WO2020156102A1 - Ic-rt-lamp reagent kit for detecting lettuce necrotic yellows virus and detection method thereof - Google Patents

Ic-rt-lamp reagent kit for detecting lettuce necrotic yellows virus and detection method thereof Download PDF

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WO2020156102A1
WO2020156102A1 PCT/CN2020/071512 CN2020071512W WO2020156102A1 WO 2020156102 A1 WO2020156102 A1 WO 2020156102A1 CN 2020071512 W CN2020071512 W CN 2020071512W WO 2020156102 A1 WO2020156102 A1 WO 2020156102A1
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lnyv
lamp
primer
lettuce
detection
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Chinese (zh)
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张玉宝
王亚军
谢忠奎
郭志鸿
邱阳
何玉惠
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中国科学院西北生态环境资源研究院
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Priority to CA3165373A priority Critical patent/CA3165373C/en
Priority to AU2020214373A priority patent/AU2020214373B2/en
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    • C12Q1/6844—Nucleic acid amplification reactions
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Definitions

  • the invention relates to an IC-RT-LAMP kit for specifically detecting lettuce necrotic yellowing virus LNYV and a detection method thereof.
  • Lettuce (Lactuca sativa) is a leaf-use type of lettuce species in the Compositae family. It is also known as goose and lettuce. It belongs to annual and biennial vegetables. It is one of the vegetables with high nutritional value, and it is also one of the vegetables loved by the people of our country. One. In recent years, lettuce has become more and more popular among people, especially after discovering some of its effects, the planting area of lettuce has gradually expanded. At the same time, growers are also facing a series of disease problems, among which virus diseases are relatively common diseases. Widespread; open field cultivation is harmful, with an incidence rate of up to 60% or even higher.
  • the virus After the virus infects the lettuce, it destroys the lettuce's nutrients and clears the channels, changes the lettuce organs or disrupts the balance of tissue metabolism, and inhibits photosynthesis. Plants have a greater impact on the onset of disease in the seedling stage, mainly showing mosaic and leaf mottled shapes, while the leaves are shrunken and crooked, and there may be open veins. In severe cases, irregular gray-brown necrotic spots may appear; adult plants are sick. The leaves are shrunken, and the bubble-like protrusions on the leaf surface are very uneven, the growth of the plant is inhibited, and obvious dwarfing appears. Even the veins become brown or brown necrotic spots appear, which seriously affects the quality and yield.
  • PVS Potatovirus S
  • LMV Lettuce mosaic virus
  • DYMV Dandelion yellow mosaic virus
  • CMV Cucumber mosaic virus
  • LNYV is a typical member of the genus Cytoplasma Rhabdovirus.
  • the virus particles are slug or multi-shaped. After fixation, they are mostly bacillus-like, with a length of 200-350nm and a diameter of 70-95nm.
  • the viral nucleic acid is a single molecule linear negative sense ssRNA, length 11 -15kb, the protein contains 5 structural proteins, namely protein L (molecular weight 160-180kDa), protein G (78kDa), protein N (57kDa), protein NS (38kDa) and protein M (19kDa).
  • the host of LNYV is limited to some plants of Chenopodiaceae, Compositae, Leguminosae, Liliaceae and Solanaceae. Aphids are the main transmission mediators. LNYV has been reported in Australia and New Zealand earlier; this time LNYV was detected on lettuce in Lanzhou. Reported for the first time in China.
  • LNYV enzyme-linked immunosorbent assay
  • RT-PCR reverse transcription polymerase chain reaction
  • the sensitivity of the ELISA method is not high enough, and non-specific reactions are prone to occur. It must also rely on professional equipment such as a microplate reader; the RT-PCR method has strong detection specificity and high sensitivity, but compared with the present invention, it needs to extract high-quality RNA and operate It is complicated, the reagents required are expensive, and the technical level of the inspectors is high. In addition, it is necessary to rely on the PCR machine and other expensive molecular biology professional instruments and equipment, and the popularization is greatly restricted.
  • LAMP loop-mediated isothermal amplification of DNA
  • This technology relies on primers that can recognize multiple specific regions on the target sequence and a Bst DNA enzyme with unwinding function and cascade amplification to rapidly and specifically amplify target genes under constant temperature conditions.
  • the product is a mixture of a stem-loop structure composed of a series of repeated target sequences and a DNA fragment with a polycyclic cauliflower-like structure, which is a special stepped band in the electrophoresis gel.
  • the pyrophosphate ions precipitated from dNTPs combine with Mg 2+ in the reaction solution to produce a by-product (magnesium pyrophosphate) to form a milky white precipitate. Add the color developing solution to determine the result by visual observation.
  • LAMP technology can also be used to detect RNA, and reverse transcriptase can be added to the reaction system for RT-LAMP.
  • LAMP technology can complete the amplification reaction in a constant temperature water bath. It has the advantages of simple operation, strong specificity, and high sensitivity. It is used in food safety monitoring, animal and plant pathogens and medical pathogen detection. widely used.
  • an object of the present invention is to provide a lettuce necrotic yellowing virus LNYV IC- RT-LAMP specific detection primer composition
  • Another object of the present invention is to provide the IC-RT-LAMP detection kit for the above-mentioned lettuce necrotic yellowing virus LNYV;
  • Another object of the present invention is to provide an IC-RT-LAMP method for detecting the above-mentioned lettuce necrotic yellowing virus LNYV.
