Molecular identification method for potato late blight of molecular marker primer

Abstract

The invention relates to the technical field of plant pathology detection, in particular to a potato late blight molecular identification method of a molecular marker primer, and the primer comprises a molecular marker primer pair for identification of Phytophthora infestans (Phytophthora infestans). The primer pair comprises specific sequences of three continuous adenine bases (AAA) in a targeted pathogenic bacterium ribosome internal transcriptional spacer (ITS), and comprises a forward primer, a reverse primer, a reverse primer and a reverse primer, the nucleotide sequence of the forward primer is shown as SEQ ID NO: 1, and the nucleotide sequence of the reverse primer is 5 '-GCTGCGTCTTCTATGATGC-3'; the nucleotide sequence of the reverse primer is as shown in SEQ ID NO: 2: 5 '-TCCTCCGCTTATTGATATGC-3'. The nucleotide sequence of the reverse primer is as shown in SEQ ID NO: 2. According to the potato late blight molecular identification method of the molecular marker primer, 100% specificity of the primer pair is achieved through base mismatch design, detection of samples such as tubers and soil can be completed within 4 hours by combining optimized CTAB / magnetic bead method extraction and 56 DEG C annealing PCR technology, the sensitivity reaches 10 pg / mu L, and the primer pair is high in specificity and high in specificity. The method is suitable for early warning and accurate prevention and control of potato late blight.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant pathology detection, in particular to a molecular marker primer-based molecular identification method for potato late blight. BACKGROUND

[0002] Potato late blight is a devastating disease caused by the pathogenic oomycete Phytophthora infestans, which can cause a 30-80% reduction in potato yield. Early and accurate detection of the disease is crucial for disease control, but existing detection techniques have the following shortcomings:

[0003] (1) Traditional morphological identification

[0004] Time-consuming and inefficient: requires pathogen isolation and culture, microscopic observation of morphological characteristics, and a detection period of up to 24-48 hours, which cannot meet the needs of rapid early warning in the field;

[0005] Dependence on experience: morphological identification of mycelium and sporangia is easily influenced by subjective judgment of the operator, especially for early infections that do not form typical symptoms;

[0006] (2) Serological detection techniques

[0007] Insufficient specificity: ELISA and other methods that use antibody-antigen reactions may have cross-reactions with closely related pathogens (such as Phytophthora erythroseptica), with a misjudgment rate as high as 15-20%;

[0008] High cost: antibody preparation takes a long time and is expensive, and the shelf life of the kit is short (usually ≤6 months), limiting its promotion at the grassroots level;

[0009] (3) Conventional molecular biology methods

[0010] Cross amplification with universal primers: traditional ITS universal primers (such as ITS1 / ITS4) target the conserved region of ribosomal DNA, which cannot distinguish between P. infestans and closely related species, and may produce false positive results in complex samples;

[0011] Limited detection sensitivity: the detection limit of ordinary PCR methods for pathogen DNA is 100 pg / μL, which cannot meet the early detection needs of seed potato infection, soil latent infection, etc.;

[0012] Poor adaptability in multiple scenarios: humic substances and polysaccharides in complex samples such as soil and tubers can inhibit PCR reactions, requiring tedious DNA purification steps, and the detection process takes more than 6 hours;

[0013] Therefore, we propose a molecular marker primer-based molecular identification method for potato late blight. SUMMARY

[0014] The main purpose of the present application is to provide a molecular marker primer for molecular identification of potato late blight, which can effectively solve the problems in the background art.

[0015] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0016] A molecular marker primer, the primer comprises a molecular marker primer pair for identification of Phytophthora infestans, the primer pair targets a specific sequence comprising three consecutive adenine bases (AAA) in the internal transcribed spacer (ITS) of the pathogenic ribosome, comprising:

[0017] The forward primer has a nucleotide sequence as shown in SEQ ID NO: 1: 5'-GCTGCGTTCTTCATCGATGC-3';

[0018] The reverse primer has a nucleotide sequence as shown in SEQ ID NO: 2: 5'-TCCTCCGCTTATTGATATGC-3';

[0019] The primer pair has ≥3 base mismatches in the binding region with the ITS sequence of the related pathogenic bacteria, can specifically amplify a 368bp ITS fragment of Phytophthora infestans, and has no cross-reaction with Phytophthora erythroseptica and Alternaria solani.

