CN111500787B - LAMP visual detection primer and kit for grape intra-berry necrosis virus and application of LAMP visual detection primer and kit - Google Patents
LAMP visual detection primer and kit for grape intra-berry necrosis virus and application of LAMP visual detection primer and kit Download PDFInfo
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Abstract
The invention discloses LAMP visual detection primers and kit for grape berry necrosis virus and application thereof. The kit comprises a primer combination set consisting of a primer 1-a primer 6; the primer 1-primer 6 are single-stranded DNA molecules shown in sequences 1-6 in a sequence table. Experiments prove that: the primer combination set has the advantages of good specificity, high sensitivity and short detection time, and can effectively detect different variant types of the grape berry necrosis viruses. The direct RT-LAMP color development method of the grape berry necrosis virus is established based on the primer combination set, and the method has the advantages of no need of purifying RNA, no need of Lai Guichong instruments, visual observation of results, accuracy, rapidness, wide application range and the like, has guiding significance for rapid detection of grape seedling viruses in China, and provides a powerful tool for rapid detection of field and detoxified grape sample GINV.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to LAMP visual detection primers and kit for grape berry necrosis virus and application of the LAMP visual detection primers and kit.
Background
Grape intra-berry necrosis virus (GINV) is one member of the genus Trichovirus (Trichovirus), has a virion size of 740X 12nm, has a total genome length of 7229nt, consists of 3 open reading frames, and encodes Replicase (RP), mobility Protein (MP) and Coat Protein (CP), respectively. GINV caused intra-berry necrosis (first known as mosaic disease) of grapes, first discovered in 1984 on kungfeng grapes, causing serious damage in the vineyard of sorbus japan at that time. Some sensitive varieties, including Kyoho, pioneer, lichuan seedless, kangbaier early growth, and the like, are infected with the disease, the growth of plants is weakened, the germination in spring is delayed, and the symptoms of stem necrosis, short section, flower leaf, small fruit grain, fruit outside color change, fruit inside necrosis, and the like are presented. GINV can be transmitted and compromised in the field by gall mites. The virus is discovered and reported for the first time in 2016 in China, and simultaneously, GINV is shown to be related to grape appearance of chlorotic mottle and ring spot symptoms. RT-PCR detection results show that the detection rate of GINV of grape samples in China is 35.9%, the detection rate of GINV on grapes showing obvious ring spot symptoms is 94.1%, and the yield and the quality of grapes in China are seriously influenced. As an important grape virus newly reported in China, a sensitive and simple detection method is necessary to be established, and a foundation is provided for further comprehensive prevention and control of the grape virus.
Currently, the detection of GINV is mainly based on the conventional RT-PCR method. However, the RT-PCR detection method still has defects in sensitivity, detection efficiency and repeatability. The real-time fluorescent quantitative RT-PCR (RT-qPCR) method can directly read the amplification signal through a fluorescent quantitative PCR instrument, avoids aerosol pollution, has sensitivity obviously higher than that of the conventional RT-PCR, but the method needs to purchase an expensive fluorescent PCR instrument, and is not suitable for basic unit detection.
Loop-mediated isothermal amplification (LAMP) is a novel isothermal nucleic acid amplification method created by doctor Notomi in 2000, and exponential amplification of nucleic acid is performed at 60-65 ℃ by using 4 primers specifically recognizing 6 regions on a target sequence and Bst DNA polymerase with strand displacement activity, and the amplification efficiency can reach 10 9 ～10 10 On the order of one copy, the entire reaction takes only 1h. The method has the advantages of constant temperature amplification, high detection speed, high sensitivity, low cost and visual result.
Disclosure of Invention
The invention aims to provide LAMP visual detection primers and kit for grape intra-berry necrosis virus and application of the LAMP visual detection primers and kit.
In a first aspect, the invention provides a set of primers for detecting or aiding in the detection of grape berry necrosis virus.