  • An IC-RT-LAMP detection primer composition for the specific detection of lettuce necrotic yellowing virus LNYV consisting of RT reverse primer LNYV-R and a LAMP primer set, the LAMP primer set is composed of forward and outward primers F3, The reverse outer primer B3, the forward inner primer FIP, the reverse inner primer BIP, the forward loop primer LF and the reverse loop primer LB are composed; the specific sequence of each primer is as follows:
  • FIP 5’-CTGTTAGATACTCCCGCCTGCGACACCTCCTAACACCTCCTT-3’;
  • An IC-RT-LAMP kit for specific detection of lettuce necrotic yellowing virus LNYV including LNYV antibody IgG, RT reverse primer LNYV-R, LAMP primer set, first-strand cDNA synthesis reagent and LAMP amplification reaction reagent,
  • the specific LAMP primer set includes forward-outer primer F3, reverse outer primer B3, forward inner primer FIP, reverse inner primer BIP, forward loop primer LF and reverse loop primer LB, the above-mentioned specific RT reverse primer
  • the sequence of LNYV-R and LAMP primer set is as follows:
  • FIP 5’-CTGTTAGATACTCCCGCCTGCGACACCTCCTAACACCTCCTT-3’;
  • the first strand cDNA synthesis reagent consists of 10mM dNTPs, 5 ⁇ M-MLV reaction buffer, 30U/ ⁇ L RNase inhibitor, 200U/ ⁇ L M-MLV reverse transcriptase and DEPC-H 2 O
  • the LAMP amplification reaction reagent is composed of 10mM dNTPs, 10 ⁇ Thermpopol reaction buffer, 100mM MgSO 4 , 8U/ ⁇ L Bst DNA polymerase and ddH 2 O.
  • the kit of the present invention also includes LNYV antibody IgG coating buffer (CB), phosphate buffer (PBS), phosphate washing buffer (PBST), fluorescent dye detection solution, negative control substance and positive control substance.
  • the antibody coating buffer CB is a carbonate buffer with a concentration of 0.05M and a pH of 9.6; the concentration of the phosphate buffer (PBS) and phosphate washing buffer (PBST) is 0.02M, pH The value is 7.4; the fluorescent dye detection solution is 1000 ⁇ SYBR Green I; the negative control is ddH 2 O; the positive control is the LNYV protein N gene standard; the sequence of the positive control is as follows:
  • An IC-RT-LAMP detection method for specifically detecting lettuce necrotic yellowing virus LNYV includes the following steps:
  • Step 1 Antibody preparation: express and purify the LNYV protein N genetic engineering fusion protein, and use it to immunize animals to obtain specific LNYV antibody IgG.
  • Step 2Immune capture IC Coat the specific LNYV antibody IgG obtained in step 1 in a reaction container to capture the LNYV particles in the sample to be tested.
  • Step 3Reverse transcription RT Using the LNYV particles captured in step 2 as a template, use LNYV's RT reverse primer LNYV-R and the first strand cDNA synthesis reagent to perform reverse transcription RT reaction to obtain the first strand cDNA.
  • Step 4LAMP amplification use the first-strand cDNA obtained in step 3 as a template, use LAMP specific primer set and LAMP amplification reaction reagents for LAMP amplification; use ddH 2 O as a negative control, and use LNYV protein N gene standard As a positive control.
  • Step 5Analyze and judge the reaction product Use the LAMP amplification reaction product obtained in Step 4 to determine whether the sample to be tested contains lettuce necrotic yellowing virus.
  • the LNYV antibody in step 1 can be a polyclonal antibody or a monoclonal antibody, which can be self-made or obtained from commercial sources;
  • Step 4 The reaction system for LAMP amplification (take the reaction system 12.5 ⁇ L as an example) is as follows: 50ng cDNA 0.5 ⁇ L, 10 ⁇ Thermpopol buffer 1.25 ⁇ L, 100mM MgSO 4 0.75 ⁇ L, 10mM dNTPs 1.75 ⁇ L, 10 ⁇ M B3 and F3Primer 0.25 each ⁇ L, 10 ⁇ M FIP and BIP Primer each 2.0 ⁇ L, 10 ⁇ M LF and LB Primer each 0.5 ⁇ L, 8U/ ⁇ L Bst DNA polymerase 0.5 ⁇ L, and ddH 2 O make up to 12.5 ⁇ L.
  • Step 4 The reaction conditions of the LAMP amplification are: amplification in a constant temperature water bath at 58-70°C for 60-100 minutes, followed by denaturation at 80°C for 10 minutes to terminate the reaction.
  • step 5 the specific method for judging whether the virus particle is lettuce necrotic yellowing virus according to the amplification result is a fluorescent dye macroscopic observation method or agarose gel electrophoresis detection technology.
  • the method of visual observation of the fluorescent dye is: take the SYBR Green I fluorescent dye reaction solution and add it to the reaction product of the LAMP amplification in step 4, and observe directly with the naked eye. If the amplified product appears green, it means that the SYBR Green I dye and double-stranded DNA Binding, it is a positive reaction, indicating that the sample contains LNYV; if the color of the amplified product is orange, it is a negative reaction, indicating that the sample does not contain LNYV.
  • the agarose gel electrophoresis detection technology is: the LAMP amplified product in step 4 is subjected to 2.0% agarose gel electrophoresis. If there is a brightly diffused ladder-like nucleic acid band in the gel, it is a positive reaction, indicating a sample Contains LNYV; if there is no nucleic acid band in the gel, it is a negative reaction, which means that the sample does not contain LNYV.
  • the invention organically combines the immune capture IC and the LAMP technology, and successfully establishes a method for specifically detecting the lettuce necrotic yellowing virus LNYV with the IC-RT-LAMP technology.
  • the detection object is a complete virus particle
  • the specific pathogen antigen is captured by a solid-phased specific antibody
  • the pathogen genome sequence specific primer is used for amplification
  • the amplified product is detected and analyzed Achieve the detection of intact pathogens.
  • the IC-RT-LAMP detection method organically combines serological methods and molecular biology methods with strong specificity and a sensitivity of 3.5 pg/mL, which is 100 times higher than ordinary PCR. It does not require RNA extraction or professional expertise. The equipment simplifies the operation process, reduces the detection difficulty, and improves the detection efficiency.
  • the present invention has the following advantages and positive effects:
  • the IC-RT-LAMP method for specific detection of lettuce necrotic yellowing virus provided by the present invention overcomes the poor specificity, low sensitivity, and high difficulty of extracting RNA in the detection method of lettuce necrotic yellowing virus in the prior art. Problems such as expensive reagents and the need for thermal cycling equipment.
  • the present invention organically combines serology and LAMP amplification technology to give full play to the advantages of the two detection methods.
