LAMP primer set for detecting jujube madness, locust tree witches' broom or cherry lethal yellowing phytoplasma, kit and application thereof

Abstract

The present invention discloses a LAMP primer set for detecting jujube madness, locust tree witches' broom disease and cherry lethal yellowing phytoplasma, as well as a kit and application thereof. The present invention designs a LAMP primer set for detecting jujube madness, locust tree witches' broom disease and cherry lethal yellowing phytoplasma based on the conservative sequence of the tuf gene of the 16SrV-B group phytoplasma. The present invention also discloses a LAMP detection kit for jujube madness, locust tree witches' broom disease and cherry lethal yellowing phytoplasma. The present invention uses the LAMP primer set to establish a LAMP detection method for jujube madness, locust tree witches' broom disease and cherry lethal yellowing phytoplasma, performs loop-mediated isothermal amplification on the DNA of the sample to be tested, and determines whether the sample to be tested is infected with the phytoplasma according to the amplification result. The detection method established by the present invention can be used for the detection of jujube madness, locust tree witches' broom disease and cherry lethal yellowing phytoplasma, and can better distinguish phytoplasmas between different groups and within a group, and has the advantages of good stability, strong specificity and high sensitivity.

Description

Technical Field

[0001] The present invention relates to the detection of phytoplasmas, in particular to a LAMP primer set for detecting jujube madness disease, locust tree witches' broom disease or cherry lethal yellowing phytoplasma, and also to the application of the LAMP primer set in the detection of jujube madness disease, locust tree witches' broom disease or cherry lethal yellowing phytoplasma, belonging to the field of LAMP detection of jujube madness disease, locust tree witches' broom disease and cherry lethal yellowing phytoplasma. Background Art

[0002] Phytoplasma (formerly known as Mycoplasma-like organism, MLO for short) is a kind of difficult-to-cultivate prokaryotes that are similar to plant pathogenic bacteria but have no cell wall. They can cause serious diseases of many important food crops, vegetables, ornamental plants, fruit trees and other economic trees. According to the 16S rDNA gene sequence of phytoplasmas, phytoplasmas can be divided into 28 groups, among which the phytoplasmas that cause jujube madness, locust tree witches' broom disease and cherry lethal yellowing all belong to the 16Sr VB subgroup. This type of disease has a wide distribution range, spreads quickly, and has strong pathogenicity. Once infected, it is difficult to cure with traditional disease prevention and control techniques. Jujube and cherry are important economic forest tree species in China. Because their fruits are deeply loved by people, they have been widely planted in China in recent years. Sophora japonica, as a greening tree species, is a common street tree in northern China. The occurrence of phytoplasma diseases has seriously hindered the industrial development of these economic trees. In order to fundamentally solve the problem of prevention and control of such diseases, it is necessary to establish an accurate, rapid, sensitive and practical pathogen detection technology system and strict plant quarantine measures to eliminate the spread and epidemic of diseases at the source.

[0003] In the early stage, the detection of phytoplasma diseases mainly relied on traditional methods combining biology, electron microscopy observation, and antibiotic testing. Later, with the development of biotechnology, detection methods based on enzyme-linked immunosorbent assay, nucleic acid hybridization, and PCR technology were established, and the sensitivity and accuracy of detection continued to improve. However, the above methods that have been established often require expensive instruments and equipment, and the operating procedures are complicated, the detection time is long, and the technical requirements for the testers are high, which greatly limits the use and promotion of such diagnostic methods.

[0004] Loop-mediated isothermal amplification is a constant temperature nucleic acid amplification method developed by Notomi et al. in 2000. It is a new type of molecular diagnostic method. This method designs specific primers for six specific regions of the target gene and uses a DNA polymerase with strand displacement activity to achieve specific amplification of the target nucleic acid at a constant temperature. Because this method has the characteristics of strong specificity, simple operation, no need for complex instruments, short detection cycle and visual results, it is very suitable for promotion and use as a disease diagnosis method at the grassroots and on-site.

[0005] Some reports have applied loop-mediated isothermal amplification technology to the detection of phytoplasmas, such as aster yellows and strawberry green petals phytoplasmas in the 16Sr I group, guayule witches' broom phytoplasmas in the 16Sr II group, jujube madness and grape golden yellow phytoplasmas in the 16Sr V group, and papaya witches' broom phytoplasmas in the 16Sr XII group. However, the target genes of these methods are mainly based on the 16S rDNA gene. The 16S rDNA gene is a double copy in phytoplasmas and has a certain degree of variability; while the protein elongation factor encoding gene tuf is a single copy in phytoplasmas and is considered to be an evolutionary label. It is slightly less conservative than the 16SrDNA gene and can further distinguish subgroups based on the 16S rDNA gene grouping. Therefore, developing a LAMP detection method with tuf gene as the target gene will have better specificity for the detection of jujube madness, locust witches' broom and cherry lethal yellow phytoplasmas. Summary of the invention

[0006] The first technical problem to be solved by the present invention is to provide a set of LAMP primers for detecting jujube madness, locust tree witches' broom and cherry lethal yellowing phytoplasma, which is a specific primer set designed for the phytoplasma tuf gene;

[0007] Another technical problem to be solved by the present invention is to provide a LAMP detection method for jujube madness, locust tree witches' broom and cherry lethal yellow phytoplasma;

[0008] The third technical problem to be solved by the present invention is to provide a LAMP detection kit for jujube madness, locust tree witches' broom disease and cherry lethal yellowing phytoplasma.

