real-time PCR quantitative detection method for the soil overwintering of the grapevine peronospora parasitica does not exist in China. The primer PV5/PV6 designed by the invention shows higher specificity to the plasmopara viticola, the Real-time PCR detection result shows that the primer pair has a unique product absorption peak to the plasmopara viticola, and the detection offline of the conventional PCR is 1.62 multiplied by 10⁵copies·μL^-1Recombinant plasmid DNA; the detection lower line of the established real-time PCR system is 1.62 multiplied by 10³copies·μL^-1Recombinant plasmid DNA, 100 times of conventional PCR.

Due to the fact that the content of the plasmopara viticola in field soil is low, high-quality DNA cannot be obtained by a conventional soil microorganism DNA kit, and the detection accuracy is directly influenced. The invention is optimized on the basis of the soil microorganism total DNA extraction method to obtain DNA with higher quality, and the minimum Real-time PCR quantitative detection result is 2.81 multiplied by 10⁴copies·μL^-1。

The Real-time PCR technology has the characteristics of simple and convenient operation, high speed, high efficiency, high sensitivity and the like, and is suitable for detecting the dynamic change of microorganisms in the environment in time and space. Compared with the conventional PCR, Real-timePCR has higher requirements on the specificity of the primer and the size of the amplified fragment, and the size of the amplified fragment of the primer is generally 100-200 bp. The size of the primer amplification fragment accords with the Real-time PCR amplification requirement, is 105bp, and has high specificity to the downy mildew of grapevine.

The above-described embodiment is only one of the preferred embodiments of the present invention, and general changes and substitutions by those skilled in the art within the technical scope of the present invention are included in the protection scope of the present invention.

Claims

1. A method for detecting the overwintering amount of plasmopara viticola soil by real-time fluorescent quantitative PCR (polymerase chain reaction), which is characterized by comprising the following steps:

step 1: collecting a soil sample and extracting soil DNA;

step 2: designing specific primers, and performing conventional PCR detection on DNA of plasmopara viticola, erysiphe necator, botrytis cinerea and botrytis cinerea by using the specific primers, wherein the blank control is ddH₂O；

And step 3: purifying PCR products amplified by the specific primers designed in the step 2 to prepare recombinant plasmids;

step 6: performing Real-time PCR detection on the DNA of the soil sample in the step 1 to obtain a Ct value; quantifying according to the linear relation between the copy number logarithm of the recombinant plasmids with different dilution gradients and the Ct value in the step 5;

and 7: and (4) detecting the overwintering amount of the plasmopara viticola in the soil according to the Real-time PCR standard curve in the step 5 and the quantitative result in the step 6.

2. The method according to claim 1, wherein in step 1, soil sampling points are set by a grid method according to the size of a field, a GPS instrument is used for dotting, and the positions of the sampling points are determined; designing 25-30 sampling points per 100 mu of land on average; collecting soil samples by adopting a cross-shaped 5-point method for each soil sample point, and uniformly mixing the soil samples to obtain soil samples of 1 sample point; the specific sampling method comprises the following steps: determining 4 points in four directions 3-5 m away from the center of the sampling point, respectively removing large blocks of dry soil with the surface of 0-5 cm including the center point, and taking a soil sample with the thickness of 2cm from each point 5-10 cm away from the ground; and (3) uniformly mixing the soil samples with the 5 points, sieving the mixture by using a 20-mesh sieve, taking 3-5 parts of 20g of soil samples, repeating the steps for 3-5 times, respectively filling the soil samples into plastic packaging bags, sealing the plastic packaging bags, putting the plastic packaging bags into an ice box, and carrying back the plastic packaging bags to be stored at-80 ℃ for later use.

3. The method according to claim 1, wherein the specific primer design in step 2 is specifically:

designing a specific primer pair PV5/PV6 with a 105bp fragment through an ITS full-length gene of the plasmopara viticola; the total length of an ITS gene of the downy grapevine is 2337 bp; wherein the content of the first and second substances,

PV5 is 5'-CGCATATTGCACTTTCGGGTT-3';

PV6 is 5'-TGGCTTTACTTCCACCGACT-3'.

