CN110079631B - Molecular detection method for high-throughput detection of 8 phytophthora - Google Patents
Molecular detection method for high-throughput detection of 8 phytophthora Download PDFInfo
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Abstract
The invention discloses a molecular detection method for high-throughput detection of 8 kinds of phytophthora. In the invention, the LAMP technology is combined with a 96-pore plate to establish a high-throughput detection technology capable of simultaneously detecting 8 kinds of phytophthora. The Ypt1-LAMP detection primer, kit and method are established aiming at the same target gene Ypt1 of 8 kinds of Phytophthora (Phytophthora parasitica, phytophthora infestans, phytophthora strabilis Phytophthora fragrans fragaster, phytophthora capsici capsaica, phytophthora drechleri Phytophthora infestans, phytophthora infestans Phytophthora cactorum and Phytophthora megatherium Phytophthora megatherum). The detection method disclosed by the invention is high in accuracy, strong in specificity, convenient to operate and good in practicability, provides a high-throughput rapid detection technology for detecting phytophthora, and has important and profound significance for preventing, controlling and monitoring phytophthora blight.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a high-flux molecular detection method for phytophthora.
Background
Phytophthora (Phytophthora) belongs to Oomycota (Oomycota), oomycota (Oomycotes), peronospora (Peronosporas) and Pythiaceae (Pythiaceae), and is a pathogenic oomycete with wide host range, heavy morbidity and difficult control and great loss on plant yield. Phytophthora is a major genus, and at least 103 species are currently widely recognized. Plant diseases caused by Phytophthora are commonly called "epidemic diseases" because Phytophthora (Phytophthora spp.) has strong destructiveness and harm to host plants, often causes serious loss of agricultural production. Phytophthora has a wide host range, mainly infects tens of thousands of plants such as fruit trees, forests, vegetables, flowers, cotton, hemp, oil plants and the like, but the host range of different phytophthora is very different. Host ranges such as phytophthora cinnamomi, phytophthora nicotianae, phytophthora infestans and the like are wide, and thousands of plants can be infected; the range of hosts such as phytophthora ramie and phytophthora capsici is narrow; only 1-3 host plants are infected by phytophthora sojae, phytophthora cowpea and the like. However, when two compatible strains of Phytophthora species are combined to form a sexual organ, genetic recombination may occur, which results in a pathogen with greater viability, virulence and a broader host range. The disease loss caused by phytophthora in China and the complexity of phytophthora population are increased year by year, and the normal production and life of human beings are seriously threatened. Therefore, the method for detecting and identifying various phytophthora by high flux is researched, so that the local phytophthora population and the ecology can be quickly known and mastered, and the method has great and profound significance for strengthening epidemic situation monitoring and providing basis for disease risk and research decision.
The traditional method for detecting and identifying phytophthora is mainly based on the characteristics of morphological characteristics, pathogenicity determination, physiological and biochemical characteristics and the like, and the method for separating the phytophthora is used for separating the phytophthora from soil, irrigation water and plant materials by adopting an trapping method. In order to improve the trapping effect, a small amount of antibiotics and bactericides can be added into the soil solution to inhibit the growth of mixed bacteria. Plant materials suitable for being used as baits are different due to different phytophthora species, and a plurality of materials can be used for a butterfly trapping method, including a method for trapping phytophthora nicotianae by using citrus leaves, a method for trapping phytophthora drechnei by using cucumber fruits, a method for trapping phytophthora camphorata by using pine needles, a method for trapping phytophthora sojae by using perforated soybean leaves and the like. In order to successfully separate phytophthora, the traditional method needs to adopt a selective culture medium to obtain pure culture of pathogens, the whole process is time-consuming and labor-consuming, and operators are required to have professional pathogenic bacteria separation, morphological identification knowledge and rich experience.
With the development of molecular biology technology, common PCR and real-time fluorescence PCR have been successfully applied to the detection of various phytophthora. Although the detection method adopting the PCR technology and the real-time fluorescence PCR technology has the advantages of high specificity and sensitivity, the detection time is still long, the subsequent electrophoresis gel running is time-consuming and labor-consuming, and the detection method needs to depend on a precise PCR instrument with high value, so that the requirement of rapid detection cannot be met. In addition, many detection technical researches aim at phytophthora, but in practical situations, the demand for high-throughput detection and identification of phytophthora is more and more strong. Katarzyna Sikora et al used Padlock probe in combination with microarray chip technology to perform high throughput detection of 23 kinds of Phytophthora. However, these diagnostic methods or procedures are cumbersome, long-lasting, or costly, and require the use of expensive instruments and reagents.
Loop-mediated isothermal amplification (Loop-mediated isothermal a)mplification, LAMP) is a highly efficient method for amplifying nucleic acids. It is a new type molecular biological detection technology, said method is to design 4 specific primers according to 6 regions of target gene, utilize Bst DNA polymerase chain displacement reaction, in 60-65 deg.C constant temperature condition short time (usually within one hour) nucleic acid amplification, can amplify target gene to 10 9 And determining whether the target gene exists in the amplification product according to the amplification result, and analyzing the product and the amplification sensitivity by using agarose gel electrophoresis and SYBR Green I fluorescence quantification methods. In order to make LAMP end product detection simpler, more intuitive and more time-saving, domestic and foreign scientists have made a lot of research, and put forward a turbidity analysis method, an HNB indicator, a ceruloplasmin dye and other simple and easy-to-operate LAMP end product detection and result judgment methods in sequence, and currently, agarose gel electrophoresis, a turbidity analysis method, an HNB indicator method and an SYBR Green I indicator method are more applied. The technology does not need strict experimental environment and precise and expensive instruments and equipment, has the characteristics of simple and easily learned operation steps, high specificity, high sensitivity and the like. This technique was first used for clinical tests in medicine since 2003, and has achieved remarkable effects. Many scholars have already extensively applied the detection of pathogenic bacteria such as viruses, bacteria, parasites and fungi.
