CN106399490B - LAMP primer group for detecting phytoplasma and kit and application thereof - Google Patents
LAMP primer group for detecting phytoplasma and kit and application thereof Download PDFInfo
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Abstract
The invention discloses an LAMP primer group for detecting phytoplasma and a kit and application thereof. According to the invention, 6 groups and 2 groups of primers are respectively designed aiming at 6 regions of the conserved sequences of 16S genes and tuf genes of 16SrI group phytoplasmas, and finally, the LAMP primer group with strong specificity and consisting of nucleotide sequences shown by SEQ ID No.2, SEQ ID No.3, SEQ ID No.4 and SEQ ID No.5 is obtained. The invention further discloses a kit for paulownia arbuscular, chinaberry arbuscular, mulberry atrophy, lettuce yellowing and catharanthus roseus greening phytoplasma prepared by using the primer group and establishes a detection method, so that the kit has the advantages of good stability, strong specificity, high sensitivity and the like, and can be applied to detection of paulownia arbuscular, chinaberry arbuscular, mulberry atrophy, lettuce yellowing and catharanthus roseus greening phytoplasma.
Description
Technical Field
The invention relates to an LAMP primer group for detecting 16SrI group phytoplasma, in particular to a method for detecting paulownia arbuscular, melia azedarach arbuscular, mulberry atrophy, lettuce yellowing and catharanthus roseus greening phytoplasma by using the LAMP primer group, and also relates to application of the LAMP primer group in early diagnosis of paulownia arbuscular, melia azedarach arbuscular, mulberry atrophy, lettuce yellowing and catharanthus roseus greening phytoplasma and monitoring and identification of pathogenic bacteria, belonging to the technical field of plant disease detection, identification and prevention and control.
Background
Phytoplasma (original name Mycoplasma-like organism for short MLO) is a kind of prokaryotic organism which is similar to plant pathogenic bacteria and has no cell wall. Can cause serious diseases of economic trees such as a plurality of important food crops, vegetables, ornamental plants, fruit trees and the like. The phytoplasmas can be divided into 28 groups according to the phytoplasma 16S rDNA gene sequence, wherein the phytoplasmas causing paulownia arbuscular, melia azedarach arbuscular, mulberry atrophy, lettuce yellowing and vinca greening all belong to the 16Sr I group. The diseases are wide in distribution range, high in propagation speed and strong in pathogenicity, and once the diseases are infected, the traditional disease control technology is difficult to cure. The paulownia is originally produced in China, has wide cultivation area in China, is an important fast-growing and high-yield timber forest, and is also an important street tree and an environment beautifying tree species. Paulownia arbuscular diseases occur in large areas in China, and the growth and development of trees are seriously influenced. Mulberry atrophy is a very dangerous disease and is distributed in Jiangsu, Zhejiang and Anhui provinces in China. The disease is increasing in recent years, particularly in the Tai lake area of the world of Jiang and Zhejiang in China, the annual incidence rate is generally 5-15%, the serious disease area reaches more than 30%, and a plurality of diseased plants die after years. Lettuce yellowing is recently reported to occur in Chinese Fujian, and the yield and quality of lettuce are seriously reduced after the disease occurs, thus causing serious loss to farmers. In order to fundamentally solve the problem of preventing and treating the diseases, the most effective measure is to establish strict plant quarantine and stop pathogeny from the source.
The detection of phytoplasma diseases mainly depends on the traditional method combining biology, electron microscope observation and antibiotic test in the early stage, and the detection method based on enzyme linked immunosorbent assay, nucleic acid hybridization and PCR technology is also established in succession along with the development of biotechnology in the later stage, so that the detection sensitivity and accuracy are continuously improved. However, these methods at present often require expensive instruments and equipment, and have complex operation procedures, long detection time and high technical requirements for detection personnel, which greatly limit the use and popularization of such diagnostic methods.
Loop-mediated isothermal amplification is a isothermal nucleic acid amplification method developed by Notomi et al in 2000, and is a novel molecular diagnostic method. The method designs specific primers aiming at 6 specific regions of a target gene, and utilizes a DNA polymerase with strand displacement activity to amplify nucleic acid at constant temperature. The method has the characteristics of strong specificity, simple operation, no need of complex instruments, short detection period and visualized results, so that the method is very suitable for being popularized and used as a disease diagnosis method of a basic level and a site.
Loop-mediated isothermal amplification is a novel molecular diagnostic method. The application of this technique to the detection of phytoplasma has been partially reported, and for example, corresponding LAMP detection methods have been developed for the detection of 16Sr I group of Severum verum and strawberry green petal phytoplasma, 16Sr II group of Parthenium argentatum, 16Sr V group of jujube witches broom and Vitis vinifera yellow phytoplasma, 16Sr XII group of Carica papaya arbuscular phytoplasma, and the like. However, the target gene for these methods is mainly the 16S rDNA gene. The 16S rDNA gene is double-copy in phytoplasma and has certain variability, while the protein elongation factor encoding gene tuf is single-copy in phytoplasma and is considered as a new evolution label. It is slightly less conserved than the 16S rDNA gene and can further distinguish subgroups based on 16S rDNA gene grouping.