The complete primer set for detecting or assisting in detecting the grape berry necrosis virus provided by the invention consists of a primer 1-a primer 6;
the primer 1 is a 1) or a 2) as follows:
a1 A single-stranded DNA molecule shown as a sequence 1 in a sequence table;
a2 A single-stranded DNA molecule obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 1 and having the same function as the sequence 1;
the primer 2 is a 3) or a 4) as follows:
a3 A single-stranded DNA molecule shown in a sequence 2 in a sequence table;
a4 A single-stranded DNA molecule obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 2 and having the same function as the sequence 2;
the primer 3 is a 5) or a 6) as follows:
a5 A single-stranded DNA molecule shown in a sequence 3 in a sequence table;
a6 A single-stranded DNA molecule obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 3 and having the same function as the sequence 3;
the primer 4 is a 7) or a 8) as follows:
a7 A single-stranded DNA molecule shown in a sequence 4 in a sequence table;
a8 A single-stranded DNA molecule obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 4 and having the same function as the sequence 4;
the primer 5 is a 9) or a 10) as follows:
a9 A single-stranded DNA molecule shown as a sequence 5 in a sequence table;
a10 A single-stranded DNA molecule obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 5 and having the same function as the sequence 5;
the primer 6 is a 11) or a 12) as follows:
a11 A single-stranded DNA molecule shown in a sequence 6 in a sequence table;
a12 Single-stranded DNA molecule obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 6 and having the same function as the sequence 6.
In the above set of primer sets, the molar ratio of the primer 1, the primer 2, the primer 3, the primer 4, the primer 5 and the primer 6 is 1.
In a second aspect, the present invention protects a novel use of the primer set described above.
The invention protects the application of the primer set in any one of the following b 1) -b 8):
b1 Preparing a product for detecting or assisting in detecting the necrotic virus in grape berries;
b2 Detecting or aiding in the detection of grape intra-berry necrotic virus;
b3 Preparing a product for detecting or assisting in detecting whether a sample to be detected is infected with grape berry necrosis virus;
b4 Detecting or assisting to detect whether the sample to be detected is infected with the grape berry necrosis virus;
b5 Preparing a product for identifying or assisting in identifying whether the pathogenic microorganism to be detected is grape intra-berry necrosis virus;
b6 Identifying or assisting in identifying whether the pathogenic microorganism to be detected is grape intra-berry necrosis virus;
b7 Preparing a product for identifying or assisting in identifying the necrotic virus in grape berries from other viruses;
b8 To identify or aid in identifying necrotic viruses within grape berries from other viruses.
In a third aspect, the present invention provides a kit comprising the above primer set;
the function of the kit is any one of the following c 1) -c 4):
c1 Detecting or aiding in the detection of grape intra-berry necrotic virus;
c2 Detecting or assisting to detect whether the sample to be detected is infected with the grape berry necrosis virus;
c3 Identifying or assisting in identifying whether the pathogenic microorganism to be detected is grape intra-berry necrosis virus;
c4 To identify or aid in identifying necrotic viruses within grape berries from other viruses.
Further, the kit can also comprise other reagents for detecting grape intra-berry necrosis virus by RT-LAMP. In the invention, the other reagent for detecting grape intra-berry necrosis virus by RT-LAMP is WarmStart Colorimetric LAMP 2 × Master Mix.
Further, the kit further comprises purified water.
In a fourth aspect, the invention provides a method for preparing the kit. The preparation method of the kit comprises the following steps of d 1) or d 2):
d1 Each primer in the primer set is packaged separately;
d2 Each primer in the primer set is mixed together in proportion.
In the above method for preparing a kit, in the d 2), the primer 1, the primer 2, the primer 3, the primer 4, the primer 5 and the primer 6 in the primer set are mixed together in a molar ratio of 1.
In a fifth aspect, the present invention provides a method for detecting or aiding in detecting whether a sample to be detected is infected with grape intra-berry necrosis virus.
The method for detecting or assisting in detecting whether the sample to be detected is infected with the grape berry necrosis virus is the following e 1) or e 2):
e1 Extracting nucleic acid of a sample to be detected, performing RT-LAMP by using the nucleic acid of the sample to be detected as a template and adopting the primer combination set, and judging whether the sample to be detected is infected with grape berry necrosis virus or not by observing the color of a reaction system by naked eyes after the RT-LAMP reaction is finished: if the color of the reaction system is yellow, infecting the sample to be detected or infecting the necrotic virus in grape berries by candidates; if the color of the reaction system is pink, the sample to be detected is not infected or candidate uninfected grape berry necrosis viruses;
e2 Taking a sample tissue fluid to be detected, directly taking the sample tissue fluid to be detected as a template, carrying out RT-LAMP by adopting the complete set of primer combination, and judging whether the sample to be detected is infected with the grape berry necrosis virus or not by observing the color of a reaction system by naked eyes after the RT-LAMP reaction is finished: if the color of the reaction system is yellow, infecting the sample to be detected or infecting the necrotic virus in grape berries by candidates; if the color of the reaction system is pink, the sample to be detected is not infected or candidate uninfected grape intra-berry necrosis virus.