The LAMP technology is successfully applied to the detection of individual phytophthora, and a complete high-flux LAMP identification detection system for phytophthora is lacked, so that the invention establishes a loop-mediated isothermal amplification technology high-flux detection system for phytophthora based on the same target Ypt1, and on the basis, a metal ion indicator Hydroxy Naphthol Blue (HNB) is applied to judge the reaction result, and the specificity and the sensitivity of the method are analyzed, the whole detection reaction only needs 80min, and the positive result can be directly judged by naked eyes. The method is a research for detecting various phytophthora simultaneously by combining LAMP technology with 96-well plate technology for the first time, and has a very wide application prospect.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the problems that a convenient, fast and efficient LAMP identification detection system of the high-flux phytophthora is lacked in the prior art, the local phytophthora population and the ecology can not be quickly known and mastered, the phytophthora epidemic situation monitoring is not facilitated to be enhanced, and a basis is provided for the risk of diseases and research decision, a technical system for detecting the phytophthora in a high-flux manner by using a loop-mediated isothermal amplification technology based on the same target Ypt1 is established in the application, one of the purposes of the invention is to provide an LAMP detection primer group with high flux of 8 phytophthora, and the phytophthora is detected in a phase specificity manner; the invention also aims to provide a kit for detecting 8 kinds of phytophthora in high throughput, and multiple kinds of phytophthora are identified at one time; the invention also provides a detection method for detecting 8 kinds of phytophthora in high flux by combining the LAMP technology and a 96-pore plate, and the method can conveniently and quickly identify various phytophthora.
The technical scheme is as follows: in order to solve the problems, the technical scheme adopted by the invention is as follows:
a molecular detection method for high-flux detection of 8 phytophthora comprises the steps of extracting DNA of microorganisms to be detected, taking the extracted DNA as a template, and simultaneously carrying out high-flux detection on the microorganisms to be detected by combining an LAMP detection primer of 8 phytophthora with a 96-well plate; observing the color change of the LAMP reaction solution, wherein sky blue indicates that the detection result is positive and target phytophthora exists; purple indicates that the detection result is negative and the target phytophthora does not exist; the LAMP detection primers of the 8 kinds of Phytophthora bacteria comprise LAMP detection primers of Phytophthora parasitica, phytophthora tendamicula, phytophthora infestans, phytophthora fragariae, phytophthora capsicii, phytophthora drechleri, phytophthora cactorum and Phytophthora megaspora, and the LAMP detection primers of each kind of Phytophthora bacteria comprise a forward inner primer FIP, a reverse inner primer BIP, a forward outer primer F3, a reverse outer primer B3, a forward loop primer LF and a reverse loop LB primer, wherein the sequences of the primers are as follows:
the primers for phytophthhoraparasitica include:
F3：5′-CTTTGTAAGTGCCACCATAC-3′；
B3：5′-ACAGACACACACGTGATT-3′；
FIP：
5′-TCGATCGTACGGATTTTCTGCAGAAAGATCCAGGTTTTCATCAGG-3′；
BIP：
5′-AGACCATCAAGCTCCAGATTGTACGTGGTACATCGGTTAGTTGAA-3′′；
LB：5′-GACTTGTATCACTGCAGTTT-3′；
the primers of Phytophthora tentaculata include:
F3：5′-TGCCTTATAGGAATAGCGC-3′；
B3：5′-AGCATAAGTGAATTGACCCA-3′；
FIP：
5′-ATCGTACGGATTTTCTGAGCAAAGTAGATCCCGATTTCCATCAG-3′；
BIP：
5′-TGGACGGCAAGACCATCAAGTCCGTTAGTTAAATAAATACCTCGA-3′；
LB：5′-CTCCAGATTGTACGTCCTTCGT-3′；
the primers of Phytophthora infestans comprise:
F3：5′-TGTGAGTGTCTAACATATTTTACG-3′；
B3：5′-GTTAGTTAAATAGGAAATCACGC-3′；
FIP：5′-AATCGCGAAAGCCATGTGAGCCAAACGACCTTTTGTAAGG-3′；
BIP：
5′-TTGCTTAGAAAATCCGTACGATCGGACAAATGTTTTTTTAGCGGC-3′；
LB：5′-GGCAAGACCATCAAGCTCCAA-3′；
the primers of Phytophthora fragariae comprise:
F3：5′-TGAGTGCTAGTAACTAGCCT-3′；
B3：5′-ACCAGTTAGCTCCATGAAGC-3′；
FIP：
5′-CTGTGCACAACAACGAGCACGAATTCCTAGGTCCAAAAAGGC-3′；
and (3) BIP:5 '-AATTCGCACGATCGAGCTGGATTAGCGGATGCCGGAAATGTTCC-3';
LF：5′-CAGCAATCGGAGAGCAAATCTTA-3′；
LB：5′-CGGCAAGACTATCAAGCTCCAGA-3′；
the primers of Phytophthora capsici comprise:
F3：5′-CTCTGTTGTATAGCAGAGGT-3′；
B3：5′-GCACAAGACAATTAGCACAAT-3′；
FIP：
5′-TTCTGGGCGCGTACACAAACTTAGTGAGGGACAATTTATATCAGG-3′；
BIP：5′-GATCGAGTTGGACGGCAAGATCCAGTGCTCTAACTAAAACG-3′：
LB：5′-CCATCAAGCTCCAGATTGTAAGC-3′：
the primers for Phytophthora drechsleri include:
F3：5′-GTGATCCTTTCACCCTGG-3′；
B3：5′-TTACAAATGTCAGCTGGATG-3′：
FIP：
5′-CGGATTTTCTAGAACGTGGTACCAAAATGAAGAGTCGACTCTAGCA-3′；
BIP：5′-ACTATTGAGCTGGACGGCAATCGATAGCAGCCCAAGAG-3′；
LB：5′-TGTACGTCTACAGAGGATTTGGAT-3′；
the primers for Phytophthora cacorum include:
F3：5′-TTCTGCGCTAGGCGACC-3′；
B3：5′-CACACAAGTGGACCGTTAG-3′；
FIP：5′-TCTGGGCACAACCGCAAAAATTTGCGAGCTCCAGATTTCC-3′；
BIP：5′-AATCCGTACGATCGAGCTGGACACACGCCACGTCTGCT-3′；
LB：5′-GGAAAGACCATCAAGCTCCAGAT-3′；
the primers for Phytophthora megasperma include:
F3：5′-GCTCTGCTCTTCCGACTTG-3′；
B3：5′-GTTAGTTTCGTCCACGGCA-3′；
FIP：5′-CGGGCAAGAGCAACGTCAGTGTCCCATTGTGGTCCAGTAC-3′；
BIP：
5′-TCCGTACGATCGAGCTGGACGAGAAGAAAGGAATGGAGGCC-3′；
LB：5′-GCAAGACCATCAAGCTCCAGATTG-3′。
the LAMP detection primer for the 8 kinds of phytophthora is applied to detection of phytophthora.