[0009] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0010] The present invention first discloses a LAMP primer set for detecting jujube madness, locust tree witches' broom disease and cherry lethal yellowing phytoplasma, comprising: a pair of outer primers, a pair of inner primers and a pair of loop primers; the LAMP primer set is selected from any one of the following five groups of primers:

[0011] Primer set I: the outer primer pair consists of an upstream outer primer with a nucleotide sequence of SEQ ID No.1 and a downstream outer primer with a nucleotide sequence of SEQ ID No.2; the inner primer pair consists of an upstream inner primer with a nucleotide sequence of SEQ ID No.3 and a downstream inner primer with a nucleotide sequence of SEQ ID No.4; the loop primer pair consists of an upstream loop primer with a nucleotide sequence of SEQ ID No.5 and a downstream loop primer with a nucleotide sequence of SEQ ID No.6;

[0012] Primer set II: the outer primer pair consists of an upstream outer primer with a nucleotide sequence of SEQ ID No.7 and a downstream outer primer with a nucleotide sequence of SEQ ID No.8; the inner primer pair consists of an upstream inner primer with a nucleotide sequence of SEQ ID No.9 and a downstream inner primer with a nucleotide sequence of SEQ ID No.10; the loop primer pair consists of an upstream loop primer with a nucleotide sequence of SEQ ID No.11 and a downstream loop primer with a nucleotide sequence of SEQ ID No.12;

[0013] Primer set III: the outer primer pair consists of an upstream outer primer with a nucleotide sequence of SEQ ID No.13 and a downstream outer primer with a nucleotide sequence of SEQ ID No.14; the inner primer pair consists of an upstream inner primer with a nucleotide sequence of SEQ ID No.15 and a downstream inner primer with a nucleotide sequence of SEQ ID No.16; the loop primer pair consists of an upstream loop primer with a nucleotide sequence of SEQ ID No.17 and a downstream loop primer with a nucleotide sequence of SEQ ID No.18;

[0014] Primer Group IV: The outer primer pair consists of an upstream outer primer with a nucleotide sequence of SEQ ID No.19 and a downstream outer primer with a nucleotide sequence of SEQ ID No.20; the inner primer pair consists of an upstream inner primer with a nucleotide sequence of SEQ ID No.21 and a downstream inner primer with a nucleotide sequence of SEQ ID No.22; the loop primer pair consists of an upstream loop primer with a nucleotide sequence of SEQ ID No.23 and a downstream loop primer with a nucleotide sequence of SEQ ID No.24;

[0015] Primer Group V: The outer primer pair consists of an upstream outer primer with a nucleotide sequence as shown in SEQ ID No.25 and a downstream outer primer with a nucleotide sequence as shown in SEQ ID No.26; the inner primer pair consists of an upstream inner primer with a nucleotide sequence as shown in SEQ ID No.27 and a downstream inner primer with a nucleotide sequence as shown in SEQ ID No.28; the loop primer pair consists of an upstream loop primer with a nucleotide sequence as shown in SEQ ID No.29 and a downstream loop primer with a nucleotide sequence as shown in SEQ ID No.30.

[0016] The present invention collects nucleic acid sequences of tuf genes of different groups of phytoplasmas in GeneBank, uses DNAMAN software to compare and align the sequences, finds out the gene difference sites and highly conservative regions of phytoplasmas of jujube madness, locust tree witches' broom and cherry lethal yellowing 16SrV-B subgroup and other phytoplasmas, and designs 5 groups of specific primers for 6 regions of conservative sequences of tuf genes of phytoplasmas of 16SrV-B subgroup phytoplasmas, including a pair of outer primers, a pair of inner primers and a pair of loop primers. The results of primer group validation tests show that primer groups of groups I, II, IV and V only generate positive signals in diseased tissue samples, and no signals appear in healthy tissue samples; while primer group III amplifies both diseased and healthy samples, and has poor specificity. The results of the specificity test of primer groups I, II, IV and V in the present invention further show that primer group I has a good amplification effect on jujube madness, locust tree branches, and cherry lethal yellowing of the 16SV-B subgroup, and has no amplification reaction on the Chinese wolfberry branches of the 16SV-H subgroup and other groups as well as the healthy control; primer group II has amplified jujube madness, locust tree branches, and cherry lethal yellowing of the 16SV-B subgroup, and Chinese wolfberry branches of the 16SV-H subgroup, and has no inter-group specificity; primer group IV has a good amplification effect on jujube madness, locust tree branches, and cherry lethal yellowing of the 16SV-B subgroup, but the comparison of primer sequences shows that the gene sequences of this group of primers and other groups of phytoplasmas have certain duplications, and there is a risk of insufficient specificity; the amplification effect of primer group V is poor, and jujube madness and locust tree branches only start to rise and amplify after 48 minutes.

[0017] In summary, primer group I has good inter-group specificity. The present invention determines that the LAMP primer group for detecting jujube madness, locust tree witches' broom disease or cherry lethal yellowing phytoplasma is primer group I, that is, the outer primer pair is composed of an upstream outer primer with a nucleotide sequence of SEQ ID No.1 and a downstream outer primer with a nucleotide sequence of SEQ ID No.2; the inner primer pair is composed of an upstream inner primer with a nucleotide sequence of SEQ ID No.3 and a downstream inner primer with a nucleotide sequence of SEQ ID No.4; the loop primer pair is composed of an upstream loop primer with a nucleotide sequence of SEQ ID No.5 and a downstream loop primer with a nucleotide sequence of SEQ ID No.6.

[0018] The LAMP primer set of the present invention can be used to detect jujube madness, locust tree witches' broom disease or cherry lethal yellowing phytoplasma.