[0020] Preferably, the AAA sequence is located at positions 120-150 of the ITS region, and has three base mismatches of A→G, A→T and A→C with the homologous sequence of Phytophthora erythroseptica.

[0021] Preferably, the GC content of the forward primer and the reverse primer is 50%, and the length of the primer is 20 nucleotides, which is suitable for annealing temperature of 56℃.

[0022] Preferably, the amplification product of the primer pair is verified by sequencing and contains a specific sequence as shown in SEQ ID NO: 3, wherein positions 120-122 are AAA.

[0023] A molecular marker primer for molecular identification of potato late blight, comprising the following steps:

[0024] Step (1) DNA extraction:

[0025] The tuber / leaf sample is subjected to CTAB method using 2% CTAB lysis solution, and is subjected to water bath at 65℃ for 30min;

[0026] The soil sample is filtered through a 200-mesh screen, and then extracted by a magnetic bead method, and a lysis solution containing 1% CTAB is used;

[0027] Step (2) PCR amplification: the reaction system is 25 μL, containing template DNA 1 μL (≥10 pg / μL), 10×PCR Buffer 2.5 μL, primers 1 μL (10 μM) each, Taq enzyme 0.5 U, and the reaction condition is 94°C pre-denaturation for 5 min, 35 cycles (94°C for 30 s→56°C for 30 s→72°C for 40 s), and finally 72°C extension for 10 min;

[0028] Step (3) agarose gel electrophoresis: 1.8% gel, 100V voltage electrophoresis for 35 min, and observing 368 bp specific bands.

[0029] Preferably, the DNA recovery rate of the magnetic bead method is ≥85%, and the detection sensitivity of the soil sample reaches 10 pg / μL of pathogenic bacteria DNA.

[0030] Preferably, the gray value of the positive band in step (3) needs to reach more than 3 times of the background value, and there is no amplification band in the sample of the close relative pathogenic bacteria.

[0031] Preferably, the yield of the CTAB method for extracting tuber DNA is 50-80 ng / mg of tissue, and the purity OD260 / OD280 is 1.8-2.0.

[0032] Preferably, the annealing temperature of the PCR reaction is in the range of 55-58°C, and when 56°C, the non-specific amplification product is reduced by 92%.

[0033] The application of a molecular marker primer pair in preparing a potato late blight detection reagent, which realizes disease screening by detecting pathogenic bacteria DNA in seed potatoes, leaves or soil.

[0034] Compared with the prior art, the application has the following beneficial effects:

[0035] 1. High-specificity detection, avoiding the risk of misjudgment

[0036] The primer pair targets the continuous AAA base marker site unique to the ITS region of potato late blight, and there are ≥3 base mismatches (such as A→G / T / C) with the homologous sequences of close relative pathogenic bacteria (such as Phytophthora erythroseptica), and after verification of 20 kinds of close relative pathogenic bacteria, the cross-reaction rate is 0, completely solving the misjudgment problem of traditional ITS universal primers (the misjudgment rate of the background technology is 15%-20%).

[0037] The agarose gel electrophoresis result shows that only the 368 bp specific band of the Phytophthora infestans sample appears, and the related pathogenic bacteria and blank control are not amplified, and the specificity is 100%.

[0038] 2. Ultra-high detection sensitivity, early warning

[0039] The optimized PCR system reduces the detection lower limit from 100 pg / μL of the traditional method to 10 pg / μL, and the sensitivity is increased by 10 times, and the trace pathogenic bacteria DNA (such as latent infection period mycelium) in seed potato and soil can be detected, and the disease can be found 5-7 days earlier than the traditional morphological method.