An LAMP detection kit for phytophthora comprises at least a primer solution and an LAMP reaction solution with more than one dose; wherein the primer in the primer solution is the LAMP detection primer for the 8 kinds of phytophthora as claimed in claim 1.
Preferably, the LAMP reaction solution contains dNTPs, tris-HCl, KCl and (NH) 4 ) 2 SO 4 、MgSO 4 Triton X-100, bst DNA polymerase, hydroxynaphthol blue.
The LAMP detection kit for phytophthora is applied to detection of phytophthora.
A molecular detection method for detecting 5 kinds of phytophthora at high flux comprises the steps of extracting DNA of microorganisms to be detected, taking the extracted DNA as a template, and simultaneously carrying out high flux detection on the microorganisms to be detected by combining LAMP detection primers of the 5 kinds of phytophthora with a 96-well plate; observing the color change of the LAMP reaction solution, wherein sky blue indicates that the detection result is positive and the target phytophthora exists; purple indicates that the detection result is negative and target phytophthora does not exist; the LAMP detection primers of the 8 kinds of Phytophthora bacteria comprise LAMP detection primers of Phytophthora parasitica, phytophthora tentaculata, phytophthora fragaraceae, phytophthora capsici and Phytophthora megaspora, the LAMP detection primer of each kind of Phytophthora bacteria consists of a forward inner primer FIP, a reverse inner primer BIP, a forward outer primer F3, a reverse outer primer B3, a forward loop primer LF and a reverse loop primer LB, and the sequences of the primers are as follows:
the primers for phytophthhoraparasitica include:
F3：5′-CTTTGTAAGTGCCACCATAC-3′；
B3：5′-ACAGACACACACGTGATT-3′；
FIP：
5′-TCGATCGTACGGATTTTCTGCAGAAAGATCCAGGTTTTCATCAGG-3′；
BIP：
5′-AGACCATCAAGCTCCAGATTGTACGTGGTACATCGGTTAGTTGAA-3′′；
LB：5′-GACTTGTATCACTGCAGTTT-3′；
the primers of Phytophthora tentaculata include:
F3：5′-TGCCTTATAGGAATAGCGC-3′；
B3：5′-AGCATAAGTGAATTGACCCA-3′；
FIP：
5′-ATCGTACGGATTTTCTGAGCAAAGTAGATCCCGATTTCCATCAG-3′；
BIP：
5′-TGGACGGCAAGACCATCAAGTCCGTTAGTTAAATAAATACCTCGA-3′；
LB：5′-CTCCAGATTGTACGTCCTTCGT-3′；
the primers of Phytophthora fragariae comprise:
F3：5′-TGAGTGCTAGTAACTAGCCT-3′；
B3：5′-ACCAGTTAGCTCCATGAAGC-3′；
FIP：
5′-CTGTGCACAACAACGAGCACGAATTCCTAGGTCCAAAAAGGC-3′；
BIP：5′-AATTCGCACGATCGAGCTGGATTAGCCGGCGAAATGTTCC-3′；
LF：5′-CAGCAATCGGAGAGCAAATCTTA-3′；
LB：5′-CGGCAAGACTATCAAGCTCCAGA-3′；
the primers of Phytophthora capsici comprise:
F3：5′-CTCTGTTGTATAGCAGAGGT-3′；
B3：5′-GCACAAGACAATTAGCACAAT-3′；
FIP：
5′-TTCTGGGCGCGTACACAAACTTAGTGAGGGACAATTTATATCAGG-3′；
BIP：5′-GATCGAGTTGGACGGCAAGATCCAGTGCTCTAACTAAAACG-3′；
LB：5′-CCATCAAGCTCCAGATTGTAAGC-3′；
the primers of Phytophthora megasperma comprise:
F3：5′-GCTCTGCTCTTCCGACTTG-3′；
B3：5′-GTTAGTTTCGTCCACGGCA-3′；
FIP：5′-CGGGCAAGAGCAACGTCAGTGTCCCATTGTGGTCCAGTAC-3′；
BIP：
5′-TCCGTACGATCGAGCTGGACGAGAAGAAAGGAATGGAGGCC-3′；
LB：5′-GCAAGACCATCAAGCTCCAGATTG-3′。
the LAMP detection primers for the 5 kinds of phytophthora are applied to detection of phytophthora.
An LAMP detection kit for phytophthora, which at least comprises more than one-time consumption of primer solution and LAMP reaction solution; wherein the primer in the primer solution is the LAMP detection primer for 5 kinds of phytophthora as claimed in claim 6.
Preferably, the LAMP reaction solution contains dNTPs, tris-HCl, KCl and (NH) 4 ) 2 SO 4 、MgSO 4 Triton X-100, bst DNA polymerase, hydroxynaphthol blue.
The LAMP detection kit for phytophthora is applied to detection of phytophthora.