Further, in e 2), the method for taking the tissue fluid of the sample to be tested comprises the following steps: after pricking a sample to be tested for several times (for example, 3 times) with a tip (for example, 10. Mu.L tip), the tip with the tissue fluid is immediately dipped for several times (for example, 3 times) in a centrifuge tube (1.5 mL) filled with RNase-free deionized water (10. Mu.L), and the liquid in the centrifuge tube is uniformly mixed to serve as a template.
In a sixth aspect, the invention provides a method for identifying or assisting in identifying whether a pathogenic microorganism to be tested is a grape intra-berry necrosis virus.
The method for identifying or assisting in identifying whether the pathogenic microorganism to be detected is grape intra-berry necrotic virus comprises the following steps: extracting nucleic acid of pathogenic microorganism to be detected, performing RT-LAMP by using the nucleic acid of the pathogenic microorganism to be detected as a template and adopting the primer combination set, and judging whether the pathogenic microorganism to be detected is the grape berry necrosis virus or not by observing the color of a reaction system through naked eyes after the RT-LAMP reaction is finished: if the color of the reaction system is yellow, the pathogenic microorganism to be detected is or is selected as grape berry necrosis virus; if the color of the reaction system is pink, the pathogenic microorganism to be detected is not or is not candidate to be the grape berry necrosis virus.
In a seventh aspect, the present invention features a method of identifying or aiding in identifying necrotic virus in grape berries from other viruses.
The method for identifying or assisting in identifying the necrotic virus in grape berries and other viruses comprises the following steps: extracting nucleic acid of a virus to be detected, performing RT-LAMP by using the virus nucleic acid to be detected as a template and adopting the primer combination set, and judging whether the virus to be detected is grape berry necrosis virus or other viruses by observing the color of a reaction system by naked eyes after the RT-LAMP reaction is finished: if the color of the reaction system is yellow, the virus to be detected is or is selected as grape berry necrosis virus; if the color of the reaction system is pink, the virus to be detected is or is selected as other viruses.
In any of the above methods, the reaction system of RT-LAMP is as follows: mu.L of WarmStart Colorimetric LAMP 2 × Master Mix (NEB), 1. Mu.L of primer mixture, 3.6. Mu.L of deionized water, and 0.4. Mu.L of RNA template. The concentrations of the primer 1, the primer 2, the primer 3, the primer 4, the primer 5 and the primer 6 in the primer mixture are respectively 2 μ M, 16 μ M, 4 μ M and 4 μ M.
The reaction conditions of the RT-LAMP are as follows: reacting at 65 ℃ for 1h.
In any of the above applications or kits or methods, the sample to be tested may be a grape sample; further, the grape samples can be tissue samples of different parts of grapes; furthermore, the tissue sample of different parts of the grape can be grape leaves (such as upper tender leaves, upper petioles, middle leaves, middle petioles, lower old leaves and lower old petioles of grape fruiting branches) or grape fruits.
In any of the above applications or kits or methods, the other virus may be at least one of the following viruses: GLRaV-2 (grape leaf-related virus 2, grape leaf-associated virus 3), GLRaV-3 (grape leaf-related virus 3, grape leaf-associated virus 3), GFkV (grape spot virus, grapevine fleck), GFABV (grape broad bean wilt virus, grapevine faba virus), GRSPaV (vitis vinifera virus, grapevine rupestris stem-associated virus), GLRaV-1 (grape leaf-related virus 1, grape leaf-associated virus 1), GVE (grape virus E, grapevine virus E), GFLV (grapevine fanleaf virus), GLRaV-4 (grapevine leaf-associated virus 4 ), GLRaV-7 (grapevine leaf-associated virus 7, grapevine leaf-associated virus 7), GVA (grapevine virus A), GVB (grapevine virus B), GPGV (Gray Birdono grapevine virus).
In any of the above uses or kits or methods, the grape berry necrosis virus may be a different variant of grape berry necrosis virus (e.g., LN-ANSJ, XJ-Ths, or LN-Beta 2).