  • the captured LNYV virus particles are directly used as the detection object for RT-LAMP amplification, which avoids RNA extraction and reduces Experiment difficulty.
  • the present invention uses high-quality LNYV antibody IgG to capture LNYV particles in the sample to be tested, which improves the specificity of detection and reduces false positives.
  • the LAMP reaction specifically recognizes 8 sequences of the target gene (LNYV protein N) through 6 primers (F3, B3, FIP, BIP, LF, LB), with strong specificity, and the sensitivity reaches 3.5 pg/mL, which is higher than ordinary PCR 100 times.
  • the present invention gets rid of the dependence on thermal cycling equipment, and the LAMP reaction only needs to occur in a constant temperature water bath, which greatly expands the application range of the method. After the reaction is over, the result can be directly judged by the naked eye through the color change, which makes the detection result more intuitive, thereby increasing the application value of the detection.
  • the present invention provides a new technology platform for lettuce virus disease detection, which can be used for rapid detection of lettuce necrotic yellowing virus LNYV, and can also be used to monitor the occurrence, spread and prevalence of LNYV virus, and is very suitable for popularization and application at the grassroots level.
  • Figure 1 is an example of the present invention IC-RT-LAMP detection of lettuce necrotic yellow virus LNYV specific color diagram;
  • Figure 2 shows the specificity of IC-RT-LAMP for detection of lettuce necrotic yellowing virus LNYV by electrophoresis analysis according to the embodiment of the present invention
  • M 600bp Marker
  • 1 negative control
  • 2-7 respectively correspond to: LSV susceptible tissue; CMV Affected tissue; LMoV susceptible tissue; ArMV susceptible tissue; JHMV susceptible tissue; 7: Lettuce LNYV susceptible tissue;
  • Figure 3 is an electrophoresis detection diagram of the results of the IC-RT-LAMP reaction temperature optimization test for lettuce necrotic yellowing virus in the embodiment of the present invention
  • Figure 4 is an electrophoresis detection diagram of the results of the lettuce necrotic yellowing virus IC-RT-LAMP reaction time optimization test in the embodiment of the present invention
  • Figure 5 shows the sensitivity and color development diagram of IC-RT-LAMP for detection of lettuce necrotic yellowing virus LNYV;
  • Figure 6 shows the sensitivity of IC-RT-LAMP to detect lettuce necrotic yellowing virus LNYV by electrophoresis analysis according to the embodiment of the present invention
  • M 600bp Marker
  • 1 negative control
  • 2-7 respectively correspond to the concentration of LNYV protein N gene standard product :10 -1 (3.5 ⁇ 10 4 ng/mL); 10 -3 (3.5 ⁇ 10 2 ng/mL); 10 -5 (3.5 ⁇ 10 0 ng/mL); 10 -7 (3.5 ⁇ 10 -2 ng /mL); 10 -8 (3.5 ⁇ 10 -3 ng/mL); 10 -9 (3.5 ⁇ 10 -4 ng/mL);
  • Figure 7 shows the sensitivity of RT-PCR to detect lettuce necrotic yellowing virus LNYV by electrophoresis analysis according to the embodiment of the present invention
  • 2-7 respectively correspond to 2-7 respectively correspond to LNYV protein N gene Standard concentration: original times (3.5 ⁇ 10 5 ng/mL); 10 -1 (3.5 ⁇ 10 4 ng/mL); 10 -3 (3.5 ⁇ 10 2 ng/mL); 10 -5 (3.5 ⁇ 10 0 ng/mL); 10 -6 (3.5 ⁇ 10 -1 ng/mL); 10 -7 (3.5 ⁇ 10 -2 ng/mL).
  • LNYV-F specific forward
  • LNYV-R reverse primers
  • the 10 ⁇ L RT reaction system is as follows: total RNA 2 ⁇ L, 10 ⁇ M LNYV specific reverse primer LNYV-R 1 ⁇ L, DEPC-H 2 O 3 ⁇ L, denatured at 70°C for 10min, quickly place on ice and chill for 2min; then add 5 ⁇ M-MLV buffer 2 ⁇ L , 10mM dNTPs 1 ⁇ L, 30U/ ⁇ L RNase inhibitor 0.34 ⁇ L, 200U/ ⁇ L M-MLV reverse transcriptase 0.35 ⁇ L and DEPC-H 2 O 0.31 ⁇ L; after mixing, 42°C water bath for 1h, 70°C for 15min, put on ice stand-by;
  • the PCR reaction system is 12.5 ⁇ L, including: 50ng cDNA 0.5 ⁇ L, 5U/ ⁇ L Ex Taq DNA polymerase 0.1 ⁇ L, 10 ⁇ PCR buffer 1.25 ⁇ L, 2.5mM dNTPs 1 ⁇ L, 10 ⁇ M forward primer LNYV-F 0.25 ⁇ L, 10 ⁇ M reverse Primer LNYV-R 0.25 ⁇ L, and ddH 2 O 9.15 ⁇ L;
  • PCR amplification conditions are: pre-denaturation at 94°C for 3min; denaturation at 94°C for 30s, annealing at 52°C for 45s, extension at 72°C for 1min, 35 cycles of amplification, and finally extension at 72°C for 10min;
  • the target fragment was recovered, and the target fragment was ligated to the pMD18-T vector using a cloning vector kit, transformed into DH5 ⁇ competent cells, and screened by blue-white plate; 3 randomly selected White spot colonies were respectively inoculated on ampicillin LB medium and shaken at 37°C for 12-16 hours; plasmids were extracted with plasmid micro-extraction kit; 2 ⁇ L of plasmids were taken respectively, and PCR amplification was carried out under the same conditions as the above PCR reaction system; The positive recombinant plasmids obtained by PCR detection were sequenced; the positive plasmids with completely correct sequence confirmed by sequencing were the standard products.