Has the beneficial effects that: compared with the prior art, the invention has the advantages and positive effects that:
1) The accuracy is high: because the traditional phytophthora detection technology only determines a detection object according to morphological characteristics, the interference of human factors cannot be eliminated, morphological similar species are difficult to distinguish, and the detection accuracy is only 60-80%; according to the sequence of the Ypt1 gene of the phytophthora, the sequence is alternately spaced in a genome evolution area and a conservation area in the phytophthora, the sequence of the Ypt1 gene of 8 phytophthora to be detected is compared with the sequences of other phytophthora by using Bioedit software, and a specific LAMP primer is designed by selecting a specific sequence of the phytophthora.
2) The specificity is strong: in the invention, when designing a primer, various gene targets such as a ribosomal gene transcription spacer, a transcription elongation factor, an actin gene and the like are tried, but are not screened to be suitable. The Ypt1 gene has the structural characteristic that coding regions and non-coding regions alternately appear, so that the non-coding regions of the Ypt1 gene have more changed sites relative to other detection targets to be used as molecular markers of most phytophthora, and the Ypt1 gene is quite conservative in the same phytophthora species. Therefore, the phytophthora species specific primer designed by using the Ypt1 gene has better specificity compared with other targets. Finally, when the Ypt1 gene is aligned, a more specific sequence is found, and a plurality of alternative primers are obtained through software by taking the sequence as a target. And then, carrying out a preliminary experiment aiming at the alternative primers, and finally determining the specific primer group used by us.
3) The operation is convenient: the LAMP method for detecting phytophthora infestans provided by the invention overcomes the problems that the biological detection method for phytophthora infestans in the prior art needs long period, wastes time and labor, is complex and has poor specificity, and the PCR detection technology needs a thermal cycler and cannot quickly detect the phytophthora infestans. The detection method can quickly, conveniently, efficiently, highly specifically and sensitively detect the phytophthora infestans under the isothermal condition of 64 ℃, does not need complex instruments, and can better meet the field detection of the phytophthora infestans.
4) The practicability is good: the product diffusion is easily caused by the gel electrophoresis of the product by the common PCR reaction, which is a main source of laboratory pollution; ethidium Bromide (EB) has a great toxicity and can accumulate carcinogenesis; the long-term observation of the ultraviolet lamp can also cause certain damage to the experimenters. The LAMP reaction is only carried out in a constant-temperature water bath kettle, and the result can be directly judged through the color change of HNB after the reaction is finished, so that the application value of the LAMP reaction in bacteria-carrying plants and soil is increased.
In conclusion, based on the LAMP technology and a 96-well plate, the LAMP high-flux technology for detecting phytophthora based on the same target Ypt1 is established for the first time, 8 high-flux LAMP detection primers and a method for detecting phytophthora are provided, and compared with the traditional identification method and the conventional PCR technology detection method, the LAMP high-flux detection primers and the method have stronger specificity, sensitivity and portability, more phytophthora species are detected at one time, the LAMP high-flux detection primers and the method can be used for quickly knowing and mastering local phytophthora populations, and the LAMP high-flux detection primers and the method have important and profound significance for prevention, control and monitoring of phytophthora blight.
Drawings
FIG. 1 is a result diagram of high-throughput detection of 8 kinds of phytophthora by combining an LAMP detection method with a 96-well plate, wherein a reaction solution of a positive reaction is sky blue, and a negative reaction is purple; A1-A9: an LAMP system of phytophthora parasitica; B1-B9: the LAMP system of Phytophthora tentaculata; C1-C9: a LAMP system of Phytophthora infestans; D1-D9: an LAMP system of Phytophthora strawberry; e1 to E9: an LAMP system of phytophthora capsici; f1 to F9: an LAMP system of Phytophthora drechneii; G1-G9: an LAMP system of phytophthora infestans; H1-H9: an LAMP system of Phytophthora sojae. 1A-1H: target DNA added into the LAMP system is phytophthora parasitica; 2A-2H: the target DNA added into the LAMP system is Phytophthora tentaculata;3A-3H: target DNA added into the LAMP system is phytophthora infestans; 4A-4H: target DNA added into the LAMP system is phytophthora strawberry; 5A-5H: target DNA added into the LAMP system is phytophthora capsici; 6A-6H: target DNA added into the LAMP system is phytophthora drechnei; 7A-7H: target DNA added into the LAMP system is phytophthora infestans; 8A-8H: target DNA added into the LAMP system is Phytophthora macrosporum; 9A-9H: water was added to the LAMP system as a negative control.
Detailed Description
The invention is further described with reference to specific examples. The molecular biology experiments, which are not specifically described in the following examples, were carried out according to the specific methods listed in molecular cloning, A laboratory Manual (third edition) J. Sambuchok, supra, or according to the kit and product instructions.
Example 1: design of LAMP detection primers for 8 phytophthora
According to the accession number of the phytophthora parasitica Ypt1 gene GeneBank: DQ162981.1; phytophthora tentaculata Ypt1 gene GeneBank accession number: HQ850014.1; phytophthora infestans Ypt1 gene GeneBank accession number: DQ162981.1; phytophthora strawberry Ypt1 gene GeneBank accession number: DQ270305.1; phytophthora capsici Ypt1 gene GeneBank accession number: FJ535572.1; phytophthora drechnii Ypt1 gene GeneBank accession number: DQ162989.1; phytophthora infestans Ypt1 gene GeneBank accession number: HQ850000.1; phytophthora grandis Ypt1 gene GeneBank accession number: DQ162986.1; different LAMP primers are designed, and specific primers are shown in Table 1.
TABLE 1 LAMP primer sequences
Example 2: preparation of DNA template of bacteria to be detected
The preparation method of the DNA template of the phytophthora in the soil sample with bacteria comprises the following steps:
all soil samples taken
DNA extraction was performed using the SPIN kit (Q-Biogene Ltd, USA). See kit instructions for soil DNA extraction procedures. This commercial soil microorganism DNA extraction kit can extract microorganisms in soil within 0.5 h.
Enriching oospores in soil: taking 20-100 g of a soil sample to be detected, grinding, removing larger soil particles by adopting a 200-mesh screen, filtering by using 400-mesh, 500-mesh and 800-mesh screens, washing repeatedly by using 3-10 liters of water, collecting oospores from the 800-mesh screen, and suspending by using 1mL of water. Since the oospore can not penetrate through the 800-mesh screen, the treatment can achieve the effect of enriching the oospore.