The invention provides a set of primer combination for RT-LAMP visual detection of grape intra-berry necrosis virus. Experiments prove that: the primer combination set has the advantages of good specificity, high sensitivity and short detection time, and can effectively detect different variant types of the grape berry necrosis viruses. The invention also establishes a direct RT-LAMP color development method for detecting the necrotic viruses in grape berries based on the primer combination set, and the method has the advantages of no need of purifying RNA, no need of Lai Guichong instruments, visual observation of results, accuracy, rapidness, wide application range and the like, has guiding significance for the rapid detection of grape seedling viruses in China, and provides a powerful tool for the rapid detection of GINV in fields and virus-free grape samples.
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FIG. 1 shows the amplification results of 6 sets of primer combinations on the same GINV positive sample. 1: primer CP set1;2: primer CP set2;3: primer CP set3;4: primer MP set1;5: primer MP set2;6: primer MP set3.
FIG. 2 shows the amplification of different variants of GINV by primer CP set3. A: and (5) amplifying the result by an RT-LAMP fluorescence method. B: and (5) detecting the result by an RT-LAMP color development method. C: and (3) an electrophoresis picture of the amplification product by an RT-LAMP color development method, wherein M: DNA Marker DL2000 (Takara); 1. 2 and 3: (ii) 3 distinct variants of GINV from different populations of GINV; 4: negative control; 5: blank control.
FIG. 3 is a GINV RT-LAMP specific assay. A: and (5) performing amplification result by using an RT-LAMP fluorescence method. B: and detecting the result by an RT-LAMP chromogenic method. C: and (3) an electrophoresis picture of the amplification product by an RT-LAMP color development method, wherein M: DNA Marker DL2000 (Takara); sample No. 1: grape leaves infected with GLAV-2, GLAV-3, GFkV, GFabV and GRSPaV; sample No. 2: grape leaves infected with GLRaV-1, GLRaV-2, GLRaV-3 and GFkV; sample No. 3: grape leaves infected with GLRaV-3, GFkV, GVE and GFLV; sample No. 4: grape leaves infected with GLRaV-4 and GLRaV-7; sample No. 5: grape leaves infected with GRSPaV, GVA and GFkV; sample No. 6: grape leaves infected with GRSPaV, GLRaV-3, GVB and GPGV; 7: positive control (grape leaves infected with GINV); 8: negative control (grape leaves not infected with GINV).
FIG. 4 shows a comparison of RT-LAMP and RT-PCR sensitivity. A: detecting the result by an RT-LAMP color development method; b: RT-LAMP color developmentElectrophoresis picture of the amplified product; c: electrophoresis picture of RT-PCR amplification product. M: DNA Marker DL2000 (Takara); 1-9:10 0 ～10 -8 Gradient diluted GINV positive sample RNA;10: and (4) water control.
FIG. 5 is a detection result of the GINV positive sample and the GINV negative sample by the direct RT-LAMP color development method. A: detecting results by a direct RT-LAMP color development method; b: and (3) amplifying the product electrophoretogram by a direct RT-LAMP color development method. M: DNA Marker DL2000 (Takara); 1-3: GINV positive samples No. 1, no. 2 and No. 3; 4-6: GINV negative samples nos. 4, 5 and 6; 7: a positive control; 8: and (5) negative control.
FIG. 6 shows the amplification results of different tissues of grape by direct RT-LAMP chromogenic method. 1: an upper blade; 2: an upper petiole; 3: a middle blade; 4: a middle petiole; 5: a lower blade; 6: a lower petiole; 7: fruits; 8: and (5) negative control.
FIG. 7 shows the results of field grape samples tested by conventional RT-PCR and direct RT-LAMP color development.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
Example 1 design and screening of primer combinations for RT-LAMP detection of GINV
1. Design and synthesis of primer combination for RT-LAMP detection of GINV
6 sets of primer combinations for RT-LAMP detection of GINV were designed based on conserved regions of Coat Protein (CP) and Mobile Protein (MP) genes of reported GINV isolates, the primer sequences are shown in Table 1, and all the primer sequences were synthesized by Biotechnology engineering (Shanghai) GmbH.
TABLE 1 primer combinations for RT-LAMP detection of GINV
2. Screening of primer combinations for RT-LAMP detection of GINV
1. Preparation of primer mixture
And (3) combining the 6 sets of primers synthesized in the step one to respectively prepare 6 sets of 10-fold primer mixed liquor (the solvent is pure water). The concentrations of the F3, B3, FIP, BIP, LF and LB primers in the primer mixture prepared from the primer combinations CP set1, CP set2, CP set3 and MP set1 are 2. Mu.M, 16. Mu.M, 4. Mu.M and 4. Mu.M respectively. The concentrations of F3, B3, FIP and BIP primers in the primer mixture prepared from the primer combinations MP set2 and MP set3 were 2. Mu.M, 16. Mu.M and 16. Mu.M, respectively.