4. The method according to claim 1, wherein the recombinant plasmid is prepared in step 3 by:

and

connecting T1 Simple Cloning Vector at 25 ℃, transferring to Trans1-T1 competent cells, culturing overnight at 37 ℃, picking white monoclonal with sterilized toothpick, transferring to LB liquid culture medium containing Amp, and culturing for 12h at 37 ℃ under 200r/min with shaking;

the plasmid DNA concentration and purity are determined by an ultramicro-spectrophotometer, and the DNA copy number concentration per unit volume of plasmid is calculated according to Moore's law.

Plasmid copy number ═ plasmid concentration (ng. mu.L)^-1) Plasmid volume (. mu.L). times.6.02X 10²³]/[ (vector length bp + fragment length bp) × 660g · mol^-1]。

5. The method according to claim 1, wherein the method for detecting sensitivity in step 4 specifically comprises: the genome DNA is diluted by 10 times after being measured by an ultramicro spectrophotometer, the detection is carried out by adopting the amplification method of conventional PCR and Real-time PCR, and the sensitivity of the primer is judged according to the dilution concentration and the fluorescence value of the DNA.

6. The method according to claim 1, wherein the Real-time PCR detection and construction of the Real-time PCR standard curve in the step 5 are specifically as follows: performing Real-time PCR detection by using a primer Pv5/Pv6, selecting a concentration range with a stable fluorescence value and good linear proportion, and constructing a Real-time PCR standard curve by taking a copy number logarithm value as an x axis and a Ct value as a y axis; verifying the validity according to the correlation coefficient, the slope, the PCR amplification efficiency and the like of the Real-time PCR standard curve; wherein the correlation coefficient R2 is more than 0.98; the slope of the curve is-3.0 to-3.5; the PCR amplification efficiency E is 0.9-1.2; the standard deviation is less than 0.2; real-time PCR amplification efficiency E is 10^(-1/slope)-1。

7. The method according to claim 1, wherein the Ct value obtained by Real-time PCR detection in step 6 is specifically: real-time PCR amplification System-20. mu.L:

10 muL of Tip Green qPCR Supermix, 0.4 muL of forward and reverse primers and 1 mu L, ddH of template₂O9.2 μ L; the reaction condition is that the pre-denaturation is carried out for 30s at 94 ℃; denaturation at 94 ℃ for 5s, annealing at 59 ℃ for 15s, and annealing at 72 DEG CCollecting fluorescence signals after 30s of extension, 40 cycles; the melting curve was established by collecting fluorescence signals 1 time every 0.5 ℃ at elevated temperatures from 60 ℃ and then increasing to 95 ℃ as 5. multidot.s-1.

8. The method according to claim 1, wherein the step 7 of detecting the soil overwintering amount specifically comprises the following steps: constructing a Real-time PCR standard curve by fluorescence quantitative software; carrying out curve simulation through statistical software; the amount of DNA of Plasmopara viticola in the soil was determined.

9. The method according to claim 1, wherein the method for extracting soil DNA in step 1 comprises:

(4) centrifuging and discarding the filtrate, and transferring the HiBind DNA Mini Colum into a new 2mL collecting pipe; adding 700 mu L of DNA WashBuffer, centrifuging for 1min, discarding the filtrate, and repeating for 1 time;

(5) centrifuging the DNA Mini Colum, transferring the DNA Mini Colum into a 1.5ml centrifuge tube, adding 50-100 mu L of ElutionBuffer in a water bath at 70 ℃, and standing for 1min at room temperature; centrifuging, eluting HiBind DNA Mini Colum for 1 time, and standing at room temperature for 1 min; after centrifugation, the eluted DNA is stored at-20 ℃; the DNA concentration and purity of the sample are determined by a ultramicro-spectrophotometer.

10. The method according to claim 1, wherein the conventional PCR detection in step 2 is specifically: conventional PCR amplification System-25. mu.L:

PCR SuperMix12.5. mu.L, positive and negative primer 0.5. mu. L, DNA template 1. mu. L, ddH2O 10.5. mu.L; the reaction condition is that the pre-denaturation is carried out for 10min at 94 ℃; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 1min, and 35 cycles; extending for 10min at 72 ℃, and storing at 4 ℃; the PCR product was subjected to electrophoresis on a 1.5% agarose gel to detect the band of interest.