[0019] The present invention further discloses a LAMP detection method for jujube madness, locust tree witches' broom disease or cherry lethal yellowing phytoplasma, comprising: (1) extracting total DNA of a sample to be tested; (2) using the extracted total DNA as a template and the LAMP primer set as an amplification primer to establish a loop-mediated isothermal amplification reaction system and perform loop-mediated isothermal amplification; (3) determining the amplification result: placing the amplification reaction tube in a constant temperature fluorescence detector or a fluorescence PCR instrument, reading the fluorescence signal in real time, and determining that the result is positive if an "S"-shaped amplification curve appears. , that is, the sample to be tested is infected with phytoplasma. If there is no "S"-shaped amplification curve, it is judged as negative, that is, the sample to be tested is not infected with phytoplasma; or, after the amplification reaction is completed, 1 μL of color developing solution (the color developing solution is a mixture of 1.25mM calcein and 12.5mM manganese chloride) is added to the amplified product, and the color change is observed by naked eyes under sunlight. If the amplified product turns emerald green, it is judged as positive, that is, the sample to be tested is infected with phytoplasma. If the amplified product is still brownish yellow, it is judged as negative, that is, the sample to be tested is not infected with phytoplasma. Negative and positive controls are set for each test. When both negative and positive controls are established, the entire experiment is valid and the results can be determined.

[0020] The loop-mediated isothermal amplification reaction system includes: the total volume of the reaction system is 25 μL, including 0.5 μL of 10 μM upstream outer primer, 0.5 μL of 10 μM downstream outer primer, 1 μL of 40 μM upstream inner primer, 1 μL of 40 μM downstream inner primer, 1 μL of 20 μM upstream loop primer, 1 μL of 20 μM downstream loop primer, 12.5 μL of 2×LAMP reaction solution RM, 1 μL of 8U / μL Bst DNA polymerase, 0.5 μL of fluorescent dye 10×SYBR GreenⅠ, 1 μL of DNA template, and the balance is ultrapure water. Preferably, the loop-mediated isothermal amplification reaction system includes: the total volume of the reaction system is 25 μL, wherein, 0.5 μL of the upstream outer primer shown in 10 μM SEQ ID No.1, 0.5 μL of the downstream outer primer shown in 10 μM SEQ ID No.2, 1 μL of the upstream inner primer shown in 40 μM SEQ ID No.3, 1 μL of the downstream inner primer shown in 40 μM SEQ ID No.4, 1 μL of the upstream loop primer shown in 20 μM SEQ ID No.5, 1 μL of the downstream loop primer shown in 20 μM SEQID No.6, 12.5 μL of 2×LAMP reaction solution RM, 1 μL of 8U / μL Bst DNA polymerase, 0.5 μL of fluorescent dye 10×SYBR GreenⅠ, 1 μL of DNA template, and the remainder is ultrapure water. The present invention has no concentration requirement for the DNA template, and generally the extracted DNA concentration can be detected between 50 and 2000 ng / μl. In addition, to avoid volatilization, 20 μL of sealing liquid can be added to the reaction system. The procedure of the loop-mediated isothermal amplification includes: constant temperature reaction at 60-65°C for 40-80 minutes, inactivation at 80°C for 5-10 minutes; preferably, constant temperature reaction at 63°C for 40-60 minutes, inactivation at 80°C for 5-10 minutes; most preferably, constant temperature reaction at 63°C for 60 minutes, inactivation at 80°C for 5 minutes.

[0021] The specificity test results show that the loop-mediated isothermal amplification detection method of the present invention has strong specificity for jujube madness, locust tree witches' branches, and cherry lethal yellowing of the 16SrV-B subgroup, and can well distinguish phytoplasmas between and within different groups. Only the diseased tissue samples of jujube madness, locust tree witches' branches, and cherry lethal yellowing of the 16SrV-B subgroup show positive reactions, while the 16SrV-H subgroup Polygonum yangyi witches' branches, as well as different groups of paulownia witches' branches, chinaberry witches' branches, mulberry atrophy, catharanthus roseus chlorosis, lettuce yellowing, peanut witches' branches, sweet potato witches' branches, and clumps of clumps of clumps and all healthy controls are negative. It proves that the LAMP detection method of the present invention can be used for the detection of jujube madness, locust tree witches' branches, and cherry lethal yellowing phytoplasmas of the 16SrV-B subgroup.

[0022] The results of the stability test showed that when the LAMP detection method of the present invention was used to simultaneously detect samples of jujube madness disease from different varieties in different regions, the samples of jujube madness disease from different places and different varieties all showed a positive reaction, an "S"-shaped amplification curve appeared in the fluorescence detection instrument, and the color changed to emerald green when observed with the naked eye, proving that the loop-mediated isothermal amplification detection method established by the present invention has good stability.

[0023] The sensitivity test results showed that conventional PCR was effective when the DNA template was diluted to 10 -2 The detection limit is reached when the DNA template is diluted to 10 -4 The detection result was still positive when the loop-mediated isothermal amplification detection method established in the present invention was proved to be 100 times more sensitive than conventional PCR.

[0024] The LAMP detection method of the present invention is applied to the detection of jujube madness, locust tree witches' broom disease and cherry lethal yellowing phytoplasma, which is of great significance for the early warning of such diseases, pathogen monitoring in epidemic areas and bacteria inspection of breeding materials such as seedlings, etc. At the same time, it can also avoid high investment in instruments and is suitable for promotion and use in grassroots production units.