  • the fragment lengths corresponding to the LNYV protein N gene of lettuce were 614bp respectively; the NanoDrop ND-1000 nucleic acid/protein analyzer was used to determine the standard products OD 260 nm and OD 280 nm values, calculate the plasmid concentration based on the OD 260 nm and OD 280 nm values;
  • Example 2 An IC-RT-LAMP detection kit for the specific detection of lettuce necrotic yellowing virus LNYV
  • the kit consists of LNYV antibody IgG, antibody coating buffer (CB), phosphate buffer (PBS), phosphate washing buffer (PBST), RT reverse primer LNYV-R, LAMP primer set, first strand cDNA synthesis reagent , LAMP amplification reaction reagent, fluorescent dye detection solution, negative control substance and positive control substance.
  • the specific LAMP primer set includes forward outer primer F3, reverse outer primer B3, forward inner primer FIP, and reverse inner primer
  • the sequences of BIP, forward loop primer LF and reverse loop primer LB, the above-mentioned specific RT reverse primer LNYV-R and LAMP primer set are as follows:
  • FIP 5’-CTGTTAGATACTCCCGCCTGCGACACCTCCTAACACCTCCTT-3’;
  • the first strand cDNA synthesis reagent is composed of 10mM dNTPs, 5 ⁇ M-MLV reaction buffer, 30U/ ⁇ L RNase inhibitor, 200U/ ⁇ L M-MLV reverse transcriptase and DEPC-H 2 O;
  • LAMP amplification reaction reagents consist of 10mM dNTPs, 10 ⁇ Thermpopol reaction buffer, 100mM MgSO 4 , 8U/ ⁇ L Bst DNA polymerase and ddH 2 O;
  • the antibody coating buffer CB is a carbonate buffer with a concentration of 0.05M and a pH of 9.6; the concentration of the phosphate buffer (PBS) and phosphate washing buffer (PBST) is 0.02M, and the pH is 7.4;
  • the fluorescent dye detection solution is 1000 ⁇ SYBR Green I; the negative control substance is ddH 2 O; the positive control substance is the lettuce LNYV protein N gene standard substance.
  • Example 3 Establishment of a method for detection of lettuce necrotic yellow virus LNYV by immunocapture IC-RT-LAMP
  • LNYV protein N gene fusion protein Total RNA was extracted from the leaves of lettuce infected with LNYV in Lanzhou area to perform reverse transcription polymerase chain reaction (RT-PCR) to amplify the protein N gene fragment of LNYV. Cloned into pET-28a vector by restriction enzyme digestion. The recombinant plasmid was transformed into E. coli BL21, cultured at 37°C, IPTG induced expression, and purified by affinity chromatography on a nickel column to obtain a 22.5kDa LNYV protein N gene fusion protein;
  • RT-PCR reverse transcription polymerase chain reaction
  • the protein antigen and Freund’s complete adjuvant are mixed in equal volumes and then injected into multiple points under the skin; two weeks later, booster immunization is performed, and the protein antigen and Freund’s incomplete adjuvant are fully mixed in equal volumes and subcutaneously Multi-point injections; booster immunization every two weeks thereafter, collect blood from the carotid artery 5-7 days after the fourth booster immunization, calm down, centrifuge, and add 0.02% sodium azide to the collected serum at -20°C Save; the obtained antiserum was crudely extracted by ammonium sulfate precipitation with three saturations of 20%, 50%, and 33%, dialyzed to pH 7.8 phosphate buffer, and then purified with DE52 anion exchange column to obtain rabbit anti-LNYV Polyclonal antibody IgG;
  • the reaction system is 12.5 ⁇ L, including 50ng cDNA 0.5 ⁇ L, 10 ⁇ Thermpopol buffer 1.25 ⁇ L, 100mM MgSO 4 0.75 ⁇ L, 10mM dNTPs 1.75 ⁇ L, 10 ⁇ M B3 And F3Primer 0.25 ⁇ L each, 10 ⁇ M FIP and BIP Primer each 2.0 ⁇ L, 10 ⁇ M LF and LB Primer each 0.5 ⁇ L, 8U/ ⁇ L Bst DNA polymerase 0.5 ⁇ L, ddH 2 O2.25 ⁇ L; and ddH 2 O as a negative control, with lettuce LNYV protein N gene standard as a positive control;
  • the reaction temperature of LAMP is shown in lanes 3-8 in Figure 3, which is 58-70°C, and the amplification time of LAMP is shown in lanes 4-6 in Figure 4, which is 60-100 minutes, followed by denaturation at 80°C for 10 minutes to terminate the reaction ;
  • the observation of the results can also use agarose gel electrophoresis detection technology: take 5 ⁇ L of the LAMP reaction product for 2.0% agarose gel electrophoresis, in 1 ⁇ TAE buffer environment, 100V stabilized electrophoresis for 30 minutes, and then use gel imaging The system observes and records the results:
  • Example 4 Detection of the specificity of lettuce necrotic yellowing virus LNYV by immunocapture IC-RT-LAMP
  • IC-RT-LAMP In order to analyze the specificity of immunocapture IC-RT-LAMP to detect the lettuce necrotic yellowing virus LNYV, the 6 main viruses that infect lily, angelica and lettuce (lily recessive virus-LSV, lily cucumber mosaic virus-CMV, lily mottle) Virus-LMoV, Arabidopsis Mosaic Virus-ArMV, Japanese Ceratophyllum Mosaic Virus-JHMV and Lettuce Necrotic Yellowing Virus-LNYV) were samples of infected leaves. The virus particles were captured with LNYV polyclonal antibody IgG, and the above implementation The IC-RT-LAMP detection system in the example reacts. With healthy lettuce leaves as a negative control, the experiment was repeated 3 times;
  • the standard LNYV protein N gene of lettuce in Example 1 was used as the sample, and the standard was determined by NanoDrop ND-1000 nucleic acid/protein analyzer After the concentration (3.5 ⁇ 10 5 ng/mL), the above standards were diluted 10-fold with DEPC-H 2 O and stored at -20°C as a template. Take 1.0 ⁇ L of each diluted solution diluted by 10 times as a template, and add the LAMP reaction reagents in the above examples to carry out LAMP amplification. The reaction procedure is 65°C for 100 min;
  • PCR amplification conditions are: pre-denaturation at 94°C for 3min; denaturation at 94°C for 30s, annealing at 52°C for 45s, extension at 72°C for 1min, cyclic amplification 35 times, and finally extension at 72°C for 10min;

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Abstract

Provided are an IC-RT-LAMP reagent kit for specifically detecting lettuce necrotic yellows virus and a detection method and use thereof. The reagent kit comprises an LNYV antibody IgG, an RT reverse primer LNYV-R, a LAMP primer set, a first strand cDNA synthesis reagent, a LAMP amplification reaction reagent, a fluorescent dye test solution, a negative control, and a positive control, the LAMP primer set being composed of a forward outer primer F3, a reverse outer primer B3, a forward inner primer FIP, a reverse inner primer BIP, a forward loop primer LF, and a reverse loop primer LB. The sensitivity of the detection method reaches 3.5 pg/mL, 100 times higher than ordinary PCR.