2、RT-LAMP
1) Extraction of RNA
Reference is made to the literature "detection of grape virus E molecules and analysis of gene sequences, plant pathology report 2014,44 (5): 455-460 "extracts total RNA from grape leaves infected with GINV.
2)RT-LAMP
Using the RNA extracted in the step 1 as a template, and respectively adopting 6 sets of primer combinations to prepare 6 primer mixed liquid for use
RT-LAMP was performed using the LAMP kit (NEB, cat. E1700 s). The RT-LAMP reaction system is 10 mu L: mu.L of WarmStart LAMP 2 × Master Mix (WarmStart LAMP 2 × Master Mix is
Reagents in LAMP kit), 1. Mu.L of 10-fold primer mixture, and 0.2. Mu.L of Fluorescent dye (50X) are
Reagents in LAMP kit), 3.4. Mu.L deionizationWater, 0.4 μ L of GINV positive sample RNA. After uniformly mixing the reaction system, placing the mixture in a fluorescent quantitative PCR instrument for reaction, wherein the reaction procedure is as follows: 30s at 65 ℃; fluorescence signals were collected at 65 ℃ for 15s,65 ℃ for 45s, and the cycle was 60 times. And determining the optimal primer combination according to the amplification curve condition of each group of primer mixed liquor.
The results show that: the primer combinations CP set1, CP set2, MP set3 gave no amplification curve, while the primer combinations CP set3, MP set1 and MP Sset2 gave an amplification curve, with CP set3 having the fastest peak onset time and higher fluorescence values (FIG. 1). Thus, CP set3 primer combination is the optimal primer combination for the detection of GINV by RT-LAMP.
Example 2 detection of different variants of GINV by RT-LAMP fluorescence and RT-LAMP visualization based on CP set3 primer combination
RT-LAMP was performed on different varieties of GINV using the optimal primer set CP set3 selected in example 1 to confirm whether the primer set can amplify different varieties of GINV.
Test RNA samples: total RNA extracted from leaves of grapes infected with the GINV variant LN-ANSJ (type 1); total RNA extracted from grape leaves infected with GINV variant XJ-Ths (type 2); total RNA extracted from grape leaves infected with GINV variant LN-Beta2 (type 3). The 3 different variants of GINV (LN-ANSJ (type 1), XJ-Ths (type 2) and LN-Beta2 (type 3)) are described in the literature: "Occurence and genetic diversity of Grapevine berry inner cross viruses in Chinese plant Disease,2017,101 (1): 144-149".
1. RT-LAMP fluorescence method
Using NEB by RT-LAMP fluorescence method
The LAMP kit performs RT-LAMP on different varieties of GINV. The reaction system and the reaction procedure were the same as in step two of example 1. The primer mixture in the reaction system is the primer mixture prepared from the optimal primer combination CP set3 screened in example 1. Total RNA extracted from grape leaves not infected with GINV was used as a negative control and water was used as a blank control.
2. RT-LAMP color development method
Using NEB by RT-LAMP color development
And (3) carrying out RT-LAMP on different variants of GINV by the LAMP allochroic premixed solution. Total RNA extracted from grape leaves not infected with GINV was used as a negative control and water was used as a blank control.
The RT-LAMP chromogenic method reaction system is 10 mu L: mu.L of WarmStart Colorimetric LAMP 2 × Master Mix (NEB, M1800L), 1. Mu.L of primer mixture, 3.6. Mu.L of deionized water, and 0.4. Mu.L of RNA template. The primer mixture in the reaction system was the primer mixture prepared from the optimal primer combination CP set3 selected in example 1.
The reaction conditions of the RT-LAMP chromogenic method are as follows: reacting at 65 ℃ for 1h. And (3) after the reaction is finished, judging whether the sample to be detected contains GINV or not by observing the color of the reaction system by naked eyes: if the color of the reaction system is yellow, the sample to be detected contains GINV; and if the color of the reaction system is pink, the sample to be detected does not contain GINV. And carrying out agarose gel electrophoresis detection on the amplification product by the RT-LAMP color development method.