Therefore, the tuf gene is used as a target gene, and the LAMP primer with better specificity is designed aiming at phytoplasmas such as paulownia arbuscular, melia azedarach arbuscular, mulberry atrophy, lettuce yellowing and catharanthus roseus greening, so that the LAMP primer has very important significance for early diagnosis of the phytoplasmas such as paulownia arbuscular, melia azedarach arbuscular, mulberry atrophy, lettuce yellowing and catharanthus roseus greening and monitoring and identification of pathogenic bacteria.
Disclosure of Invention
One of the objects of the present invention is to provide a LAMP primer set for paulownia arbuscular, melia azedarach arbuscular, mulberry atrophy, lettuce yellowing and vinca green-changing phytoplasma;
the other purpose of the invention is to provide an LAMP detection method for paulownia arbuscular, chinaberry arbuscular, mulberry atrophy, lettuce yellowing and vinca green phytoplasma;
the third purpose of the invention is to provide an LAMP detection kit for paulownia arbuscular, chinaberry arbuscular, mulberry atrophy, lettuce yellowing and vinca green phytoplasma;
the fourth purpose of the invention is to provide an LAMP primer group and application of the detection method thereof in early diagnosis of paulownia arbuscular, chinaberry arbuscular, mulberry atrophy, lettuce yellowing and vinca green phytoplasma and monitoring and identification of pathogenic bacteria.
In order to achieve the purpose, the invention is realized by the following technical scheme:
the invention firstly discloses an LAMP primer group for detecting paulownia arbuscular, chinaberry arb arbuscular, mulberry atrophy, lettuce yellowing and vinca green change phytoplasma, which comprises a pair of inner primer pairs and a pair of outer primer pairs, wherein the outer primer pairs (PaWB3-F3/PaWB3-B3) consist of nucleotide sequences shown in SEQ ID No.2 and SEQ ID No. 3; the inner primer pair (PaWB3-FIP/PaWB3-BIP) consists of nucleotide sequences shown in SEQ ID No.4 and SEQ ID No. 5.
The invention also discloses an LAMP kit for detecting paulownia arbuscular, chinaberry arbuscular, mulberry atrophy, lettuce yellowing and vinca green change phytoplasma, which comprises the following components in parts by weight: the kit comprises LAMP primers, LAMP reaction liquid RM, Bst DNA polymerase, fluorescent dye 10 xSYBRGreen I, DNA of a sample to be detected, positive and negative controls, ultrapure water and sealing liquid, wherein the LAMP primers are the LAMP primer group.
The invention further discloses a detection method of the LAMP primer group in paulownia arbuscular, chinaberry arbuscular, mulberry atrophy, lettuce yellowing and vinca green phytoplasma, which comprises the following steps:
(1) extracting DNA of a sample to be detected; (2) taking the DNA of a sample to be detected as a template, and establishing an LAMP reaction system by taking the LAMP primer group as an amplification primer to carry out isothermal gene amplification; (3) and judging the result of the amplification product.
Wherein, the LAMP reaction system comprises: an outer primer pair consisting of nucleotide sequences shown by SEQ ID No.2 and SEQ ID No.3 and a primer group consisting of an inner primer pair consisting of nucleotide sequences shown by SEQ ID No.4 and SEQ ID No.5, LAMP reaction liquid RM, Bst DNA polymerase, fluorescent dye 10 × SYBR Green I, sample DNA to be detected, ultrapure water and sealing liquid;
preferably, the LAMP reaction system is 45 μ L, comprising: 10 μ M PaWB 3-F30.5 μ L, 10 μ M PaWB 3-B30.5 μ L, 40 μ M PaWB3-FIP 1 μ L, 40 μ M PaWB3-BIP 1 μ L, LAMP reaction solution RM (2X) 12.5 μ L, 8U/μ L BstDNA polymerase 1 μ L, fluorescent dye 10 × SYBR Green I0.5 μ L, test sample DNA 0.5 μ L, ultrapure water 7.5 μ L and 20 μ L sealing solution.
The isothermal gene amplification is carried out at the constant temperature of 63 ℃ for 40-60 min, and inactivation is carried out at 80 ℃ for 5-10 min; preferably, the reaction is carried out at constant temperature of 63 ℃ for 60min, and inactivation is carried out at 80 ℃ for 5 min.
And (3) judging the result of the amplification product: placing the amplified product in a constant-temperature fluorescence detector or a fluorescence PCR instrument, reading a fluorescence signal in real time, judging that the sample is positive reaction if an S-type amplification curve appears, namely the sample is infected with phytoplasma, and judging that the sample is negative if no S-type amplification curve exists; or, adding 1 μ L of a color developing solution to the amplification product, wherein the color developing solution is a mixed solution of 1.25mM of calcein and 12.5mM of manganese chloride, observing the color change in the reaction tube by naked eyes under sunlight, judging that the amplification product is a positive reaction if the amplification product is changed into emerald green, namely that the sample is infected with phytoplasma, and judging that the amplification product is negative if the amplification product is still brownish yellow.