Description

一种检测莴苣坏死黄化病毒的IC-RT-LAMP试剂盒及其检测方法An IC-RT-LAMP kit for detecting lettuce necrotic yellowing virus and its detection method 技术领域Technical field
本发明涉及一种特异性检测莴苣坏死黄化病毒LNYV的IC-RT-LAMP试剂盒及其检测方法。The invention relates to an IC-RT-LAMP kit for specifically detecting lettuce necrotic yellowing virus LNYV and a detection method thereof.
背景技术Background technique
生菜(Lactuca sativa)属菊科莴苣种的叶用类型，别名鹅仔菜、莴仔菜，属一年生和二年生蔬菜，是具有很高营养价值的蔬菜之一，也是深受我国人民喜爱的蔬菜之一。近年来，生菜越来越受到人们的喜欢，特别是发现它的一些功效后，生菜的种植面积逐渐扩大，同时，种植户也面临着一系列病害问题，其中病毒病是比较常见的病害，分布广泛；露地种植危害重，发病率高达60％，甚至更高。Lettuce (Lactuca sativa) is a leaf-use type of lettuce species in the Compositae family. It is also known as goose and lettuce. It belongs to annual and biennial vegetables. It is one of the vegetables with high nutritional value, and it is also one of the vegetables loved by the people of our country. One. In recent years, lettuce has become more and more popular among people, especially after discovering some of its effects, the planting area of lettuce has gradually expanded. At the same time, growers are also facing a series of disease problems, among which virus diseases are relatively common diseases. Widespread; open field cultivation is harmful, with an incidence rate of up to 60% or even higher.
病毒侵染生菜后，破坏生菜的养分疏通通道，改变生菜器官或打乱组织代谢平衡，抑制光合作用。植株在幼苗期发病影响较大，主要表现出花叶斑驳状，同时叶片皱缩、歪扭，还有可能出现明脉，在严重的时候还会出现不规则灰褐色坏死斑；成株发病，叶片皱缩，叶面泡状突起很不平整，植株的生长受到抑制，出现明显的矮缩，甚至叶脉变成褐色或出现褐色坏死斑，严重影响着品质和产量。近年来，植物病毒侵染导致生菜大面积受害现象越来越严重，文献报道的感染生菜的主要病毒有马铃薯病毒属的马铃薯S病毒(Potato virus S,PVS)、马铃薯Y病毒属的莴苣花叶病毒(Lettuce mosaic virus,LMV)、蒲公英黄花叶病毒(Dandelion yellow mosaic virus,DYMV)和黄瓜花叶病毒(Cucumber mosaic virus,CMV)等。After the virus infects the lettuce, it destroys the lettuce's nutrients and clears the channels, changes the lettuce organs or disrupts the balance of tissue metabolism, and inhibits photosynthesis. Plants have a greater impact on the onset of disease in the seedling stage, mainly showing mosaic and leaf mottled shapes, while the leaves are shrunken and crooked, and there may be open veins. In severe cases, irregular gray-brown necrotic spots may appear; adult plants are sick. The leaves are shrunken, and the bubble-like protrusions on the leaf surface are very uneven, the growth of the plant is inhibited, and obvious dwarfing appears. Even the veins become brown or brown necrotic spots appear, which seriously affects the quality and yield. In recent years, plant virus infection has caused more and more serious damage to lettuce on a large scale. The main viruses that infect lettuce reported in the literature are Potatovirus S (Potatovirus S, PVS) and Lettuce mosaic of Potatovirus Y Virus (Lettuce mosaic virus, LMV), Dandelion yellow mosaic virus (DYMV) and Cucumber mosaic virus (CMV), etc.
2018年，中国科学院寒区旱区环境与工程研究所植物病毒研究组在甘肃兰州地区的生菜田里发现了一种新的感染生菜的植物病毒-莴苣坏死黄化病毒(Lettuce necrotic yellows virus,LNYV)。LNYV为细胞质弹状病毒属的典型成员，病毒粒子呈弹状或多形状，经过固定后大多为杆菌状，长200-350nm，直径70-95nm，病毒核酸为单分子线形负义ssRNA，长11-15kb，蛋白质包含5种结构蛋白，分别为蛋白L(分子质量160-180kDa)、蛋白G(78kDa)、蛋白N(57kDa)、蛋白NS(38kDa)和蛋白M(19kDa)。LNYV的寄主限于藜科、菊科、豆科、百合科和茄科的一些植物，蚜虫是其主要的传播介体，LNYV早先在澳大利亚和新西兰有报道；此次在兰州生菜上检测到的LNYV为中国首次报道。In 2018, the Plant Virus Research Group of the Cold and Arid Regions Environmental and Engineering Research Institute of the Chinese Academy of Sciences discovered a new plant virus that infects lettuce in the lettuce field in Lanzhou, Gansu Province-Lettuce necrotic yellows virus (LNYV) ). LNYV is a typical member of the genus Cytoplasma Rhabdovirus. The virus particles are slug or multi-shaped. After fixation, they are mostly bacillus-like, with a length of 200-350nm and a diameter of 70-95nm. The viral nucleic acid is a single molecule linear negative sense ssRNA, length 11 -15kb, the protein contains 5 structural proteins, namely protein L (molecular weight 160-180kDa), protein G (78kDa), protein N (57kDa), protein NS (38kDa) and protein M (19kDa). The host of LNYV is limited to some plants of Chenopodiaceae, Compositae, Leguminosae, Liliaceae and Solanaceae. Aphids are the main transmission mediators. LNYV has been reported in Australia and New Zealand earlier; this time LNYV was detected on lettuce in Lanzhou. Reported for the first time in China.