[0040] The DNA recovery rate of the magnetic bead method is greater than or equal to 85%, combined with soil screen filter pretreatment, even if the content of the pathogenic bacteria is as low as 10 pg / μL (equivalent to 10 ng mycelium in 1 g soil), a clear band can still be stably amplified.

[0041] 3. The detection efficiency is significantly improved, and it is suitable for rapid detection in the field

[0042] From sample processing to result determination, the whole process is less than or equal to 4 hours, which is 6-12 times higher than the traditional morphological method (24-48 hours), and more than 30% shorter than the conventional molecular biology method (more than 6 hours).

[0043] The premixed PCR reaction liquid and standardized electrophoresis parameters are adopted, and the operation can be carried out without professional molecular biology background, and the primary agricultural technology personnel can complete the detection in the field after simple training, so that the demand of 'detection and control' in the disease epidemic period is met.

[0044] 4. Strong adaptability in multiple scenes, covering whole growth period monitoring

[0045] Tuber detection: the improved CTAB method effectively removes tuber polysaccharide impurities, the DNA yield is 50-80 ng / mg tissue, the purity OD260 / OD280 is 1.8-2.0, and the problem that the polysaccharide inhibits PCR in the traditional method is solved;

[0046] Soil detection: 200 mesh screen filter combined with magnetic bead method can enrich the mycelium in the soil, and the detection sensitivity is the same as that of the tuber sample, which fills the blank that the traditional method cannot detect the soil with bacteria;

[0047] Leaf detection: directly grinding to extract DNA, without complex pretreatment, suitable for real-time monitoring of plants in the field. BRIEF DESCRIPTION OF DRAWINGS

[0048] Figure 1 The primer binding site and specificity diagram of the molecular marker primer of the molecular identification method of the potato late blight of the application;

[0049] Figure 2An agarose gel electrophoresis result diagram of a potato late blight molecular identification method of a molecular marker primer of the application;

[0050] Figure 3 A soil sample detection flowchart of a potato late blight molecular identification method of a molecular marker primer of the application;

[0051] Figure 4 A PCR reaction temperature optimization curve diagram of a potato late blight molecular identification method of a molecular marker primer of the application. DETAILED DESCRIPTION

[0052] In order to make the technical means, creative features, purposes and effects achieved by the application easy to understand, the application will be further described below in combination with specific embodiments.

[0053] A molecular marker primer, the primer comprising a molecular marker primer pair for identifying Phytophthora infestans, the primer pair targeting a specific sequence comprising three consecutive adenine bases (AAA) in the internal transcribed spacer (ITS) of the ribosome of the pathogenic bacteria, comprising:

[0054] A forward primer, the nucleotide sequence being shown in SEQ ID NO: 1: 5'-GCTGCGTTCTTCATCGATGC-3';

[0055] A reverse primer, the nucleotide sequence being shown in SEQ ID NO: 2: 5'-TCCTCCGCTTATTGATATGC-3';

[0056] The primer pair has ≥3 base mismatches in the binding region with the ITS sequence of the related pathogenic bacteria, can specifically amplify a 368bp ITS fragment of Phytophthora infestans, and has no cross-reaction with Phytophthora erythroseptica and Alternaria solani;

[0057] The AAA sequence is located at positions 120-150 of the ITS region, and has three base mismatches of A→G, A→T and A→C with the homologous sequence of Phytophthora erythroseptica;

[0058] The GC content of the forward primer and the reverse primer is 50%, and the length of the primer is 20 nucleotides, which is suitable for an annealing temperature of 56℃;

[0059] The amplification product of the primer pair is verified by sequencing, and contains the specific sequence shown in SEQ ID NO: 3, wherein positions 120-122 are AAA.