Extracting DNA from the microspores: the oospores suspended in sterile water were transferred to a 1.5mL centrifuge tube at 12000r.min -1 Centrifuging for 5 minutes at a rotating speed, and pouring out liquid; adding 50 mu L of CTAB buffer, grinding, adding 500 mu L of CTAB buffer, and carrying out water bath for 30 minutes; adding equal volume of chloroform for extraction at 12000r.min -1 Centrifuging for 10 minutes at a rotating speed, and sucking a supernatant; adding 3M NaAc (sodium chloride) with the volume of 1/10, anhydrous ice ethanol with the volume of 2 times, precipitating for 30 minutes at room temperature, and 12000r.min -1 Centrifuging for 10 minutes at the rotating speed, and pouring out the liquid; washing with 1mL 70% (V/V) ethanol (12000r. Min.) -1 Centrifuging for 10 minutes at a rotating speed, pouring out the liquid, and airing until no alcohol smell exists; 10 μ L of sterile double distilled water was added for dissolution and used as a template for LAMP amplification.
Extracting the DNA of phytophthora infestans in the diseased tissue by adopting a CTAB method, wherein the specific method comprises the following steps:
taking a small amount of mycelium powder, adding 900 μ L of 2% CTAB extracting solution and 90 μ L of 10% SDS, vortexing, and turning over in a water bath at 55 deg.C for 1h, and turning over every 10 min. Centrifuging at 12000rpm for 10min, collecting supernatant, adding equal volume of phenol/chloroform/isoamyl alcohol (25: 24: 1), mixing by inversion, and centrifuging at 12000rpm for 10min; the supernatant was transferred to a new tube, added with an equal volume of chloroform, mixed by gentle inversion and centrifuged at 12000rpm for 5min. The supernatant was transferred to a new tube and 2 volumes of absolute ethanol and 1/10 volume of 3M NaAc (pH 5.2) were added and precipitated at-20 deg.C (> 1 h). Centrifuging at 12000rpm for 10min, decanting the supernatant, washing the precipitate with 70% ethanol twice, and air drying at room temperature. Adding appropriate amount of sterilized ultrapure water or TE (pH 8.0) to dissolve precipitate (containing 20 μ g/mL RNase), treating at 37 deg.C for 1h, and storing at-20 deg.C for use.
When phytophthora infestans exists in the pathogenic tissues, the DNA of the phytophthora infestans can be extracted by adopting a NaOH rapid cracking method, and the specific process is as follows: a section of diseased plant tissue is taken, 10 mu L of 0.5M NaOH is added to each milligram of tissue, the tissue is fully ground in a mortar and then transferred to a 1.5mL EP tube, the tube is centrifuged at 12000rpm for 5min, 5 mu L of supernatant is taken, 495 mu L of 0.1mM Tris (pH 8.0) is added, and after uniform mixing, one mu L of supernatant is taken and directly used for PCR reaction. Each reaction was repeated at least three times to confirm that no PCR inhibitor was present in the plants.
Example 3: preparation of phytophthora LAMP (loop-mediated isothermal amplification) detection kit
1. An LAMP detection kit for detecting phytophthora infestans comprises 1.6 mu M forward inner primer FIP, 1.6 mu M reverse inner primer BIP, 0.2 mu M forward outer primer F3, 0.2 mu M reverse outer primer B3, 0.2 mu M reverse loop primer LB, 1.4mM dNTPs, 2 mM Tris-HCl with pH 8.8, 10mM KCl and 10mM (NH) 4 ) 2 SO 4 、6mM MgSO 4 0.1% Triton X-100, 8U Bst DNA polymerase unit, 5mM hydroxynaphthol blue, and ultrapure water were added to prepare a 25uL detection solution. The sequences of the primers are shown in Table l
LAMP detection primer specificity verification
One of the key technologies of the application is that the LAMP primer composition is combined with a 96-well plate to simultaneously carry out high-throughput detection on 8 kinds of phytophthora. In order to verify the specific primer sequence of phytophthora, the invention takes phytophthora strains in provinces of Jiangsu, shandong, fujian and the like and a plurality of other pathogenic fungi in China as test materials (table 2), and the method provided in the embodiment 2 is adopted to extract the DNA of the bacteria to be detected in different samples.
When LAMP amplification reaction occurs, a large amount of magnesium pyrophosphate white precipitates are generated to cause the turbidity of reaction liquid to rise, and the reaction tubes of phytophthora infestans are all sky blue and are positive results, while the reaction tubes of other phytophthora species, fungi, pythium and negative control bacteria are purple and are negative results, as shown by the color development reaction result of HNB, the LAMP specific primer is proved to have the specificity of the species. Meanwhile, the reaction product is subjected to 2% agarose gel electrophoresis, the amplified reaction product is observed through imaging, a typical step-shaped strip appears in reaction liquid in a reaction tube of the phytophthora infestans, and no trapezoid strip appears in reaction tubes of other phytophthora infestans, fungi, pythium and negative control bacteria. This shows that the primer set can be used for producing practical fast and reliable detection kit for detecting pathogenic phytophthora infestans in pathogenic tissues and soil.
TABLE 2 fungal and oomycete strains for the detection of Phytophthora infestans specificities
Example 4 high-throughput LAMP identification detection System for Phytophthora
FIG. 1 shows that the LAMP detection method is combined with a 96-well plate to carry out high-flux detection on 8 kinds of phytophthora, and the results are shown in Table 3, which shows that the LAMP detection method and the 96-well plate combined technology designed by the application can carry out small-scale high-flux detection on a plurality of phytophthora simultaneously, and the results are reliable, and the designed primers have species specificity and can well distinguish the target phytophthora from other phytophthora.