The invention collects the nucleic acid sequences of different groups of phytoplasma 16S genes and tuf genes in GeneBank, and uses DNAMAN software to compare and similarity the sequencesAnd (2) comparing the gene difference sites and highly conserved regions of paulownia arbuscular, chinaberry arbuscular, mulberry atrophy, lettuce yellowing and vinca rosea greening (16Sr I group) phytoplasma and other groups of phytoplasma, and designing specific primers aiming at 6 regions of conserved sequences of 16S genes and tuf genes (consisting of nucleotide sequences shown in SEQ ID No. 1) of the 16SrI group phytoplasma by utilizing LAMP Primer online design software Primer Explorer V4. 6 sets of primer groups (I-VI) are designed for the 16S gene of the phytoplasma, two sets of primer groups (seventy and eighty) are designed for the tuf gene, isothermal gene amplification is carried out by utilizing the primer groups, the I group and the eighty group can not realize amplification from the amplification result, and the negative control of the III group also shows amplification. Thus discarding the group I, group III and group VIII primers. Carrying out specificity analysis on the primers of the group IV, the group V, the group VI and the group seventeen, and meanwhile, carrying out disease onset and healthy tissue samples of paulownia fortunei branches, chinaberry branches, mulberry atrophy, vinca greenchange and lettuce yellowing of the group 16 SI; 16 sII group of diseased and healthy tissue samples of peanut, sweet potato and swiss chard; 16 Sv group of diseased and healthy tissue samples of jujube witches broom, locust tree branches, cherry induced death and yellowing and hardy anomala branches; a 16SXIV group of chestnut yellowing onset and health tissue sample; and with sterilization of ddH2And O is used as a negative control, and specific tests of loop-mediated isothermal amplification of paulownia arbuscular, chinaberry arbuscular, mulberry tree atrophy, vinca greening and lettuce yellows phytoplasma are carried out, and the results show that the amplification reactions of the primers in the IV group, the V group and the VI group are extremely sensitive, and all samples are amplified and comprise diseased samples and healthy controls. In addition, from multiple replicates of primer design for the phytoplasma 16S gene, it was shown that: since the results were extremely unstable and contamination of healthy controls was very likely to occur, primers designed for the 16S gene were discarded. The diseased tissue samples of Paulownia fortunei, Chinaberry fortunes, mulberry atrophy, vinca greening and lettuce yellowing of which the primer group is only 16Sr I are shown as positive reaction, an S-shaped amplification curve appears in a fluorescence detector, the color is changed into verdant green by visual observation, and peanut tress, sweet potato tress, stinky vegetable tress, jujube witches broom, locust tree tress, cherry yellowing, sun-cured wood tress, Chinese chestnut yellowing and all health controls of different genera are all positive reactionsAnd (4) negativity. Therefore, the loop-mediated isothermal amplification detection method established by the primer set (the outer primer pair consisting of the nucleotide sequences shown by SEQ ID No.2 and SEQ ID No.3 and the inner primer pair consisting of the nucleotide sequences shown by SEQ ID No.4 and SEQ ID No. 5) has stronger specificity on the paulownia fortunei, melia azedarach, mulberry atrophy, vinca greenchange and lettuce yellowing of the 16Sr group, can better distinguish phytoplasmas among different groups, and can be used for detecting the paulownia fortunei, melia mulberry atrophy, vinca greenchange and lettuce yellowing phytoplasmas of the 16Sr group.
The invention carries out detection and inspection on the stability and sensitivity of the loop-mediated isothermal amplification detection method for paulownia arbuscular, chinaberry arbuscular, mulberry atrophy, lettuce yellowing and vinca greenchange plants established by the LAMP primer group (an outer primer pair consisting of nucleotide sequences shown by SEQ ID No.2 and SEQ ID No.3 and an inner primer pair consisting of nucleotide sequences shown by SEQ ID No.4 and SEQ ID No. 5).
The stability detection shows that: samples from different places with paulownia witches broom can show positive reaction, an S-shaped amplification curve appears in a fluorescence detector, and the visible color is changed into emerald green by visual observation, so that the loop-mediated isothermal amplification detection method established by the invention has better stability.
The sensitivity test shows that: routine PCR is carried out by detecting sample after being diluted to 2% by agarose gel electrophoresis-10The detection limit is reached; the loop-mediated isothermal amplification detection method is diluted to 2-13The detection result is still positive (S-shaped amplification curve appears in a fluorescence detector, and the visible color changes into emerald green when observed by naked eyes). Therefore, the sensitivity of the loop-mediated isothermal amplification detection method established by the invention is 8 times higher than that of the conventional PCR.
The invention also discloses application of the LAMP primer group in early diagnosis of paulownia arbuscular, chinaberry arbuscular, mulberry atrophy, lettuce yellowing and vinca green phytoplasma and monitoring and identification of pathogenic bacteria.
Compared with the prior art, the technical scheme of the invention has the following beneficial effects:
(1) fast and efficient: the whole amplification can be completed only in 40-60 min.