[0025] The present invention also discloses a LAMP detection kit for jujube madness, locust tree witches' broom disease or cherry lethal yellowing phytoplasma, comprising: LAMP primers, LAMP reaction solution RM, Bst DNA polymerase, fluorescent dye 10×SYBR GreenⅠ and ultrapure water, wherein the LAMP primers are primer group I, comprising: an outer primer pair consisting of an upstream outer primer with a nucleotide sequence as shown in SEQ ID No.1 and a downstream outer primer with a nucleotide sequence as shown in SEQ ID No.2, an inner primer pair consisting of an upstream inner primer with a nucleotide sequence as shown in SEQ ID No.3 and a downstream inner primer with a nucleotide sequence as shown in SEQ ID No.4, and a loop primer pair consisting of an upstream loop primer with a nucleotide sequence as shown in SEQ ID No.5 and a downstream loop primer with a nucleotide sequence as shown in SEQ ID No.6.

[0026] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0027] The LAMP primer set for detecting jujube madness, locust tree witches' broom or cherry lethal yellow phytoplasma of the present invention has strong specificity for jujube madness, locust tree witches' broom and cherry lethal yellow phytoplasma of 16SrV-B subgroup, but cannot generate amplification reaction for Paulownia witches' broom of 16Sr I group, peanut witches' broom of 16Sr II group and Dianthus yangyi witches' broom of 16Sr VH subgroup. The LAMP detection method for jujube madness, locust tree witches' broom or cherry lethal yellow phytoplasma established by the present invention has good stability and high sensitivity. Compared with ordinary PCR, its detection sensitivity is improved by more than 100 times; it is also easy to operate, does not require complex instruments, does not require special reagents, does not require pre-double-stranded DNA denaturation and other steps, and only requires a constant temperature instrument to complete the detection. The entire amplification can be completed in only 40-60 minutes, which is fast and efficient. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The real-time fluorescence detection results of the loop-mediated isothermal amplification products of the jujube madness phytoplasma; among them, positive is the positive control that comes with the LAMP amplification kit, negative is the negative control that comes with the LAMP amplification kit, JWB is the jujube madness sample, JH is the healthy jujube tree sample, and CK is the sterilized ddH 2 O represents control; N represents no sample;

[0029] Figure 2 The color reaction test results of the loop-mediated isothermal amplification product of the jujube madness disease phytoplasma; among them, positive is the positive control that comes with the LAMP amplification kit, negative is the negative control that comes with the LAMP amplification kit, JWB is the jujube madness disease sample, JH is the healthy jujube tree sample, and CK is the sterilized ddH 2 O control;

[0030] Figure 3 Real-time fluorescence detection results for LAMP amplification specificity verification;

[0031] Figure 4 The color reaction detection results for LAMP amplification specificity verification; among them, 1: jujube madness, 2: Sophora japonica branches, 3: cherry lethal yellowing, 4: Polygonum chinense branches, 5: Paulownia branches, 6: Melia azedarach branches, 7: mulberry shrinkage, 8: Catharanthus roseus chlorosis, 9: lethal yellowing of lettuce, 10: peanut branches, 11: sweet potato branches, 12: Skunk cabbage branches, 13: chestnut yellowing, 14: healthy jujube trees, 15: healthy Sophora japonica trees, 16: healthy cherry, 17: healthy Polygonum chinense, 18: healthy Paulownia, 19: healthy Melia azedarach, 20: healthy mulberry, 21: healthy Catharanthus roseus, 22: healthy lettuce, 23: healthy peanut, 24: healthy sweet potato, 25: healthy Skunk cabbage, 26: healthy chestnut, CK: blank control;

[0032] Figure 5The real-time fluorescence detection results for LAMP amplification stability verification; among them, positive is the positive control that comes with the LAMP amplification kit, negative is the negative control that comes with the LAMP amplification kit, 8: Liaoning Dalian jujube disease sample, 10: Liaoning Huludao jujube disease sample, 12: Beijing sour jujube disease sample, 20: Jiangsu Zhenjiang jujube disease sample, 21: Anhui Hefei jujube disease sample, 22: Beijing jujube disease sample, 23 Henan Nanyang jujube disease sample, JH is a healthy jujube tree sample, CK is sterilized ddH 2 O means control; N means no sample;

[0033] Figure 6 The color reaction test results for LAMP amplification stability verification; among them, positive is the positive control that comes with the LAMP amplification kit, negative is the negative control that comes with the LAMP amplification kit, 8: Liaoning Dalian jujube disease sample, 10: Liaoning Huludao jujube disease sample, 12: Beijing sour jujube disease sample, 20: Jiangsu Zhenjiang jujube disease sample, 21: Anhui Hefei jujube disease sample, 22: Beijing jujube disease sample, 23 Henan Nanyang jujube disease sample, JH is a healthy jujube tree sample, CK is sterilized ddH 2 O control;

[0034] Figure 7 It is the detection sensitivity of conventional PCR 1% agarose gel electrophoresis; Lane M is the 2000bp DNAMaker, and the other lanes are the amplification conditions using ten-fold gradient dilution DNA as template;

[0035] Figure 8 The real-time fluorescence detection results of the LAMP amplification sensitivity test; -1 to -9 are the amplification results using ten-fold gradient dilution of DNA as template, and CK is sterilized ddH 2 O means control; N means no sample;

[0036] Fig. 9 is the color reaction detection result of LAMP amplification sensitivity test; -1 ~10 -9 The amplification results are shown in Figure 10 using ten-fold serial dilution of DNA as template. CK is sterilized ddH 2 O control;

[0037] Fig.10 The validation results of five sets of LAMP primers are shown;

[0038] Fig.11 is the sequence alignment result of the JWB I group LAMP primer set; A is the sequence alignment result of primers JWB1-F3; B is the sequence alignment result of primers JWB1-B3;

[0039] Fig.12The specificity verification results of the JWB I group LAMP primer set; A1: jujube madness; A2: Sophora japonica clumps; A3: Cherry lethal yellowing; A4: Sophora japonica clumps; A5: Paulownia japonica clumps; A6: Melia azedarach clumps; A7: Mulberry atrophy; A8: Catharanthus roseus chlorosis; B1: Peanut clumps; B2: Sweet potato clumps; B3: Clumps of clumps; B4: Jujube tree health; B5: Sophora japonica health; B6: Cherry health; B7: Sophora japonica health; B8: blank control;