3. Results of RT-LAMP fluorescence method and RT-LAMP chromogenic method for detecting different variants of GINV
The results show that: distinct amplification curves were seen for 3 different variants of GINV in RT-LAMP fluorescence (FIG. 2A), yellow in RT-LAMP chromogenic (FIG. 2B), no amplification curve and no yellowing for the negative and blank controls. The results of the RT-LAMP color-rendering amplification products showed that the RT-LAMP amplification products of 3 different varieties of GINV all showed a ladder-shaped amplification band, while the negative control and the blank control showed no amplification band (FIG. 2C). The above results indicate that RT-LAMP reaction based on primer combination CP set3 can effectively detect 3 different variants of GINV.
Example 3 specific detection
RT-LAMP was performed on grape samples infected with different viruses by RT-LAMP fluorescence method and RT-LAMP chromogenic method in example 2, and the specificity of CP set3, a primer combination of the present invention, was examined.
Test RNA samples: total RNA extracted from grape leaf samples infected with different viruses No. 1-8, respectively.
Sample No. 1: grape leaves infected with GLRaV-2, GLRaV-3, GFkV, GFabV and GRSPaV.
Sample No. 2: grape leaves infected with GLRaV-1, GLRaV-2, GLRaV-3 and GFkV.
Sample No. 3: grape leaves infected with GLRaV-3, GFkV, GVE and GFLV.
Sample No. 4: grape leaves infected with GLRaV-4 and GLRaV-7.
Sample No. 5: grape leaves infected with GRSPaV, GVA and GFkV.
Sample No. 6: grape leaves infected with GRSPaV, GLRaV-3, GVB and GPGV.
Sample No. 7 (positive control): leaves of grapes infected with GINV.
Sample No. 8 (negative control): grape leaves not infected with GINV.
The results show that: only the leaves of grapes infected with GINV showed normal amplification curve and color change, while none of the samples infected with other viruses showed significant amplification curve and no color change (fig. 3A, B, C). The primer combination CP set3 of the invention has good specificity and can specifically detect GINV.
Example 4 sensitivity detection
RT-LAMP was performed on RNA samples with different concentrations of GINV by the RT-LAMP chromogenic method and the conventional RT-PCR method in example 2, and the sensitivity of 2 detection methods was compared.
Test RNA samples: taking total RNA extracted from leaves of grapes infected with GINV as stock solution, diluting the stock solution with sterilized pure water in a gradient manner by 10 times, and diluting 10 times 0 、10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 、10 -8 The stock solution was used as the test RNA sample.
1. RT-LAMP color development method
Using NEB by RT-LAMP color development
LAMP color-changing premix liquid pair differenceThe concentration of the test RNA sample was subjected to RT-LAMP. By ddH 2 O as a control. The reaction system and reaction conditions of the RT-LAMP color development method are the same as those in the second step of the example 2.
2. Conventional RT-PCR method
The conventional RT-PCR method is to perform RT-PCR reaction using a One-step RT-PCR Kit (PrimerScript One step RT-PCR Kit Ver.2, TAKARA). The reaction system (total volume 25. Mu.L) was as follows: 2X 1step Buffer 12.5. Mu.L, 10mmol/L of each primer 0.5. Mu.L (primers GINV MP1 a/1b. The reaction procedure is as follows: 30min at 50 ℃; 5min at 94 ℃; circulation is carried out for 35 times at 94 ℃ 40sec,56 ℃ 30sec and 72 ℃ 1 min; 7min at 72 ℃; the reaction was terminated at 4 ℃.
The results show that: RNA samples were diluted to 10 -5 In time, GINV was still detected by RT-LAMP chromogenic assay (FIGS. 4A and 4B), whereas the conventional RT-PCR assay only detected dilution to 10 -3 The RNA sample of (1) (FIG. 4C). Therefore, the sensitivity of the RT-LAMP chromogenic method established by the invention is 100 times that of the conventional RT-PCR method.
Example 5 detection of GINV in tissues of different parts of Vitis vinifera by direct RT-LAMP color development
The invention establishes a direct RT-LAMP color development method without RNA extraction, and verifies the method through different grape samples infected with GINV.
A first sample to be tested: 3 GINV infected grape leaves, respectively numbered 1, 2 and 3; 3 grape leaves not infected with GINV, numbered 4, 5 and 6, respectively.
A second sample to be tested: upper young leaves, upper petioles, middle leaves, middle petioles, lower old leaves, lower old petioles and grape fruits of the grape bearing branch infected with GINV.