(2) The operation is simple and convenient: the detection can be completed only by a constant temperature instrument without complicated instruments, special reagents, steps such as denaturation of double-stranded DNA and the like.
(3) High specificity: the specific primers are designed for 6 different regions in the original tuf gene, have strong specificity, and cannot amplify the jujube witches broom of 16Sr V group, the peanut witches broom of 16Sr II group, the Chinese chestnut yellowing of 16Sr XIV group and the like.
(4) The sensitivity is high: compared with the common PCR, the detection sensitivity is improved by more than 8 times.
In a word, the LAMP isothermal amplification technology is applied to detection of paulownia arbuscular, chinaberry arbuscular, mulberry atrophy, lettuce yellowing and vinca green phytoplasma, has important significance on early warning of diseases, pathogen monitoring of epidemic areas and the like, can avoid high instrument investment, and is suitable for basic popularization and use.
Definitions of terms to which the invention relates
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The term "primer" means a small piece of single-stranded DNA that serves as a starting point for DNA replication and serves as a starting point for extension of each polynucleotide strand during a nucleic acid synthesis reaction.
The term "polymerase chain reaction", PCR for short, means a molecular biology technique for amplifying and amplifying a specific DNA fragment, wherein the PCR is performed by using the principle that DNA becomes single-stranded at high temperature of 95 ℃ in vitro, a primer is combined with the single-stranded at low temperature (usually about 60 ℃) according to the base complementary pairing principle, the temperature is adjusted to the optimal reaction temperature (about 72 ℃) of DNA polymerase, and the DNA polymerase synthesizes a complementary strand along the direction from phosphate to pentose (5'→ 3').
The term "loop-mediated isothermal amplification method", LAMP for short, is a novel isothermal nucleic acid amplification method developed in 2000, and is characterized in that 4 specific primers are designed for 6 regions of a target gene, and a strand displacement DNA polymerase is utilized to carry out heat preservation for 30-60 minutes under an isothermal condition (about 63 ℃) so as to complete the nucleic acid amplification reaction.
Drawings
FIG. 1 shows the real-time fluorescence detection results of loop-mediated isothermal amplification products of Paulownia witches broom phytoplasma: wherein the positive is a self-contained positive control of the LAMP amplification kit, the negative is a self-contained negative control of the LAMP amplification kit, PaWB is a paulownia witches broom sample, and PH is a healthy paulownia sample;
FIG. 2 shows the result of color reaction detection of loop-mediated isothermal amplification products of Paulownia witches broom phytoplasma: wherein the positive is a self-contained positive control of the LAMP amplification kit, the negative is a self-contained negative control of the LAMP amplification kit, PaWB is a paulownia witches broom sample, and PH is a healthy paulownia sample;
FIG. 3 shows the real-time fluorescence detection results of LAMP amplification products of the primer sets (I-VI) and (c) and (b);
FIG. 4 is the sequence alignment of group II primers; wherein A is the sequence alignment result of the primer PaWBII-F3; b is the sequence alignment result of the primer PaWBII-B3;
FIG. 5 shows the sequence alignment of group IV primers; wherein A is the sequence comparison result of the primer PaWB IV-F3; b is the sequence alignment result of the primer PaWB IV-B3;
FIG. 6 shows the real-time fluorescence detection results of LAMP amplification specificity verification for the primer set IV;
FIG. 7 shows the alignment of the sequences of the primers in group V; wherein A is the sequence alignment result of the primer PaWBV-F3; b is the sequence alignment result of the primer PaWBV-B3;
FIG. 8 is a real-time fluorescence detection result of LAMP amplification specificity verification of the primer group V;
FIG. 9 shows the results of sequence alignment of group VI primers; wherein A is the sequence alignment result of the primer PaWBVI-F3; b is the sequence alignment result of the primer PaWBVI-B3;
FIG. 10 shows the real-time fluorescence detection results of LAMP amplification specificity verification for primer group VI;
FIG. 11 shows the alignment of the primers; wherein A is the sequence alignment result of the primer PaWBVI-F3; b is the sequence alignment result of the primer PaWBVI-B3;
FIG. 12 shows the real-time fluorescence detection result of LAMP amplification specificity verification for primer set (c);
FIG. 13 shows the LAMP amplification specificity verification color reaction detection results for the primer set (c): wherein, 1: paulownia twigs, 2: melia azedarach branchlet, 3: mulberry atrophy, 4: vinca greening, 5: lettuce yellowing, 6: peanut branchy, 7: sweet potato twigs, 8: cleft arrow vegetable clump, 9: jujube witches broom, 10: branches of Chongyang wood, 11: cherry lethal yellowing, 12: maackia amurensis branches, 13: chinese chestnut yellowing, 14: healthy paulownia, 15: healthy neem, 16: healthy mulberry, 17: healthy vinca, 18: healthy lettuce, 19: healthy peanuts, 20: healthy sweet potato, 21: healthy cleft arrow vegetable, 22: healthy jujube tree, 23: healthy yang-qi wood, 24: healthy cherries, 25: healthy locust tree, 26: healthy chestnuts, CK: blank control;