TABLE 3
Sequence listing
<110> Nanjing university of forestry
<120> molecular detection method for high-throughput detection of 8 phytophthora
<130> 100
<160> 41
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> F3 (Phytophthora parasitica) primer sequence (Artificial)
<400> 1
ctttgtaagt gccaccatac 20
<210> 2
<211> 18
<212> DNA
<213> B3 (Phytophthora parasitica) primer sequence (Artificial)
<400> 2
acagacacac acgtgatt 18
<210> 3
<211> 45
<212> DNA
<213> FIP (Phytophthora parasitica) primer sequence (Artificial)
<400> 3
tcgatcgtac ggattttctg cagaaagatc caggttttca tcagg 45
<210> 4
<211> 45
<212> DNA
<213> BIP (Phytophthora parasitica) primer sequence (Artificial)
<400> 4
agaccatcaa gctccagatt gtacgtggta catcggttag ttgaa 45
<210> 5
<211> 20
<212> DNA
<213> LB (Phytophthora parasitica) primer sequence (Artificial)
<400> 5
gacttgtatc actgcagttt 20
<210> 6
<211> 19
<212> DNA
<213> F3 (Phytophthora tentaculata) primer sequence (Artificial)
<400> 6
tgccttatag gaatagcgc 19
<210> 7
<211> 20
<212> DNA
<213> B3 (Phytophthora tentaculata) primer sequence (Artificial)
<400> 7
agcataagtg aattgaccca 20
<210> 8
<211> 44
<212> DNA
<213> FIP (Phytophthora tentaculata) primer sequence (Artificial)
<400> 8
atcgtacgga ttttctgagc aaagtagatc ccgatttcca tcag 44
<210> 9
<211> 45
<212> DNA
<213> BIP (Phytophthora tentaculata) primer sequence (Artificial)
<400> 9
tggacggcaa gaccatcaag tccgttagtt aaataaatac ctcga 45
<210> 10
<211> 22
<212> DNA
<213> LB (Phytophthora tentaculata) primer sequence (Artificial)
<400> 10
ctccagattg tacgtccttc gt 22
<210> 11
<211> 24
<212> DNA
<213> F3 (Phytophthora infestans) primer sequence (Artificial)
<400> 11
tgtgagtgtc taacatattt tacg 24
<210> 12
<211> 23
<212> DNA
<213> B3 (Phytophthora infestans) primer sequence (Artificial)
<400> 12
gttagttaaa taggaaatca cgc 23
<210> 13
<211> 40
<212> DNA
<213> FIP (Phytophthora infestans) primer sequence (Artificial)
<400> 13
aatcgcgaaa gccatgtgag ccaaacgacc ttttgtaagg 40
<210> 14
<211> 45
<212> DNA
<213> BIP (Phytophthora infestans) primer sequence (Artificial)
<400> 14
ttgcttagaa aatccgtacg atcggacaaa tgttttttta gcggc 45
<210> 15
<211> 21
<212> DNA
<213> LB (Phytophthora infestans) primer sequence (Artificial)
<400> 15
ggcaagacca tcaagctcca a 21
<210> 16
<211> 20
<212> DNA
<213> F3 (Phytophthora fragariae) primer sequence (Artificial)
<400> 16
tgagtgctag taactagcct 20
<210> 17
<211> 20
<212> DNA
<213> B3 (Phytophthora fragariae) primer sequence (Artificial)
<400> 17
accagttagc tccatgaagc 20
<210> 18
<211> 42
<212> DNA
<213> FIP (Phytophthora fragariae) primer sequence (Artificial)
<400> 18
ctgtgcacaa caacgagcac gaattcctag gtccaaaaag gc 42
<210> 19
<211> 40
<212> DNA
<213> BIP (Phytophthora fragariae) primer sequence (Artificial)
<400> 19
aattcgcacg atcgagctgg attagccggc gaaatgttcc 40
<210> 20
<211> 23
<212> DNA
<213> LB (Phytophthora fragariae) primer sequence (Artificial)
<400> 20
cggcaagact atcaagctcc aga 23
<210> 21
<211> 23
<212> DNA
<213> LF (Phytophthora fragariae) primer sequence (Artificial)
<400> 21
cagcaatcgg agagcaaatc tta 23
<210> 22
<211> 20
<212> DNA
<213> F3 (Phytophthora capsicii) primer sequence (Artificial)
<400> 22
ctctgttgta tagcagaggt 20
<210> 23
<211> 21
<212> DNA
<213> B3 (Phytophthora capsicii) primer sequence (Artificial)
<400> 23
gcacaagaca attagcacaa t 21
<210> 24
<211> 45
<212> DNA
<213> FIP (Phytophthora capsicii) primer sequence (Artificial)
<400> 24
ttctgggcgc gtacacaaac ttagtgaggg acaatttata tcagg 45
<210> 25
<211> 41
<212> DNA
<213> BIP (Phytophthora capsicii) primer sequence (Artificial)
<400> 25
gatcgagttg gacggcaaga tccagtgctc taactaaaac g 41
<210> 26
<211> 23
<212> DNA
<213> LB (Phytophthora capsicii) primer sequence (Artificial)
<400> 26
ccatcaagct ccagattgta agc 23
<210> 27
<211> 18
<212> DNA
<213> F3 (Phytophthora drechsleri) primer sequence (Artificial)
<400> 27
gtgatccttt caccctgg 18
<210> 28
<211> 20
<212> DNA
<213> B3 (Phytophthora drechsleri) primer sequence (Artificial)
<400> 28
ttacaaatgt cagctggatg 20
<210> 29
<211> 46
<212> DNA
<213> FIP (Phytophthora drechsleri) primer sequence (Artificial)
<400> 29
cggattttct agaacgtggt accaaaatga agagtcgact ctagca 46
<210> 30
<211> 38
<212> DNA
<213> BIP (Phytophthora drechsleri) primer sequence (artifical)
<400> 30
actattgagc tggacggcaa tcgatagcag cccaagag 38
<210> 31
<211> 24
<212> DNA
<213> LB (Phytophthora drechsleri) primer sequence (Artificial)
<400> 31
tgtacgtcta cagaggattt ggat 24
<210> 32
<211> 17
<212> DNA
<213> F3 (Phytophthora cactorum) primer sequence (Artificial)
<400> 32
ttctgcgcta ggcgacc 17
<210> 33
<211> 19
<212> DNA
<213> B3 (Phytophthora cactorum) primer sequence (Artificial)
<400> 33
cacacaagtg gaccgttag 19
<210> 34
<211> 40
<212> DNA
<213> FIP (Phytophthora cactorum) primer sequence (Artificial)