[0040] Fig.13 is the sequence alignment result of the JWB II group LAMP primer set; A is the sequence alignment result of primer JWB Ⅱ-F3; B is the sequence alignment result of primer JWB Ⅱ-B3;

[0041] Fig.14 The specificity verification results of the JWB II group LAMP primer set; A1: jujube madness; A2: Sophora japonica clumps; A3: Cherry lethal yellowing; A4: Sophora japonica clumps; A5: Paulownia japonica clumps; A6: Melia azedarach clumps; A7: Mulberry atrophy; A8: Catharanthus roseus chlorosis; B1: Peanut clumps; B2: Sweet potato clumps; B3: Clumps of clumps; B4: Jujube tree health; B5: Sophora japonica health; B6: Cherry health; B7: Sophora japonica health; B8: blank control;

[0042] Fig.15 The specificity verification results of the JWB IV group LAMP primer set; A1: jujube madness; A2: Sophora japonica clumps; A3: Cherry lethal yellowing; A4: Sophora japonica clumps; A5: Paulownia japonica clumps; A6: Melia azedarach clumps; A7: Mulberry atrophy; A8: Catharanthus roseus chlorosis; B1: Peanut clumps; B2: Sweet potato clumps; B3: Clumps of clumps; B4: Jujube tree health; B5: Sophora japonica health; B6: Cherry health; B7: Sophora japonica health; B8: blank control;

[0043] Fig.16 The sequence alignment results of the JWB IV group LAMP primer set; A is the sequence alignment result of primer JWB IV-F3; B is the sequence alignment result of primer JWB IV-B3;

[0044] Fig.17 is the sequence alignment result of the JWB V group LAMP primer set; A is the sequence alignment result of primer JWB V-F3; B is the sequence alignment result of primer JWB V-B3;

[0045] Fig.18The results of specificity verification of the JWB V group LAMP primer set are shown in Figure 1. A1: jujube madness; A2: Sophora japonica branches; A3: Cherry lethal yellowing; A4: Polygonum aviculare branches; A5: Paulownia tomentosa branches; A6: Melia azedarach branches; A7: Mulberry atrophy; A8: Catharanthus roseus chlorosis; B1: Peanut branches; B2: Sweet potato branches; B3: Clumping sedge branches; B4: Jujube tree health; B5: Sophora japonica health; B6: Cherry health; B7: Polygonum aviculare health; B8: blank control. DETAILED DESCRIPTION

[0046] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, it should be understood that the embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0047] 1. Experimental drugs

[0048] Plant Genomic DNA Kit (Plant Genomic DNA Kit, Adlai Biotechnology Co., Ltd.), Nucleic Acid Amplification Kit (which includes reaction solution, water, Bst DNA polymerase, fluorescent dye, but no primers) (Guangzhou Diao Biotechnology Co., Ltd.); 2× PCR premix (Bomade Biotechnology Co., Ltd.).

[0049] Example 1 Preparation of primers

[0050] 1. Primer design and synthesis

[0051] The nucleic acid sequences of tuf genes of different groups of phytoplasmas were collected in GeneBank, and the sequences were compared and aligned for similarity using DNAMAN software to find out the genetic difference sites and highly conserved regions between 16SrV-B subgroup phytoplasmas of jujube madness, locust tree witches' broom and cherry lethal yellowing and other groups of phytoplasmas. A set of specific primers was designed for six regions of the conserved sequence of tuf genes of 16SrV-B subgroup phytoplasmas using LAMP primer online design software PrimerExplorer V4, including a pair of outer primers, a pair of inner primers and a pair of loop primers. The primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd. according to HPLC purity level.

[0052] The primer sequences are as follows:

[0053] JWB1-F3:5'-TGATGGTGATAACATACCTATT-3' (SEQ ID No. 1);

[0054] JWB1-B3:5'-TTGTGGACGATAATTATCTCCG-3' (SEQ ID No. 2);

[0055] JWB1-FIP:5'-CGTCCTGTAGCAACAGTTCCTCTTGGACACATATGTTGAGGAC-3' (SEQ IDNo. ​​3);

[0056] JWB1-BIP:5'-TTGCCCGGCGATAATATTGGTATACTTGACCACGTTGAATATCTT-3'(SEQ IDNo.4);

[0057] JWB1-LoopF:5'-CGTTAATAACGCCTTCGATAGG-3' (SEQ ID No. 5);

[0058] JWB1-LoopB:5'-TTGTTACGTGGTGTCAATTACA-3' (SEQ ID No. 6);

[0059] 2. Primer preparation

[0060] After primer synthesis, dry powdered primers were mixed with sterilized ddH 2 O were diluted to 10 μM for JWB1-F3 and JWB1-B3, 40 μM for JWB1-FIP and JWB1-BIP, and 20 μM for JWB1-LoopF ​​and JWB1-LoopB, respectively, and the primers were stored in a -20°C refrigerator away from light.

[0061] Example 2 Establishment of a rapid detection method for phytoplasma of date madness by loop-mediated isothermal amplification

[0062] 1. Extraction of total plant DNA

[0063] The total DNA of the plants to be tested was extracted using a plant genomic DNA kit (Plant Genomic DNA Kit, Adelaide Biotechnology Co., Ltd.) and stored in a -20°C refrigerator for future use.