对于LNYV在我国生菜中分布、感病率以及对我国生菜的影响等基本信息，目前都不清楚。解决以上问题，急需开发一种特异、灵敏、实用且易于推广的分子检测技术。目前国际上针对LNYV的检测方法主要有酶联免疫吸附法(ELISA)和逆转录聚合酶链式反应(RT-PCR)，但这两种方法都存在一些缺陷。The basic information about the distribution of LNYV in Chinese lettuce, the susceptibility rate, and the impact on Chinese lettuce are currently unclear. To solve the above problems, there is an urgent need to develop a specific, sensitive, practical and easy to popularize molecular detection technology. The current international detection methods for LNYV mainly include enzyme-linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR), but both of these methods have some defects.
ELISA法的灵敏度不够高，易出现非特异性反应，还必须依赖酶标仪等专业设备；RT-PCR方法检测特异性强、灵敏度高、但与本发明相比，需要提取高质量的RNA，操作复杂，所需试剂昂贵，对检测人员的技术水平要求高，除此之外，还必须依赖PCR仪等昂贵的分子生物学专业仪器设备，推广普及受到很大限制。The sensitivity of the ELISA method is not high enough, and non-specific reactions are prone to occur. It must also rely on professional equipment such as a microplate reader; the RT-PCR method has strong detection specificity and high sensitivity, but compared with the present invention, it needs to extract high-quality RNA and operate It is complicated, the reagents required are expensive, and the technical level of the inspectors is high. In addition, it is necessary to rely on the PCR machine and other expensive molecular biology professional instruments and equipment, and the popularization is greatly restricted.
相比之下，DNA环介导等温扩增技术(loop-mediated isothermal amplification of DNA,LAMP)是一种新型的核酸扩增技术。该技术依赖于能够识别靶序列上多个特异性区域的引物和一种具解旋功能且呈瀑布式扩增的Bst DNA酶在恒温条件下快速、高特异性地扩增靶基因，扩增产物是一系列反复重复的靶序列构成的茎环结构和多环花椰菜样结构的DNA片段的混合物，在电泳凝胶中呈阶梯状的特殊条带。在LAMP反应过程中，从dNTP析出的焦磷酸根离子与反应溶液中的Mg 2+结合，产生副产物(焦磷酸镁)形成乳白色沉淀，加入显色液，即可通过肉眼观察判定结果。LAMP技术也可以用来对RNA进行检测，而且可以在反应体系中加入反转录酶进行RT-LAMP。 In contrast, loop-mediated isothermal amplification of DNA (LAMP) is a new type of nucleic acid amplification technology. This technology relies on primers that can recognize multiple specific regions on the target sequence and a Bst DNA enzyme with unwinding function and cascade amplification to rapidly and specifically amplify target genes under constant temperature conditions. The product is a mixture of a stem-loop structure composed of a series of repeated target sequences and a DNA fragment with a polycyclic cauliflower-like structure, which is a special stepped band in the electrophoresis gel. During the LAMP reaction, the pyrophosphate ions precipitated from dNTPs combine with Mg 2+ in the reaction solution to produce a by-product (magnesium pyrophosphate) to form a milky white precipitate. Add the color developing solution to determine the result by visual observation. LAMP technology can also be used to detect RNA, and reverse transcriptase can be added to the reaction system for RT-LAMP.
与常规检测的PCR方法相比，LAMP技术在恒温水浴中即能完成扩增反应，具有操作简便、特异性强、灵敏度高等优势，在食品安全监测、动植物病原物和医学病原物检测中被广泛应用。Compared with conventional PCR methods, LAMP technology can complete the amplification reaction in a constant temperature water bath. It has the advantages of simple operation, strong specificity, and high sensitivity. It is used in food safety monitoring, animal and plant pathogens and medical pathogen detection. widely used.
但是，目前未见到将免疫捕获IC与RT-LAMP技术有机结合，成功建立IC-RT-LAMP技术特异检测莴苣坏死黄化病毒LNYV的方法。However, there is no organic combination of immune capture IC and RT-LAMP technology to successfully establish a method for specifically detecting lettuce necrotic yellowing virus LNYV with IC-RT-LAMP technology.
发明内容Summary of the invention
针对现有技术中莴苣坏死黄化病毒LNYV检测方法操作复杂、对操作人员技术水平要求高、所需仪器设备昂贵等问题，本发明的一个目的是提供一种莴苣坏死黄化病毒LNYV的IC-RT-LAMP特异性检测引物组合物；Aiming at the problems of complicated operation of the detection method of lettuce necrotic yellowing virus LNYV in the prior art, high technical requirements for operators, and expensive equipment and equipment required, an object of the present invention is to provide a lettuce necrotic yellowing virus LNYV IC- RT-LAMP specific detection primer composition;
本发明的另一目的是提供上述莴苣坏死黄化病毒LNYV的IC-RT-LAMP检测试剂盒；Another object of the present invention is to provide the IC-RT-LAMP detection kit for the above-mentioned lettuce necrotic yellowing virus LNYV;
本发明的又一目的是提供上述莴苣坏死黄化病毒LNYV的IC-RT-LAMP检测方 法。Another object of the present invention is to provide an IC-RT-LAMP method for detecting the above-mentioned lettuce necrotic yellowing virus LNYV.