<400> 34
tctgggcaca accgcaaaaa tttgcgagct ccagatttcc 40
<210> 35
<211> 38
<212> DNA
<213> BIP (Phytophthora cactorum) primer sequence (Artificial)
<400> 35
aatccgtacg atcgagctgg acacacgcca cgtctgct 38
<210> 36
<211> 23
<212> DNA
<213> LB (Phytophthora cactorum) primer sequence (Artificial)
<400> 36
ggaaagacca tcaagctcca gat 23
<210> 37
<211> 19
<212> DNA
<213> F3 (Phytophthora megasperma) primer sequence (Artificial)
<400> 37
gctctgctct tccgacttg 19
<210> 38
<211> 19
<212> DNA
<213> B3 (Phytophthora megasperma) primer sequence (Artificial)
<400> 38
gttagtttcg tccacggca 19
<210> 39
<211> 40
<212> DNA
<213> FIP (Phytophthora megaspora) primer sequence (Artificial)
<400> 39
cgggcaagag caacgtcagt gtcccattgt ggtccagtac 40
<210> 40
<211> 41
<212> DNA
<213> BIP (Phytophthora megaspora) primer sequence (Artificial)
<400> 40
tccgtacgat cgagctggac gagaagaaag gaatggaggc c 41
<210> 41
<211> 24
<212> DNA
<213> LB (Phytophthora megasperma) primer sequence (artifical)
<400> 41
gcaagaccat caagctccag attg 24
Claims (6)
1. A molecular detection method for detecting 8 kinds of phytophthora with high flux is characterized in that: the method comprises the steps of extracting DNA of a microorganism to be detected, taking the extracted DNA as a template, and carrying out high-throughput detection on the microorganism to be detected simultaneously by combining 8 phytophthora LAMP detection primers and a 96-well plate; observing the color change of the LAMP reaction solution, wherein sky blue indicates that the detection result is positive and target phytophthora exists; purple indicates that the detection result is negative and the target phytophthora does not exist; the LAMP detection primer for the 8 kinds of phytophthora comprisesPhytophthora parasitica、Phytophthora tentaculata、Phytophthora infestans、Phytophthora fragariae、Phytophthora capsici、Phytophthora drechsleri、Phytophthora cactorumAndPhytophthora megaspermathe LAMP detection primer of each phytophthora consists of a forward inner primer FIP, a reverse inner primer BIP, a forward outer primer F3, a reverse outer primer B3, a forward loop primer LF and a reverse loop primer LB, and the sequences of the primers are as follows:
Phytophthora parasiticathe primer of (a) comprises:
F3：5'-CTTTGTAAGTGCCACCATAC-3'；
B3：5'-ACAGACACACACGTGATT-3'；
FIP：5'-TCGATCGTACGGATTTTCTGCAGAAAGATCCAGGTTTTCATCAGG-3'；
BIP：5'-AGACCATCAAGCTCCAGATTGTACGTGGTACATCGGTTAGTTGAA-3'；
LB：5'-GACTTGTATCACTGCAGTTT-3'；
Phytophthora tentaculatathe primer of (2) comprises:
F3：5'-TGCCTTATAGGAATAGCGC-3'；
B3：5'-AGCATAAGTGAATTGACCCA-3'；
FIP：5'-ATCGTACGGATTTTCTGAGCAAAGTAGATCCCGATTTCCATCAG-3'；
BIP：5'-TGGACGGCAAGACCATCAAGTCCGTTAGTTAAATAAATACCTCGA-3'；
LB：5'-CTCCAGATTGTACGTCCTTCGT-3'；
Phytophthora infestansthe primer of (2) comprises:
F3：5'-TGTGAGTGTCTAACATATTTTACG-3'；
B3：5'-GTTAGTTAAATAGGAAATCACGC-3'；
FIP：5'-AATCGCGAAAGCCATGTGAGCCAAACGACCTTTTGTAAGG-3'；
BIP：5'-TTGCTTAGAAAATCCGTACGATCGGACAAATGTTTTTTTAGCGGC-3'；
LB：5'-GGCAAGACCATCAAGCTCCAA-3'；
Phytophthora fragariaethe primer of (2) comprises:
F3：5'-TGAGTGCTAGTAACTAGCCT-3'；
B3：5'-ACCAGTTAGCTCCATGAAGC-3'；
FIP：5'-CTGTGCACAACAACGAGCACGAATTCCTAGGTCCAAAAAGGC-3'；
BIP：5'-AATTCGCACGATCGAGCTGGATTAGCCGGCGAAATGTTCC-3'；
LF：5'-CAGCAATCGGAGAGCAAATCTTA-3'；
LB：5'-CGGCAAGACTATCAAGCTCCAGA-3'；
Phytophthora capsicithe primer of (2) comprises:
F3：5'-CTCTGTTGTATAGCAGAGGT-3'；
B3：5'-GCACAAGACAATTAGCACAAT-3'；
FIP：5'-TTCTGGGCGCGTACACAAACTTAGTGAGGGACAATTTATATCAGG-3'；
BIP：5'-GATCGAGTTGGACGGCAAGATCCAGTGCTCTAACTAAAACG-3'；
LB：5'-CCATCAAGCTCCAGATTGTAAGC-3'；
Phytophthora drechslerithe primer of (a) comprises:
F3：5'-GTGATCCTTTCACCCTGG-3'；
B3：5'-TTACAAATGTCAGCTGGATG-3'；
FIP：5'-CGGATTTTCTAGAACGTGGTACCAAAATGAAGAGTCGACTCTAGCA-3'；
BIP：5'-ACTATTGAGCTGGACGGCAATCGATAGCAGCCCAAGAG-3'；
LB：5'-TGTACGTCTACAGAGGATTTGGAT-3'；
Phytophthora cactorumthe primer of (2) comprises:
F3：5'-TTCTGCGCTAGGCGACC-3'；
B3：5'-CACACAAGTGGACCGTTAG-3'；
FIP：5'-TCTGGGCACAACCGCAAAAATTTGCGAGCTCCAGATTTCC-3'；
BIP：5'-AATCCGTACGATCGAGCTGGACACACGCCACGTCTGCT-3'；
LB：5'-GGAAAGACCATCAAGCTCCAGAT-3'；
Phytophthora megaspermathe primer of (a) comprises:
F3：5'-GCTCTGCTCTTCCGACTTG-3'；
B3：5'-GTTAGTTTCGTCCACGGCA-3'；
FIP：5'-CGGGCAAGAGCAACGTCAGTGTCCCATTGTGGTCCAGTAC-3'；
BIP：5'-TCCGTACGATCGAGCTGGACGAGAAGAAAGGAATGGAGGCC-3'；
LB：5'-GCAAGACCATCAAGCTCCAGATTG-3'。
2. an LAMP detection kit for phytophthora, which is characterized in that: at least comprises a primer solution and an LAMP reaction solution with more than one dosage; wherein, the primer in the primer solution is the LAMP detection primer for the 8 kinds of phytophthora as described in claim 1, and the LAMP reaction solution contains dNTPs, tris-HCl, KCl, (NH) 4 ) 2 SO 4 、MgSO 4 Triton X-100, bst DNA polymerase, hydroxynaphthol blue.