FIG. 14 is the real-time fluorescence detection results of LAMP amplification stability verification: wherein, the positive is the self-contained positive control of the LAMP amplification kit, the negative is the self-contained negative control of the LAMP amplification kit, and 28: sample of paulownia fortunei bush disease, 84: samples of paulownia witches broom disease in Fujian province, 95: liaoning big paulownia witches broom sample, 96: sample of paulownia witches broom bayberry branch disease, 97: guizhou Guiyang paulownia arbuscular disease sample, 98: kalopanax fortunei witches broom sample in Henan, pH: is a healthy paulownia sample;
FIG. 15 shows the result of color reaction detection for LAMP amplification stability verification: wherein, the positive is the self-contained positive control of the LAMP amplification kit, the negative is the self-contained negative control of the LAMP amplification kit, and 28: sample of paulownia fortunei bush disease, 84: samples of paulownia witches broom disease in Fujian province, 95: liaoning big paulownia witches broom sample, 96: sample of paulownia witches broom bayberry branch disease, 97: guizhou Guiyang paulownia arbuscular disease sample, 98: kalopanax fortunei witches broom sample in Henan, pH: is a healthy paulownia sample;
FIG. 16 shows the detection sensitivity of conventional PCR 1% agarose gel electrophoresis: wherein, the lane M is 4500bp DNA marker, and the other lanes are amplification conditions adopting double-gradient diluted DNA as a template;
FIG. 17 shows the real-time fluorescence detection results of the LAMP amplification sensitivity test: wherein-10 to-18 is the amplification condition using double gradient diluted DNA as template, CK is sterilized ddH2O control;
FIG. 18 shows the results of color reaction detection in the LAMP amplification sensitivity test: therein, 2-10～2-18For amplification using double-gradient diluted DNA as template, CK is sterilized ddH2And (4) performing O control.
Detailed Description
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. It is to be understood that the described embodiments are exemplary only and are not limiting upon the scope of the invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.
1. Sources of reagents
Plant genome extraction Kit (Plant Genomic DNA Kit) was purchased from Edley Biotech, Inc.;
the nucleic acid amplification kit is purchased from Guangzhou Diaoao Biotechnology Co., Ltd;
the 2 XPCR premix was purchased from Bomaide Biotech Ltd.
Example 1 establishment of Loop-mediated isothermal amplification (LAMP) Rapid detection method for Paulownia witches broom phytoplasma
1. Extraction of plant Total DNA
The total DNA of the Plant to be tested is extracted by a Plant genome extraction Kit (Plant Genomic DNA Kit, Edley Biotechnology Co., Ltd.), and is stored in a refrigerator at-20 ℃ for later use.
LAMP amplification
Reaction system: total volume 45 μ L, containing: mu.L of PaWB 3-F30.5. mu.L, PaWB 3-B30.5. mu.L, PaWB3-FIP 1. mu.L, PaWB3-BIP 1. mu.L, LAMP reaction mixture RM (2X) 12.5. mu.L, 8U/. mu.LBst DNA polymerase 1. mu.L, fluorescent dye 10 XSSYBR Green I0.5. mu.L, sample DNA to be tested 0.5. mu.L, and the amount of the sample DNA to be tested was adjusted to 25. mu.L with ultrapure water and 20. mu.L of the sealing solution was added.
Reaction conditions are as follows: mixing the prepared reaction tubes, centrifuging, reacting at 63 deg.C for 60min, and inactivating at 80 deg.C for 5 min.
3. Determination of amplification results
And (3) setting a negative control and a positive control for each detection, and when the negative control and the positive control are both established, the whole experiment is effective and the result can be judged.
Judging the LAMP reaction result: as shown in FIG. 1, the reaction tube is placed in a constant temperature fluorescence detector or a fluorescence PCR instrument, and a fluorescence signal is read in real time, and if an "S" -type amplification curve appears, the reaction tube is judged to be positive, namely, the sample is infected with the phytoplasma, and if the "S" -type amplification curve does not appear, the reaction tube is judged to be negative. Alternatively, the color change in the reaction tube was visually observed (FIG. 2), and after completion of the reaction, 1. mu.L of a color-developing solution was added to the reaction mixture, and when the amplification product became emerald green, a positive reaction was judged, that is, when the sample was infected with phytoplasma, and when the amplification product remained brown yellow, a negative reaction was judged. The result is consistent with the common PCR detection result, and the data of the method is proved to be reliable.
Experimental example 2 preparation of primers and preliminary screening
1. Experimental methods
1.1 primer design and Synthesis
The nucleic acid sequences of the 16S gene and the tuf gene of different groups of phytoplasmas are collected in GeneBank, DNAMAN software is used for comparing and comparing similarity of the sequences to find out gene difference sites and highly conserved regions of paulownia arbuscular, melia azedarach arbuscular, mulberry atrophy, lettuce yellowing and vinca green change (16Sr I group) phytoplasma and other groups of phytoplasma, and LAMP Primer online design software Primer Explorer V4 is used for designing specific primers aiming at 6 regions of the conserved sequences of the 16S gene and the tuf gene of the 16SrI group of phytoplasma. Wherein 6 sets of primer sets (I-VI) are designed aiming at the phytoplasma 16S gene, two sets of primer sets (seventy and eighty) are designed aiming at the tuf gene, the result is shown in table 1, and the primers are synthesized by Shanghai bio-engineering company according to the HPLC purity level.