为了实现上述发明目的，本发明采用的技术方案为：In order to achieve the above-mentioned purpose of the invention, the technical solution adopted by the present invention is:
一种用于特异性检测莴苣坏死黄化病毒LNYV的IC-RT-LAMP检测引物组合物：由RT反向引物LNYV-R和LAMP引物组组成，所述LAMP引物组由正向外引物F3、反向外引物B3、正向内引物FIP、反向内引物BIP、正向环引物LF和反向环引物LB组成；各引物序列具体如下：An IC-RT-LAMP detection primer composition for the specific detection of lettuce necrotic yellowing virus LNYV: consisting of RT reverse primer LNYV-R and a LAMP primer set, the LAMP primer set is composed of forward and outward primers F3, The reverse outer primer B3, the forward inner primer FIP, the reverse inner primer BIP, the forward loop primer LF and the reverse loop primer LB are composed; the specific sequence of each primer is as follows:
LNYV-R：5’-ATACCATGCCGCGAATCTGT-3’；LNYV-R: 5’-ATACCATGCCGCGAATCTGT-3’;
F3：5’-ACCTAAGCCAGCAATGACAT-3’；F3: 5’-ACCTAAGCCAGCAATGACAT-3’;
B3：5’-TGCCCAATCCAGCCTCTT-3’；B3: 5’-TGCCCAATCCAGCCTCTT-3’;
FIP：5’-CTGTTAGATACTCCCGCCTGCGACACCTCCTAACACCTCCTT-3’；FIP: 5’-CTGTTAGATACTCCCGCCTGCGACACCTCCTAACACCTCCTT-3’;
BIP：5’-CTCTGGACGCAACCCGGAAGATTGCTTCGTACCTAGCCCT-3’；BIP: 5’-CTCTGGACGCAACCCGGAAGATTGCTTCGTACCTAGCCCT-3’;
LF：5’-CGAGCCTAGTACCTTGATCTGAAG-3’；LF: 5’-CGAGCCTAGTACCTTGATCTGAAG-3’;
LB：5’-AATTTGTTGACTGAGACTGATGAGG-3’。LB: 5’-AATTTGTTGACTGAGACTGATGAGG-3’.
所述的IC-RT-LAMP检测引物组合物在检测莴苣坏死黄化病毒中的应用。The application of the IC-RT-LAMP detection primer composition in the detection of lettuce necrotic yellowing virus.
一种特异性检测莴苣坏死黄化病毒LNYV的IC-RT-LAMP试剂盒，包括LNYV抗体IgG、RT反向引物LNYV-R、LAMP引物组、第一链cDNA合成试剂和LAMP扩增反应试剂，其中特异性LAMP引物组包括正向外引物F3、反向外引物B3、正向内引物FIP、反向内引物BIP、正向环引物LF和反向环引物LB，上述特异性RT反向引物LNYV-R以及LAMP引物组的序列如下：An IC-RT-LAMP kit for specific detection of lettuce necrotic yellowing virus LNYV, including LNYV antibody IgG, RT reverse primer LNYV-R, LAMP primer set, first-strand cDNA synthesis reagent and LAMP amplification reaction reagent, The specific LAMP primer set includes forward-outer primer F3, reverse outer primer B3, forward inner primer FIP, reverse inner primer BIP, forward loop primer LF and reverse loop primer LB, the above-mentioned specific RT reverse primer The sequence of LNYV-R and LAMP primer set is as follows:
LNYV-R：5’-ATACCATGCCGCGAATCTGT-3’；LNYV-R: 5’-ATACCATGCCGCGAATCTGT-3’;
F3：5’-ACCTAAGCCAGCAATGACAT-3’；F3: 5’-ACCTAAGCCAGCAATGACAT-3’;
B3：5’-TGCCCAATCCAGCCTCTT-3’；B3: 5’-TGCCCAATCCAGCCTCTT-3’;
FIP：5’-CTGTTAGATACTCCCGCCTGCGACACCTCCTAACACCTCCTT-3’；FIP: 5’-CTGTTAGATACTCCCGCCTGCGACACCTCCTAACACCTCCTT-3’;
BIP：5’-CTCTGGACGCAACCCGGAAGATTGCTTCGTACCTAGCCCT-3’；BIP: 5’-CTCTGGACGCAACCCGGAAGATTGCTTCGTACCTAGCCCT-3’;
LF：5’-CGAGCCTAGTACCTTGATCTGAAG-3’；LF: 5’-CGAGCCTAGTACCTTGATCTGAAG-3’;
LB：5’-AATTTGTTGACTGAGACTGATGAGG-3’。LB: 5’-AATTTGTTGACTGAGACTGATGAGG-3’.
除此之外，所述第一链cDNA合成试剂由10mM dNTPs、5×M-MLV反应缓冲液、30U/μL RNase抑制剂、200U/μL M-MLV反转录酶和DEPC-H 2O组成；所述LAMP扩增反应试剂由10mM dNTPs、10×Thermpopol反应缓冲液、100mM MgSO 4、8U/μL Bst DNA polymerase和ddH 2O组成。 In addition, the first strand cDNA synthesis reagent consists of 10mM dNTPs, 5×M-MLV reaction buffer, 30U/μL RNase inhibitor, 200U/μL M-MLV reverse transcriptase and DEPC-H 2 O The LAMP amplification reaction reagent is composed of 10mM dNTPs, 10×Thermpopol reaction buffer, 100mM MgSO 4 , 8U/μL Bst DNA polymerase and ddH 2 O.
本发明的试剂盒还包括LNYV抗体IgG包被缓冲液(CB)、磷酸缓冲液(PBS)、磷酸洗涤缓冲液(PBST)、荧光染料检测液、阴性对照品和阳性对照品。其中，所述抗体包被缓冲液CB为碳酸盐缓冲液，其浓度为0.05M，PH为9.6；所述磷酸缓冲液(PBS)和磷酸洗涤缓冲液(PBST)的浓度为0.02M，PH值为7.4；所述荧光染料检测液为1000×SYBR Green I；所述阴性对照品为ddH 2O；所述阳性对照品为LNYV蛋白N基因标准品；所述阳性对照品序列如下： The kit of the present invention also includes LNYV antibody IgG coating buffer (CB), phosphate buffer (PBS), phosphate washing buffer (PBST), fluorescent dye detection solution, negative control substance and positive control substance. Wherein, the antibody coating buffer CB is a carbonate buffer with a concentration of 0.05M and a pH of 9.6; the concentration of the phosphate buffer (PBS) and phosphate washing buffer (PBST) is 0.02M, pH The value is 7.4; the fluorescent dye detection solution is 1000×SYBR Green I; the negative control is ddH 2 O; the positive control is the LNYV protein N gene standard; the sequence of the positive control is as follows:
生菜LNYV蛋白N基因标准品：Lettuce LNYV protein N gene standard:
Figure PCTCN2020071512-appb-000001
Figure PCTCN2020071512-appb-000001
所述的特异性检测莴苣坏死黄化病毒LNYV的IC-RT-LAMP试剂盒在检测莴苣坏死黄化病毒中的应用。The application of the IC-RT-LAMP kit for specifically detecting lettuce necrotic yellowing virus LNYV in detecting lettuce necrotic yellowing virus.