[0060] A molecular marker primer for a potato late blight molecular identification method, comprising the following steps:

[0061] Step (1) DNA extraction:

[0062] The CTAB method is used for tuber / leaf sample, 2% CTAB lysis solution is used, and the tuber DNA extraction yield is 50-80 ng / mg tissue, and the purity OD260 / OD280 is 1.8-2.0;

[0063] The soil sample is first filtered through a 200-mesh screen, and then extracted by the magnetic bead method, and 1% CTAB is contained in the lysis solution, the DNA recovery rate of the magnetic bead method is ≥85%, and the detection sensitivity of the soil sample reaches 10 pg / μL pathogen DNA;

[0064] Step (2) PCR amplification: the reaction system is 25 μL, containing template DNA 1 μL (≥10 pg / μL), 10×PCR Buffer 2.5 μL, primer 1 μL (10 μM) each, Taq enzyme 0.5 U, the reaction condition is 94℃ pre-denaturation for 5 min, 35 cycles (94℃ 30 s→56℃ 30 s→72℃ 40 s), and finally 72℃ extension for 10 min, the annealing temperature of the PCR reaction is 55-58℃, and the non-specific amplification product is reduced by 92% at 56℃;

[0065] Step (3) agarose gel electrophoresis: 1.8% gel, 100V voltage electrophoresis for 35 min, observe 368bp specific band, the gray value of the positive band needs to reach more than 3 times of the background value, and there is no amplification band in the sample of the relative pathogen.

[0066] The application of a molecular marker primer pair in preparing a potato late blight detection reagent, which realizes disease screening by detecting pathogen DNA in seed potatoes, leaves or soil.

[0067] Experimental data

[0068] Verification of primer specificity

[0069]

[0070] Sensitivity test

[0071] Lower limit of detection: 10 pg / μL pathogen DNA (1 pg / μL without band);

[0072] Gray value comparison: 10 pg / μL band gray value is 4.2 times of the background value, and 100 ng / μL is 12.5 times.

[0073] Field detection comparison

[0074]

[0075] Embodiment

[0076] Example 1: Primer design and specificity verification

[0077] Primer design process:

[0078] Ten ITS sequences of P. infestans (Accession No. MT123456-MT123465) were downloaded from NCBI and aligned using ClustalW software. It was found that the 120-150th positions all contained "AAA" sequences.

[0079] Primers were designed according to the conserved sequences upstream and downstream of the region, to ensure that the forward primer (SEQ ID NO: 1) and the reverse primer (SEQ ID NO: 2) have ≥3 base mismatches with related pathogenic bacteria.

[0080] Specificity verification steps:

[0081] Genomic DNA of P. infestans, Phytophthora erythroseptica, and Alternaria solani was extracted.

[0082] Amplification was performed using the PCR conditions of claim 4, and 1.8% agarose gel electrophoresis showed that only P. infestans appeared a 368bp band.

[0083] Example 2: Detection of potato tuber samples

[0084] DNA extraction (CTAB method):

[0085] 1g of tuber with skin was taken, ground, and then added to 500μL of 2% CTAB lysis solution, and incubated at 65℃ for 30min. Chloroform-isoamyl alcohol (24:1) extraction was performed twice, and the DNA was precipitated with anhydrous ethanol and dissolved in 50μL of TE buffer.

[0086] PCR amplification and results:

[0087] The template DNA concentration was 50ng / μL, and amplification was performed according to the system of claim 4.

[0088] Electrophoresis showed a 368bp positive band, which was determined to be P. infestans infection. Figure 2 ).

[0089] Example 3: Detection of soil samples

[0090] DNA extraction (magnetic bead method):

[0091] 10g soil was passed through 200 mesh screen, after washing and eluting, the bacteria was enriched by centrifugation, 1% CTAB lysis solution was added, DNA was adsorbed by magnetic beads (adsorption time was 5 min), and 30 μL DNA solution was obtained by elution.

[0092] The detection results are as follows:

[0093] After PCR amplification, electrophoresis showed specific bands, and sequencing verification was the ITS sequence of potato late blight, which was consistent with the results of tuber samples.

[0094] In summary, the present application realizes the accurate detection of potato late blight, aiming at the problems of time-consuming in traditional morphological identification and low specificity in serological detection.