TABLE 1 PCR primer sequences
1.2 preliminary screening of primers
1.2.1 extraction of Total DNA from plants
Refer to example 1.
1.2.2LAMP amplification
Refer to example 1.
1.2.3 determination of amplification results
Refer to example 1.
2. Results of the experiment
The amplification results are shown in FIG. 3, from which it can be seen that: group I and group VIII did not amplify, and group III negative controls also amplified. Thus discarding the group I, group III and group VIII primers. While the primers of group II, group IV, group V, group VI and group seventeen all have positive amplification, and the negative control has no amplification.
Example 3 specificity test
1. Test method
The loop-mediated isothermal amplification reaction system established in the above example 1 was used to simultaneously treat the onset and healthy tissue samples of paulownia arbuscular, melia azedarach arbuscular, mulberry atrophy, vinca greening and lettuce yellowing of 16 SI group; 16 sII group of diseased and healthy tissue samples of peanut, sweet potato and swiss chard; 16 Sv group of diseased and healthy tissue samples of jujube witches broom, locust tree branches, cherry induced death and yellowing and hardy anomala branches; a 16SXIV group of chestnut yellowing onset and health tissue sample; and with sterilization of ddH2O as a negative control, specific assays were performed for paulownia arbuscular, melia azedarach, mulberry atrophy, vinca greening, and lettuce yellows phytoplasma loop-mediated isothermal amplification.
Respectively extracting the pathogenic and healthy tissue samples of 16 SI group paulownia arbuscular, chinaberry arbuscular, mulberry tree atrophy, vinca greening and lettuce yellowing; 16 Spanish group of peanutsDiseased and healthy tissue samples of potato and swiss chard branchlets; 16 Sv group of diseased and healthy tissue samples of jujube witches broom, locust tree branches, cherry induced death and yellowing and hardy anomala branches; 16SXIV group of Chinese chestnut yellowing onset and healthy tissue sample plant total DNA and sterilized ddH2And O is used as a template.
The loop-mediated isothermal amplification reaction was carried out according to the reaction system and reaction conditions provided in example 1 above. The primers used were respectively: group IV, group V, group VI and group seventeen primers.
The result determination was performed according to the LAMP amplification result determination method provided in example 1 above.
2. Test results
The results are shown in fig. 6, 8 and 10 for group IV (alignment of primer sequences is shown in fig. 5), group V (alignment of primer sequences is shown in fig. 7) and group VI primers (alignment of primer sequences is shown in fig. 9), and can be seen from the figures: the amplification reaction was extremely sensitive, with amplification occurring in all samples, including diseased samples and healthy controls.
In addition, from multiple replicates of primer design for the phytoplasma 16S gene, it was shown that: since the results were extremely unstable and contamination of healthy controls was very likely to occur, primers designed for the 16S gene were discarded.
For the primer of group (alignment of primer sequences is shown in FIG. 11), the results are shown in FIGS. 12 and 13, from which it can be seen that: only the pathogenic tissue samples of 16Sr I group paulownia boughs, chinaberry boughs, mulberry atrophy, vinca greening and lettuce yellowing show positive reaction, an S-shaped amplification curve appears in a fluorescence detector, and the visible color is changed into emerald green by visual observation, while peanut branchy branches, sweet potato branchy branches, osmyl vegetable branchy branches, jujube witches broom, locust tree branchy branches, cherry dying and yellowing, sun-cured wood branchy branches, Chinese chestnut yellowing and all health controls of different genera are negative, therefore, the loop-mediated isothermal amplification detection method established by the primer set of the invention has stronger specificity on paulownia arbuscular, melia azedarach arbuscular, mulberry atrophy, vinca greening and lettuce yellowing of 16Sr I subgroups, can better distinguish phytoplasmas among different groups, can be used for detecting paulownia arbuscular, chinaberry arbuscular, mulberry tree atrophy, vinca greening disease and lettuce yellows phytoplasma in 16Sr I group.
Example 4 stability test
1. Test method
According to the loop-mediated isothermal amplification reaction system established in the above example 1, the stability test of loop-mediated isothermal amplification of paulownia arbuscular mycosis is performed on samples from different areas with paulownia fortunei bush diseases and with healthy paulownia as negative control.
Respectively extracting the total DNA of plant tissues of paulownia fortunei and paulownia fortunei bush disease onset samples from different areas as templates.
The loop-mediated isothermal amplification reaction was carried out according to the reaction system and reaction conditions provided in example 1 above.
The result determination was performed according to the LAMP amplification result determination method provided in example 1 above.
2. Test results
The judgment results are shown in fig. 14 and fig. 15, samples from different places with paulownia witches broom can show positive reaction, an S-shaped amplification curve appears in a fluorescence detector, and the visible color is changed into emerald green by visual observation, so that the loop-mediated isothermal amplification detection method established by the invention has better stability.