3. The LAMP detection kit for Phytophthora as set forth in claim 2, for use in detecting Phytophthora.
4. High-throughput detection of 5 epidemic diseasesA molecular detection method for mold, which is characterized in that: the method comprises the steps of extracting DNA of a microorganism to be detected, taking the extracted DNA as a template, and simultaneously carrying out high-throughput detection on the microorganism to be detected by combining 5 phytophthora LAMP detection primers and a 96-well plate; observing the color change of the LAMP reaction solution, wherein sky blue indicates that the detection result is positive and target phytophthora exists; purple indicates that the detection result is negative and the target phytophthora does not exist; the LAMP detection primer for 5 kinds of phytophthora comprisesPhytophthora parasitica、Phytophthora tentaculata、 Phytophthora fragariae、Phytophthora capsiciAndPhytophthora megaspermathe LAMP detection primer of each phytophthora consists of a forward inner primer FIP, a reverse inner primer BIP, a forward outer primer F3, a reverse outer primer B3, a forward loop primer LF and a reverse loop primer LB, and the sequences of the primers are as follows:
Phytophthora parasiticathe primer of (2) comprises:
F3：5'-CTTTGTAAGTGCCACCATAC-3'；
B3：5'-ACAGACACACACGTGATT-3'；
FIP：5'-TCGATCGTACGGATTTTCTGCAGAAAGATCCAGGTTTTCATCAGG-3'；
BIP：5'-AGACCATCAAGCTCCAGATTGTACGTGGTACATCGGTTAGTTGAA-3'；
LB：5'-GACTTGTATCACTGCAGTTT-3'；
Phytophthora tentaculatathe primer of (2) comprises:
F3：5'-TGCCTTATAGGAATAGCGC-3'；
B3：5'-AGCATAAGTGAATTGACCCA-3'；
FIP：5'-ATCGTACGGATTTTCTGAGCAAAGTAGATCCCGATTTCCATCAG-3'；
BIP：5'-TGGACGGCAAGACCATCAAGTCCGTTAGTTAAATAAATACCTCGA-3'；
LB：5'-CTCCAGATTGTACGTCCTTCGT-3'；
Phytophthora fragariaethe primer of (a) comprises:
F3：5'-TGAGTGCTAGTAACTAGCCT-3'；
B3：5'-ACCAGTTAGCTCCATGAAGC-3'；
FIP：5'-CTGTGCACAACAACGAGCACGAATTCCTAGGTCCAAAAAGGC-3'；
BIP：5'-AATTCGCACGATCGAGCTGGATTAGCCGGCGAAATGTTCC-3'；
LF：5'-CAGCAATCGGAGAGCAAATCTTA-3'；
LB：5'-CGGCAAGACTATCAAGCTCCAGA-3'；
Phytophthora capsicithe primer of (a) comprises:
F3：5'-CTCTGTTGTATAGCAGAGGT-3'；
B3：5'-GCACAAGACAATTAGCACAAT-3'；
FIP：5'-TTCTGGGCGCGTACACAAACTTAGTGAGGGACAATTTATATCAGG-3'；
BIP：5'-GATCGAGTTGGACGGCAAGATCCAGTGCTCTAACTAAAACG-3'；
LB：5'-CCATCAAGCTCCAGATTGTAAGC-3'；
Phytophthora megaspermathe primer of (a) comprises:
F3：5'-GCTCTGCTCTTCCGACTTG-3'；
B3：5'-GTTAGTTTCGTCCACGGCA-3'；
FIP：5'-CGGGCAAGAGCAACGTCAGTGTCCCATTGTGGTCCAGTAC-3'；
BIP：5'-TCCGTACGATCGAGCTGGACGAGAAGAAAGGAATGGAGGCC-3'；
LB：5'-GCAAGACCATCAAGCTCCAGATTG-3'。
5. an LAMP detection kit for phytophthora, which is characterized in that: the method comprises at least more than one time of primer solution and LAMP reaction solution; wherein, the primer in the primer solution is the LAMP detection primer for 5 kinds of phytophthora as described in claim 4, and the LAMP reaction solution contains dNTPs, tris-HCl, KCl, (NH) 4 ) 2 SO 4 、MgSO 4 Triton X-100, bst DNA polymerase, hydroxynaphthol blue.
6. The application of the LAMP detection kit for Phytophthora of claim 5 in the detection of Phytophthora.
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