[0064] 2. LAMP amplification

[0065] LAMP primers were prepared as in Example 1. Each tube of loop-mediated isothermal amplification reaction solution was 45 μL, including: 0.5 μL of 10 μM JWB1-F3, 0.5 μL of 10 μM JWB1-B3, 1 μL of 40 μM JWB1-FIP, 1 μL of 40 μM JWB1-BIP, 1 μL of 20 μM JWB1-LoopF, 1 μL of 20 μM JWB1-LoopB, 12.5 μL of LAMP reaction solution RM (2×), 1 μL of 8 U / μL Bst DNA polymerase, 0.5 μL of fluorescent dye 10×SYBR GreenⅠ, 1 μL of sample DNA to be tested (DNA concentration between 50 and 2000 ng / μl can be detected), which was made up to 25 μL with ultrapure water, and 20 μL of sealing solution was added at the same time. The prepared reaction tube was mixed and centrifuged, and then placed in a constant temperature reaction at 63°C for 60 min and inactivated at 80°C for 5 min.

[0066] 3. Determination of amplification results

[0067] For each test, negative and positive controls are set up. When both negative and positive controls are established, the entire experiment is valid and the results can be determined.

[0068] Determination of LAMP reaction results: Figure 1 As shown, place the reaction tube in a constant temperature fluorescence detector or fluorescence PCR instrument, read the fluorescence signal in real time, and if an "S"-shaped amplification curve appears, it is determined to be a positive reaction, that is, the sample is infected with phytoplasma, and if there is no "S"-shaped amplification curve, it is determined to be negative. Alternatively, observe the color change in the reaction tube with the naked eye ( Figure 2 ), after the reaction is completed, 1 μL of color developing solution (the color developing solution is a mixture of 1.25 mM calcein and 12.5 mM manganese chloride) is added to the reaction mixture. If the amplified product turns emerald green, it is judged as a positive reaction, that is, the sample is infected with phytoplasma. If the amplified product is still brownish yellow, it is judged as negative. This result is consistent with the result of ordinary PCR detection, proving that the data of this method is reliable.

[0069] Example 3 Specificity test

[0070] According to the loop-mediated isothermal amplification reaction system established in Example 2, the diseased and healthy tissue samples of paulownia witches' broom, chinaberry witches' broom, mulberry stunting, vinca roseus chlorosis and lettuce yellowing in the 16SⅠ group were detected simultaneously; the diseased and healthy tissue samples of peanut witches' broom, sweet potato witches' broom and clump of ... 2 O was used as negative control to conduct specificity test of ring-mediated isothermal amplification of jujube madness, locust tree witches' broom and cherry lethal yellows phytoplasma.

[0071] The total plant DNA and sterilized ddH2O were extracted from the diseased and healthy tissue samples of paulownia witches' broom, chinaberry witches' broom, mulberry stunt, vinca roseus chlorosis and lettuce yellows in the 16SⅠ group, the diseased and healthy tissue samples of peanut witches' broom, sweet potato witches' broom and clover witches' broom in the 16SⅡ group, and the diseased and healthy tissue samples of jujube madness, locust witches' broom, cherry lethal yellows and Chinese fern witches' broom in the 16SⅤ group. 2 O as a template.

[0072] The loop-mediated isothermal amplification reaction was carried out according to the reaction system and reaction conditions provided in the above Example 2. The result determination was carried out according to the LAMP amplification result determination method provided in Example 2.

[0073] The judgment result is as follows Figure 3 , Figure 4 As shown, only the diseased tissue samples of jujube madness, Sophora japonica bushes, and cherry lethal yellowing of the 16SrV-B subgroup showed positive reactions, and an "S"-shaped amplification curve appeared in the fluorescence detection instrument. At the same time, the color changed to emerald green when observed with the naked eye, while the 16SrV-H subgroup of Dianthus yangyi bushes, as well as the different genera of Paulownia bushes, Chinaberry bushes, mulberry atrophy, Catharanthus roseus chlorosis, lettuce yellowing, peanut bushes, sweet potato bushes, and clumps of clumps of clumps and all healthy controls were negative. It can be seen that the loop-mediated isothermal amplification detection method established by the present invention has strong specificity for jujube madness, Sophora japonica bushes, and cherry lethal yellowing of the 16SrV-B subgroup, can better distinguish phytoplasmas between and within different groups, and can be used for the detection of jujube madness, Sophora japonica bushes, and cherry lethal yellowing phytoplasmas of the 16SrV-B subgroup.

[0074] Example 4 Stability Test

[0075] According to the loop-mediated isothermal amplification reaction system established in Example 2, samples of jujube disease from different regions and varieties (respectively: jujube disease samples from Dalian, Liaoning, jujube disease samples from Huludao, Liaoning, jujube disease samples from Beijing, jujube disease samples from Zhenjiang, Jiangsu, jujube disease samples from Hefei, Anhui, jujube disease samples from Beijing, and jujube disease samples from Nanyang, Henan) and samples of healthy jujube trees and sterilized ddH 2 O was used as negative control to conduct stability test of loop-mediated isothermal amplification of jujube madness phytoplasma.

[0076] Total DNA and sterilized ddH2O were extracted from samples of jujube madness and healthy jujube trees from different regions and different varieties. 2 O as a template.

[0077] The loop-mediated isothermal amplification reaction was performed according to the reaction system and reaction conditions provided in the above Example 2. The result was determined according to the LAMP amplification result determination method provided in the above Example 2.

[0078] The judgment result is as follows Figure 5 , Figure 6 As shown, samples of jujube madness from different places and different varieties can all show positive reactions, and an "S"-shaped amplification curve appears in the fluorescence detection instrument, and the color changes to emerald green when observed with the naked eye. This shows that the loop-mediated isothermal amplification detection method established by the present invention has good stability.