Direct RT-LAMP color development: immediately after puncturing the sample 3 with a 10. Mu.L tip of a pipette tip, the tip of the pipette tip with the tissue fluid was gently dipped into a 1.5mL centrifuge tube containing 10. Mu.L of RNase-free deionized water for 3 times. And (3) uniformly mixing the liquid in the centrifugal tube, and performing RT-LAMP as a template for RT-LAMP chromogenic reaction. The reaction system and reaction conditions of the RT-LAMP color development method are the same as those in the second step of example 2. Taking total RNA extracted from grape leaves infected with GINV as a positive control; total RNA extracted from grape leaves that were not infected with GINV was used as a negative control.
The detection result of the first test sample shows that: all 3 positive samples and positive controls turned pink to yellow, while none of the 3 negative samples and negative controls changed (fig. 5A). The electrophoresis result of the RT-LAMP amplification product is consistent with the color development result (FIG. 5B). The direct RT-LAMP color development method established by the invention can be used for effectively and rapidly detecting the GINV in the grape sample.
The detection result of the second sample shows that: the upper young leaf, upper petiole, middle leaf, middle petiole, lower old petiole, and grape fruit samples of the grape bearing shoot all turned pink to yellow, while the negative control sample was pink (fig. 6). The direct RT-LAMP color development method established by the invention can effectively detect tissues of different parts of grape fruiting branches, such as upper tender leaves, upper petioles, middle leaves, middle petioles, lower old petioles, grape fruits and the like, and has a wide application range.
Example 6 field sample testing
The direct RT-LAMP color development method in the example 5 and the conventional RT-PCR method in the example 4 are respectively adopted to carry out GINV detection on the grape samples collected in the field, and the effects of the 2 methods in field actual sample detection are compared.
A sample to be tested: 44 field harvested grape leaves.
The results show that: the detection rate of the direct RT-LAMP color development method is 61.4% (27/44) (FIG. 7A), which is higher than the detection rate of 52.3% (23/44) of the conventional RT-PCR method (FIG. 7B). The direct RT-LAMP color development method established by the invention has better applicability.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, it is possible to make various improvements and modifications without departing from the technical principle of the present invention, and these improvements and modifications should also be considered as the protection scope of the present invention.
Sequence listing
<110> research institute for fruit trees of Chinese academy of agricultural sciences
LAMP visual detection primer and kit for grape intra-berry necrosis virus and application of LAMP visual detection primer and kit
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Claims (10)
1. The complete set of primer group for detecting or assisting in detecting the grape intra-berry necrosis virus consists of a primer 1-a primer 6;
the primer 1 is a single-stranded DNA molecule shown as a sequence 1 in a sequence table;
the primer 2 is a single-stranded DNA molecule shown in a sequence 2 in a sequence table;
the primer 3 is a single-stranded DNA molecule shown as a sequence 3 in a sequence table;
the primer 4 is a single-stranded DNA molecule shown as a sequence 4 in a sequence table;
the primer 5 is a single-stranded DNA molecule shown as a sequence 5 in a sequence table;
the primer 6 is a single-stranded DNA molecule shown as a sequence 6 in a sequence table.
2. The set of primers according to claim 1, wherein: the molar ratio of the primer 1, the primer 2, the primer 3, the primer 4, the primer 5 and the primer 6 is 1.
3. The use of the primer set of claim 1 or 2 in any one of the following b 1) to b 8):
b1 Preparing a product for detecting or assisting in detecting the grape berry necrosis virus;
b2 Detecting or aiding in the detection of grape intra-berry necrotic virus;
b3 Preparing a product for detecting or assisting in detecting whether a sample to be detected is infected with the grape berry intra-necrosis virus or not;
b4 Detecting or assisting to detect whether the sample to be detected is infected with the grape berry necrosis virus;
b5 Preparing a product for identifying or assisting in identifying whether the pathogenic microorganism to be detected is grape intra-berry necrosis virus;
b6 Identifying or assisting in identifying whether the pathogenic microorganism to be detected is grape intra-berry necrotic virus;
b7 Preparing a product for identifying or assisting in identifying the necrotic virus in grape berries from other viruses;
b8 To identify or aid in identifying necrotic viruses within grape berries from other viruses.
4. A kit comprising the primer set according to claim 1 or 2;
the kit has the functions of any one of c 1) to c 4) as follows:
c1 Detecting or aiding in the detection of grape intra-berry necrotic virus;
c2 Detecting or assisting to detect whether the sample to be detected is infected with the grape berry necrosis virus;
c3 Identifying or assisting in identifying whether the pathogenic microorganism to be detected is grape intra-berry necrotic virus;
c4 To identify or aid in identifying necrotic viruses within grape berries from other viruses.