Example 5 sensitivity test
1. Test method
Extracting total DNA of leaves of a Plant suffering from the paulownia witches broom disease by using a Plant genome extraction Kit (Plant Genomic DNA Kit, Edley Biotech Co., Ltd.), and sterilizing ddH (ddH) by using the total DNA as a template2O was diluted in two-fold gradient, and subjected to loop-mediated isothermal amplification according to the reaction system and reaction conditions described above in example 2, and simultaneously subjected to conventional PCR for sensitivity comparison.
And (3) conventional PCR detection: PCR amplification was performed using DNA diluted in two-fold gradients as template with the phytoplasma universal primers R16mF2/R16mR 1. The conventional PCR amplification system was 25. mu.L, the amplification reaction system contained 1. mu.L of the prepared DNA template, 0.5. mu.L (10. mu.M) of each forward and reverse primer, and 2 XPCR premix (0.05U/. mu.L of DNA polymerase, 4mM MgCl2And 0.4mM dNTPs) 12.5. mu.L, ddH2And O is supplemented to 25 mu L. Reaction procedure: 5min at 94 ℃; at 94 ℃ for 45s, at 52 ℃ for 45s and at 72 ℃ for 1min, for 35 cycles; extension at 72 ℃ for 10 min. The PCR product was detected by electrophoresis on a 1% agarose gel.
2. Test results
The results of conventional PCR detection by 1% agarose gel electrophoresis are shown in FIG. 16, where the sample is diluted to 2-10The detection limit is reached. The loop-mediated isothermal amplification detection method is diluted to 2 as shown in FIG. 17 and FIG. 18-13The detection result is still positive (S-shaped amplification curve appears in a fluorescence detector, and the visible color changes into emerald green when observed by naked eyes). Therefore, the sensitivity of the loop-mediated isothermal amplification detection method established by the invention is 8 times higher than that of the conventional PCR.
Claims (10)
1. The LAMP primer group for detecting paulownia arbuscular, chinaberry arbuscular, mulberry atrophy, lettuce yellowing and vinca green change phytoplasma is characterized in that: consists of a pair of inner primer pairs and outer primer pairs;
wherein, the outer primer pair consists of nucleotide sequences shown as SEQ ID No.2 and SEQ ID No. 3; the inner primer pair consists of nucleotide sequences shown by SEQ ID No.4 and SEQ ID No. 5.
2. A LAMP kit for detecting paulownia arbuscular, chinaberry arbuscular, mulberry atrophy, lettuce yellowing and vinca green change phytoplasma comprises: LAMP primer, LAMP reaction liquid RM, Bst DNA polymerase, fluorescent dye 10 XSSYBR Green I, sample DNA to be detected, positive control, negative control, ultrapure water and sealing liquid, and is characterized in that: the LAMP primer is the LAMP primer group of claim 1.
3. A method for detecting Paulownia witches, Melia azedarach witches, Mulberry atrophy, lettuce yellowing and Catharanthus roseus greening phytoplasma by using the LAMP kit according to claim 2, comprising the steps of:
(1) extracting DNA of a sample to be detected; (2) establishing an LAMP reaction system for isothermal gene amplification by using DNA of a sample to be detected as a template and the LAMP primer group of claim 1 as an amplification primer; (3) the result of the amplification product is judged.
4. A method according to claim 3, characterized by: the LAMP reaction system comprises: the LAMP primer set according to claim 1, the LAMP reaction solution RM, Bst DNA polymerase, fluorescent dye 10 × SYBR Green I, sample DNA to be detected, ultrapure water and sealing liquid.
5. The method of claim 4, wherein: the LAMP reaction system is 45 mu L and comprises: mu.L of each of the primers shown in SEQ ID No.2 and SEQ ID No.3 at 10. mu.M, 1. mu.L of each of the primers shown in SEQ ID No.4 and SEQ ID No.5 at 40. mu.M, 12.5. mu.L of 2 × LAMP reaction solution RM at 8U/. mu.L Bst 1 mu L of DNA polymerase, 0.5 mu L of fluorescent dye 10 XSSYBR Green I, 0.5 mu L of sample DNA to be detected, 7.5 mu L of ultrapure water and 20 mu L of sealing liquid.
6. The method of claim 3, wherein the isothermal gene amplification is isothermal reaction at 63 ℃ for 40 ~ 60min and inactivation at 80 ℃ for 5 ~ 10 min.
7. The method of claim 6, wherein: the isothermal gene amplification is constant temperature reaction at 63 ℃ for 60min and inactivation at 80 ℃ for 5 min.
8. A method according to claim 3, characterized by: the step (3) of determining the result of the amplification product: placing the amplified product in a constant-temperature fluorescence detector or a fluorescence PCR instrument, reading a fluorescence signal in real time, judging that the sample is positive reaction if an S-type amplification curve appears, namely the sample is infected with phytoplasma, and judging that the sample is negative if no S-type amplification curve exists; or, the color developing solution in the amplification product is used to visually observe the color change in the reaction tube under the sunlight, if the amplification product is changed into emerald green, the amplification product is judged to be positive reaction, namely, the sample is infected with phytoplasma, and if the amplification product is still brown yellow, the amplification product is judged to be negative.