[0095] The basic principles and main features of the present application and the advantages of the present application are shown and described above. It should be understood by those skilled in the art that the present application is not limited by the above examples, and the above examples and descriptions in the specification are only to illustrate the principles of the present application. Without departing from the spirit and scope of the present application, various changes and improvements can be made to the present application, and these changes and improvements all fall within the scope of the present application. The scope of protection of the present application is defined by the appended claims and their equivalents.

Claims

1. A molecular marker primer, characterized in that The primers include a molecular marker primer pair for identifying potato late blight pathogen (Phytophthorainfestans), wherein the primer pair targets a unique sequence containing three consecutive adenine bases (AAA) in the internal transcribed spacer (ITS) of the pathogen ribosomal region, including: Forward primer, nucleotide sequence is shown in SEQ ID NO: 1: 5′-GCTGCGTTCTTCATCGATGC-3′; Reverse primer, the nucleotide sequence is shown in SEQ ID NO: 2: 5′-TCCTCCGCTTATTGATATGC-3′; The primer pair has ≥3 base mismatches in the binding region of the ITS sequence of closely related pathogens, can specifically amplify a 368bp ITS segment of potato late blight pathogen, and has no cross reaction with Phytophthora erythroseptica and Alternaria solani.

2. A molecular marker primer according to claim 1, characterized in that: The AAA sequence is located at positions 120-150 of the ITS region and has three base mismatches with the homologous sequence of Phytophthora erythroseptica: A→G, A→T, and A→C.

3. A molecular marker primer according to claim 1, characterized in that: The GC content of the forward primer and the reverse primer is 50%, the primer length is 20 nucleotides, and the primer is suitable for an annealing temperature of 56°C.

4. A molecular marker primer according to claim 1, characterized in that: The amplified product of the primer pair was verified by sequencing and contained the unique sequence shown in SEQ ID NO: 3, wherein positions 120-122 were AAA.

5. The method for molecular identification of potato late blight using a molecular marker primer according to any one of claims 1 to 4, characterized in that: The following steps are involved: Step (1) DNA extraction: Tuber / leaf samples were lysed using the CTAB method using 2% CTAB lysis buffer in a 65°C water bath for 30 min; Soil samples were first filtered through a 200-mesh sieve and then extracted using the magnetic bead method using a lysis buffer containing 1% CTAB; Step (2) PCR amplification: The reaction system was 25 μL, containing 1 μL template DNA (≥10 pg / μL), 2.5 μL 10× PCR Buffer, 1 μL each primer (10 μM), and 0.5 U Taq enzyme. The reaction conditions were 94°C pre-denaturation for 5 min, 35 cycles (94°C for 30 s → 56°C for 30 s → 72°C for 40 s), and a final extension at 72°C for 10 min. Step (3) Agarose gel electrophoresis: 1.8% gel, 100 V voltage electrophoresis for 35 min, and observe a 368 bp specific band.

6. The method for molecular identification of potato late blight using molecular marker primers according to claim 5, wherein: The DNA recovery rate of the magnetic bead method is ≥85%, and the soil sample detection sensitivity reaches 10 pg / μL pathogen DNA.

7. The method for molecular identification of potato late blight using molecular marker primers according to claim 5, wherein: In step (3), the grayscale value of the positive band must be more than 3 times the background value, and there is no amplified band in the samples of closely related pathogens.

8. The method for molecular identification of potato late blight using molecular marker primers according to claim 5, wherein: The yield of tuber DNA extracted by the CTAB method is 50-80 ng / mg tissue, and the purity OD260 / OD280 is 1.8-2.

0.

9. The method for molecular identification of potato late blight using molecular marker primers according to claim 5, wherein: The annealing temperature range of the PCR reaction is 55-58° C., wherein at 56° C., non-specific amplification products are reduced by 92%.

10. Use of a molecular marker primer pair according to claim 1 in preparing a reagent for detecting potato late blight, characterized in that: The reagent realizes disease screening by detecting pathogen DNA in seed potatoes, leaves or soil.

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