5. The method for preparing the kit according to claim 4, wherein the method is d 1) or d 2):
d1 Separately packaging each primer in the primer set of claim 1 or 2;
d2 The primers in the primer set of claim 1 or 2 are mixed together in proportion.
6. The method of claim 5, wherein: the primer 1, the primer 2, the primer 3, the primer 4, the primer 5 and the primer 6 in the set of primer sets are mixed together in a molar ratio of 1.
7. A method for detecting or assisting in detecting whether a sample to be detected is infected with grape intra-berry necrotic virus is the following e 1) or e 2):
e1 Extracting nucleic acid of a sample to be detected, performing RT-LAMP by using the nucleic acid of the sample to be detected as a template and the primer set combination according to claim 1, and judging whether the sample to be detected is infected with grape berry necrosis virus or not by observing the color of a reaction system by naked eyes after the RT-LAMP reaction is finished: if the color of the reaction system is yellow, infecting the sample to be detected or infecting the necrotic virus in grape berries by candidates; if the color of the reaction system is pink, the sample to be detected is not infected or candidate uninfected grape berry necrosis viruses;
e2 Taking a sample tissue fluid to be detected, directly taking the sample tissue fluid to be detected as a template, carrying out RT-LAMP by adopting the primer set combination set according to claim 1, and judging whether the sample to be detected is infected with the grape berry necrosis virus or not by observing the color of a reaction system by naked eyes after the RT-LAMP reaction is finished: if the color of the reaction system is yellow, infecting the sample to be detected or infecting the necrotic virus in grape berries by candidates; if the color of the reaction system is pink, the sample to be detected is not infected or candidate uninfected grape berry necrosis virus.
8. A method for identifying or assisting in identifying whether a pathogenic microorganism to be detected is grape intra-berry necrosis virus or not comprises the following steps: extracting nucleic acid of pathogenic microorganism to be detected, performing RT-LAMP by using the nucleic acid of the pathogenic microorganism to be detected as a template and the primer set combination set according to claim 1, and judging whether the pathogenic microorganism to be detected is grape intra-berry necrosis virus or not by observing the color of a reaction system by naked eyes after the RT-LAMP reaction is finished: if the color of the reaction system is yellow, the pathogenic microorganism to be detected is or is selected as grape berry necrosis virus; if the color of the reaction system is pink, the pathogenic microorganism to be detected is not or is not candidate to be the grape berry necrosis virus.
9. A method for identifying or assisting in identifying grape berry necrosis virus from other viruses, comprising the steps of: extracting nucleic acid of a virus to be detected, performing RT-LAMP by using the nucleic acid of the virus to be detected as a template and the primer set combination according to claim 1, and judging whether the virus to be detected is the grape berry necrosis virus or other viruses by observing the color of a reaction system by naked eyes after the RT-LAMP reaction is finished: if the color of the reaction system is yellow, the virus to be detected is or is selected as grape berry necrosis virus; if the color of the reaction system is pink, the virus to be detected is or is selected as other viruses.
10. The method according to any one of claims 7-9, wherein: the reaction conditions of the RT-LAMP are as follows: reacting at 65 ℃ for 1h.
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| CN104894125A (en) * | 2015-06-19 | 2015-09-09 | 中国检验检疫科学研究院 | RT-LAMP (reverse transcription loop-mediated isothermal amplification) reagent box detecting grapevine virus A and special primer thereof |
| CN109266643A (en) * | 2018-09-30 | 2019-01-25 | 中国农业科学院果树研究所 | Necrosis virus infectious clone and its construction method in grape berry |
| CN109295256A (en) * | 2018-10-14 | 2019-02-01 | 中国农业科学院果树研究所 | The real-time fluorescence quantitative RT-PCR detection method and kit of Grapevine virus A |
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| CN104894125A (en) * | 2015-06-19 | 2015-09-09 | 中国检验检疫科学研究院 | RT-LAMP (reverse transcription loop-mediated isothermal amplification) reagent box detecting grapevine virus A and special primer thereof |
| CN109266643A (en) * | 2018-09-30 | 2019-01-25 | 中国农业科学院果树研究所 | Necrosis virus infectious clone and its construction method in grape berry |
| CN109295256A (en) * | 2018-10-14 | 2019-02-01 | 中国农业科学院果树研究所 | The real-time fluorescence quantitative RT-PCR detection method and kit of Grapevine virus A |
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