9. The LAMP primer set of claim 1, for use in early diagnosis of Paulownia fortunei, Melia azedarach, Mulberry atrophy, lettuce yellowing and Catharanthus roseus greening phytoplasma and monitoring and identification of pathogenic bacteria.
10. The LAMP kit of claim 2, for use in early diagnosis of Paulownia fortunei, Melia azedarach, Mulberry atrophy, lettuce yellowing and Catharanthus roseus phyroplasma and in monitoring and identification of pathogenic bacteria.
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| CN106755601A (en) * | 2017-03-07 | 2017-05-31 | 江苏省农业科学院 | Using RT-LAMP method quick detection MRDV cause of diseases |
| CN109652503B (en) * | 2019-02-02 | 2019-12-03 | 云南省农业科学院甘蔗研究所 | A kind of PCR-RFLP discrimination method of Sugarcane white leaf phytoplasma difference subgroup |
| CN111088391A (en) * | 2020-02-25 | 2020-05-01 | 中国热带农业科学院椰子研究所 | LAMP primer group for detecting 16SrI group betelnut etiolating phytoplasma in China, kit and application |
| CN111850151B (en) * | 2020-08-18 | 2021-05-07 | 中国热带农业科学院椰子研究所 | Betel nut yellowed phytoplasma micro-drop type digital PCR detection kit, method and application |
| CN112210614B (en) * | 2020-11-05 | 2021-07-30 | 中国热带农业科学院环境与植物保护研究所 | Real-time fluorescent quantitative LAMP (Loop-mediated isothermal amplification) primer group for detecting purple sisal leaf roll disease phytoplasma and application thereof |
| KR102496891B1 (en) * | 2021-03-08 | 2023-02-07 | 대한민국 | Primer set for loop-mediated isothermal amplification reaction for phytoplasma detection |
| CN113444819A (en) * | 2021-06-04 | 2021-09-28 | 中国热带农业科学院湛江实验站 | Primer, method and application for detecting peanut arbuscular phytoplasma |
| CN113999924A (en) * | 2021-11-29 | 2022-02-01 | 菏泽学院 | Primer, kit and method for amplification and typing of thyA gene of paulownia arbuscular phytoplasma |
| CN114085919B (en) * | 2021-12-17 | 2022-08-23 | 中国热带农业科学院椰子研究所 | Coconut lethal phytoplasma universal rapid visual detection kit and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101691616A (en) * | 2009-09-03 | 2010-04-07 | 湖州市农业科学研究院 | Method for detecting pathogen of shrinking disease of mulberry floral leaves |
| CN102492773A (en) * | 2011-12-07 | 2012-06-13 | 中国检验检疫科学研究院 | Chip for screening 16SrI group phytoplasmas and application thereof |
| CN102776295A (en) * | 2012-08-03 | 2012-11-14 | 中国农业大学 | Kit and method for detecting TYLCV (tomato yellow leaf curl virus) carried by tomato seedlings |
| CN103497998A (en) * | 2013-09-30 | 2014-01-08 | 新疆农业大学 | Loop-mediated isothermal amplification (LAMP) rapid detection method of jujube witches broom phytoplasma |
| CN103525923A (en) * | 2013-09-30 | 2014-01-22 | 新疆农业大学 | Primer, probe and method for qualitatively and quantitatively detecting witches' broom phytoplasma |
-
2016
- 2016-09-04 CN CN201610801184.0A patent/CN106399490B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101691616A (en) * | 2009-09-03 | 2010-04-07 | 湖州市农业科学研究院 | Method for detecting pathogen of shrinking disease of mulberry floral leaves |
| CN102492773A (en) * | 2011-12-07 | 2012-06-13 | 中国检验检疫科学研究院 | Chip for screening 16SrI group phytoplasmas and application thereof |
| CN102776295A (en) * | 2012-08-03 | 2012-11-14 | 中国农业大学 | Kit and method for detecting TYLCV (tomato yellow leaf curl virus) carried by tomato seedlings |
| CN103497998A (en) * | 2013-09-30 | 2014-01-08 | 新疆农业大学 | Loop-mediated isothermal amplification (LAMP) rapid detection method of jujube witches broom phytoplasma |
| CN103525923A (en) * | 2013-09-30 | 2014-01-22 | 新疆农业大学 | Primer, probe and method for qualitatively and quantitatively detecting witches' broom phytoplasma |
Non-Patent Citations (2)
| Title |
|---|
| 以tuf 基因为靶标的5 种16SrⅠ组植原体环介导恒温扩增技术;王圣洁等;《林业科学》;20170831;第53卷(第8期);54-63 * |
| 泡桐丛枝和枣疯病植原体tuf 基因上游序列结构、功能和遗传变异比较分析;于少帅等;《微生物学通报》;20160531;第43卷(第5期);第2页左栏第2段 * |
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