[0079] Example 5 Sensitivity Test

[0080] The total DNA of the jujube leaf of the jujube mad disease-infected plants was extracted using a Plant Genomic DNA Kit (Adlai Biotechnology Co., Ltd.) and used as a template. 2 O was diluted tenfold, and loop-mediated isothermal amplification was performed according to the reaction system and reaction conditions in Example 2, and conventional PCR was performed at the same time for sensitivity comparison.

[0081] Conventional PCR detection: Using ten-fold serial dilution of DNA as template, PCR amplification was performed with universal primers for phytoplasma R16mF2 / R16mR1 (upstream primer, R16m F2: 5′-CAT GCA AGT CGA ACG GA-3′; downstream primer, R16m R1: 5′-CTT AAC CCC AAT CAT CGA-3′). The conventional PCR amplification system was 25 μL, and the amplification reaction system contained 1 μL of prepared DNA template, 0.5 μL of each forward and reverse primer (10 μM), 12.5 μL of 2×PCR premix (0.05 U / μL Taq DNA polymerase, 4 mM MgCl2 and 0.4 mM dNTPs), and ddH 2 O to 25 μL. Reaction procedure: 94℃5min; 94℃45s, 52℃45s, 72℃1min, 35 cycles; 72℃ extension for 10min. PCR products were detected by 1% agarose gel electrophoresis.

[0082] The results of conventional PCR were detected by 1% agarose gel electrophoresis. Figure 7 As shown, the samples were diluted to 10 -2 The detection limit was reached when the sample was diluted to 10 -4 The test result is still positive (an "S"-shaped amplification curve appears in the fluorescence detection instrument, and the color changes to emerald green when observed with the naked eye) Figure 8 , Fig. 9As shown). This shows that the sensitivity of the loop-mediated isothermal amplification detection method established by the present invention is 100 times higher than that of conventional PCR.

[0083] Experimental Example 1: Design of LAMP primer set validation test for jujube madness, locust tree witches' broom and cherry lethal yellow phytoplasma

[0084] The present invention designed a total of five sets of LAMP primer sets for the tuf gene, and the specific information is shown in Table 1.

[0085] Table 1 Sequence information of five sets of LAMP primer sets

[0086]

[0087]

[0088] Preparation of LAMP reaction system: The reaction system is 45μL, including: 10μM JWB-F3 0.5μL, 10μM JWB-B3 0.5μL, 40μM JWB-FIP 1μL, 40μM JWB-BIP 1μL, 20μM JWB-LoopF ​​1μL, 20μM JWB-LoopB1μL, LAMP reaction solution RM (2×) 12.5μL, 8U / μL Bst DNA polymerase 1μL, fluorescent dye 10×SYBR GreenⅠ0.5μL, sample DNA to be tested 1μL, make up to 25μL with ultrapure water, and add 20μL sealing liquid.

[0089] Isothermal gene amplification: Mix the prepared reaction tubes and centrifuge them, place them at 63°C for 40 to 60 minutes, and inactivate them at 80°C for 5 to 10 minutes.

[0090] Five sets of LAMP primers were used to perform loop-mediated isothermal amplification on the samples to be tested. Fig.10 , Table 2.

[0091] Table 2 Results of loop-mediated isothermal amplification using five sets of LAMP primers

[0092] CH A1 A2 A3 A4 A5 A6 A7 A8 Name 1+ 1- 2+ 2- 3+ 3- 4+ 4- + / - + - + - + + + - Tt 19：00 10：30 17：00 11：00 16：00

[0093] CH B1 B2 B3 B4 B5 B6 B7 B8 Name 5+ 5- sample sample sample sample sample sample + / - + - - - - - - - Tt 25：30

[0094] Note: 1+ represents the positive sample of primer set I, 1- represents the negative sample of primer set I; 2+ represents the positive sample of primer set II, 2- represents the negative sample of primer set II, and so on; there is no sample after B3.

[0095] From the results, it can be seen that the primer sets of groups I, II, IV and V only produced positive signals in diseased tissue samples (jujube madness samples), and no signals appeared in healthy tissue samples; while primer set III showed amplification in both diseased tissue samples and healthy tissue samples.

[0096] The present invention further performs specificity verification on primer sets of groups I, II, IV and V.

[0097] Among them, JWB I group LAMP primer set (primer sequence alignment see Fig.11 ) were used to perform loop-mediated isothermal amplification on diseased tissue samples of jujube madness, locust tree clumps, cherry lethal yellowing, Chinese wolfberry clumps, paulownia clumps, chinaberry clumps, mulberry atrophy, catharanthus roseus chlorosis, peanut clumps, sweet potato clumps, and clumps of clumps, as well as healthy samples of jujube, locust, cherry, and Chinese wolfberry. The results are shown in Fig.12 The results showed that the amplification effect of 16SV-B subgroup on the lethal yellowing of Ziziphus jujuba, Sophora japonica, and Prunus persica was better; the amplification reaction of 16SV-H subgroup on the lethal yellowing of Dianthus chinensis, other groups, and healthy controls was not found.

[0098] JWB II LAMP primer set (for primer sequence alignment, see Fig.13 ) were used to perform loop-mediated isothermal amplification on samples of jujube madness, locust tree branches, cherry lethal yellowing, Chinese wolfberry branches, paulownia branches, chinaberry branches, mulberry atrophy, catharanthus roseus chlorosis, peanut branches, sweet potato branches, and clumps of clumps, as well as healthy tissues of jujube, locust, cherry, and Chinese wolfberry. The results are shown in Fig.14 The results showed that the 16SV-B subgroup of Jujube Crassula, Sophora japonica, and Cherry Lethal Yellowing, and the 16SV-H subgroup of Sophora japonica were amplified. Therefore, the JWB II group LAMP primer set was not specific between groups.

[0099] The JWB IV LAMP primer set was used to perform loop-mediated isothermal amplification on samples of diseased jujube madness, Sophora japonica bushes, cherry lethal yellowing, Chinese wolfberry bushes, Paulownia bushes, Chinaberry bushes, mulberry atrophy, Catharanthus roseus chlorosis, peanut bushes, sweet potato bushes, and clumps of clumps, as well as healthy tissues of jujube, Sophora japonica, cherry, and Chinese wolfberry. The results are shown in the table. Fig.15 The results showed that the JWB IV group LAMP primer set had a good amplification effect on the lethal yellowing of 16SV-B subgroup of Zaofeng, Sophora japonica, and Cherry; however, the comparison of primer sequences showed that it had some duplication with other groups ( Fig.16 ), there is a risk of insufficient specificity.

[0100] JWB V group LAMP primer set (primer sequence alignment see Fig.17) were used to perform loop-mediated isothermal amplification on samples of jujube madness, locust tree branches, cherry lethal yellowing, Chinese wolfberry branches, paulownia branches, chinaberry branches, mulberry atrophy, catharanthus roseus chlorosis, peanut branches, sweet potato branches, and clumps of clumps, as well as healthy tissues of jujube, locust, cherry, and Chinese wolfberry. The results are shown in Fig.18 The experimental results showed that the amplification effect of this set of primers was not very good; the amplification of Jujube Madness and Sophora japonica branches only began to rise after 48 minutes.

[0101] Based on the above experimental results, the present invention believes that the JWB I group LAMP primer set has good inter-group specificity and is the best LAMP amplification primer set for detecting jujube madness, locust tree witches' broom disease and cherry lethal yellowing (16SrV-B group) phytoplasmas.

Claims

1. A LAMP primer set for detecting jujube madness, locust tree witches' broom or cherry lethal yellowing phytoplasma, characterized in that: The LAMP primer set consists of a pair of outer primers, a pair of inner primers and a pair of loop primers; the outer primer pair consists of an upstream outer primer with a nucleotide sequence as shown in SEQ ID No.1 and a downstream outer primer with a nucleotide sequence as shown in SEQ ID No.2; the inner primer pair consists of an upstream inner primer with a nucleotide sequence as shown in SEQ ID No.3 and a downstream inner primer with a nucleotide sequence as shown in SEQ ID No.4; the loop primer pair consists of an upstream loop primer with a nucleotide sequence as shown in SEQ ID No.5 and a downstream loop primer with a nucleotide sequence as shown in SEQ ID No.

6.

2. Use of the LAMP primer set according to claim 1 in detecting jujube madness, locust tree witches' broom or cherry lethal yellowing phytoplasma.

3. A LAMP detection kit for jujube madness, locust tree witches' broom or cherry lethal yellowing phytoplasma, comprising: LAMP primers, LAMP reaction solution RM, Bst DNA polymerase, fluorescent dye 10×SYBR Green I and ultrapure water, characterized in that the LAMP primers are the LAMP primer set according to claim 1.

4. A method for detecting jujube madness, locust tree witches' broom or cherry lethal yellowing phytoplasma using the LAMP detection kit according to claim 3, characterized in that: include: (1) extracting total DNA from the sample to be tested; (2) using the extracted total DNA as a template and the LAMP primer set described in claim 1 as an amplification primer, establishing a loop-mediated isothermal amplification reaction system and performing loop-mediated isothermal amplification; The procedure of the loop-mediated isothermal amplification includes: constant temperature reaction at 60-65°C for 40 minutes, inactivation at 80°C for 5-10 minutes; (3) Determine the amplification result: Place the amplification reaction tube in a constant temperature fluorescence detector or fluorescence PCR instrument and read the fluorescence signal in real time. If an "S"-shaped amplification curve appears, it is determined to be positive; if no "S"-shaped amplification curve is present, it is determined to be negative. Alternatively, add a color developing solution to the amplification product. If the amplification product turns emerald green, it is determined to be positive; if the amplification product turns brownish yellow, it is determined to be negative.

5. The method according to claim 4, characterized in that The loop-mediated isothermal amplification reaction system includes: the total volume of the reaction system is 25 μL, wherein, 0.5 μL of 10 μM upstream outer primer, 0.5 μL of 10 μM downstream outer primer, 1 μL of 40 μM upstream inner primer, 1 μL of 40 μM downstream inner primer, 1 μL of 20 μM upstream loop primer, 1 μL of 20 μM downstream loop primer, 12.5 μL of 2×LAMP reaction solution RM, 1 μL of 8U / μL Bst DNA polymerase, 0.5 μL of fluorescent dye 10×SYBR GreenⅠ, 1 μL of DNA template, and the balance is ultrapure water.

6. The method according to claim 4 or 5, characterized in that The loop-mediated isothermal amplification reaction system includes: the total volume of the reaction system is 25 μL, wherein, 0.5 μL of the upstream outer primer shown in 10 μM SEQ ID No.1, 0.5 μL of the downstream outer primer shown in 10 μM SEQ ID No.2, 1 μL of the upstream inner primer shown in 40 μM SEQ ID No.3, 1 μL of the downstream inner primer shown in 40 μM SEQ ID No.4, 1 μL of the upstream loop primer shown in 20 μM SEQ ID No.5, 1 μL of the downstream loop primer shown in 20 μM SEQ ID No.6, 12.5 μL of 2×LAMP reaction solution RM, 1 μL of 8U / μL Bst DNA polymerase, 0.5 μL of fluorescent dye 10×SYBR GreenⅠ, 1 μL of DNA template, and the balance is ultrapure water.

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