一种特异性检测莴苣坏死黄化病毒LNYV的IC-RT-LAMP检测方法：包括以下步骤：An IC-RT-LAMP detection method for specifically detecting lettuce necrotic yellowing virus LNYV: It includes the following steps:
步骤①抗体制备：表达并纯化LNYV蛋白N基因工程融合蛋白，用其免疫动物，获得特异性的LNYV抗体IgG。 Step ① Antibody preparation: express and purify the LNYV protein N genetic engineering fusion protein, and use it to immunize animals to obtain specific LNYV antibody IgG.
步骤②免疫捕获IC：将步骤①获得的特异性的LNYV抗体IgG包被在反应容器中，捕获待测样品中的LNYV粒子。Step ②Immune capture IC: Coat the specific LNYV antibody IgG obtained in step ① in a reaction container to capture the LNYV particles in the sample to be tested.
步骤③反转录RT：以步骤②捕获的LNYV粒子为模板，利用LNYV的RT反向引物LNYV-R和第一链cDNA合成试剂进行反转录RT反应获得第一链cDNA。Step ③Reverse transcription RT: Using the LNYV particles captured in step ② as a template, use LNYV's RT reverse primer LNYV-R and the first strand cDNA synthesis reagent to perform reverse transcription RT reaction to obtain the first strand cDNA.
步骤④LAMP扩增：以步骤③得到的第一链cDNA作为模板，用LAMP特异性引物组和LAMP扩增反应试剂进行LAMP扩增；并以ddH 2O作为阴性对照，以LNYV 蛋白N基因标准品作为阳性对照。 Step ④LAMP amplification: use the first-strand cDNA obtained in step ③ as a template, use LAMP specific primer set and LAMP amplification reaction reagents for LAMP amplification; use ddH 2 O as a negative control, and use LNYV protein N gene standard As a positive control.
步骤⑤分析判断反应产物：以步骤④中所得LAMP扩增反应产物判断待测样品中是否含有莴苣坏死黄化病毒。Step ⑤Analyze and judge the reaction product: Use the LAMP amplification reaction product obtained in Step ④ to determine whether the sample to be tested contains lettuce necrotic yellowing virus.
其中：步骤①所述LNYV抗体可以为多克隆抗体或单克隆抗体，可以自制或从商业途径获得；Wherein: the LNYV antibody in step ① can be a polyclonal antibody or a monoclonal antibody, which can be self-made or obtained from commercial sources;
步骤④所述LAMP扩增的反应体系(以反应体系12.5μL为例)如下：50ng cDNA0.5μL、10×Thermpopol buffer 1.25μL、100mM MgSO 4 0.75μL、10mM dNTPs 1.75μL、10μM B3和F3Primer各0.25μL、10μM FIP和BIP Primer各2.0μL、10μM LF和LB Primer各0.5μL、8U/μL Bst DNA polymerase 0.5μL、和ddH 2O补至12.5μL。 Step ④ The reaction system for LAMP amplification (take the reaction system 12.5μL as an example) is as follows: 50ng cDNA 0.5μL, 10×Thermpopol buffer 1.25μL, 100mM MgSO 4 0.75μL, 10mM dNTPs 1.75μL, 10μM B3 and F3Primer 0.25 each μL, 10μM FIP and BIP Primer each 2.0μL, 10μM LF and LB Primer each 0.5μL, 8U/μL Bst DNA polymerase 0.5μL, and ddH 2 O make up to 12.5μL.
步骤④所述LAMP扩增的反应条件为：58-70℃恒温水浴中扩增60-100min，随后80℃变性10min，终止反应。 Step ④ The reaction conditions of the LAMP amplification are: amplification in a constant temperature water bath at 58-70°C for 60-100 minutes, followed by denaturation at 80°C for 10 minutes to terminate the reaction.
步骤⑤所述根据扩增结果判断病毒粒子是否为莴苣坏死黄化病毒的具体方法为荧光染料肉眼观察法或琼脂糖凝胶电泳检测技术。In step ⑤, the specific method for judging whether the virus particle is lettuce necrotic yellowing virus according to the amplification result is a fluorescent dye macroscopic observation method or agarose gel electrophoresis detection technology.
其中荧光染料肉眼观察法为：取SYBR Green I荧光染料反应液加入步骤④所述LAMP扩增的反应产物中，采用肉眼直接观察，若扩增产物出现绿色，说明SYBR Green I染料与双链DNA结合，则为阳性反应，表示样品中含有LNYV；若扩增产物颜色为橙色则为阴性反应，表示样品中不含LNYV。The method of visual observation of the fluorescent dye is: take the SYBR Green I fluorescent dye reaction solution and add it to the reaction product of the LAMP amplification in step ④, and observe directly with the naked eye. If the amplified product appears green, it means that the SYBR Green I dye and double-stranded DNA Binding, it is a positive reaction, indicating that the sample contains LNYV; if the color of the amplified product is orange, it is a negative reaction, indicating that the sample does not contain LNYV.
其中琼脂糖凝胶电泳检测技术为：将步骤④所述LAMP扩增的产物进行2.0％琼脂糖凝胶电泳，若凝胶中存在明亮的弥散阶梯状核酸泳带，则为阳性反应，表示样品中含有LNYV；若凝胶中无核酸泳带，则为阴性反应，表示样品中不含LNYV。Among them, the agarose gel electrophoresis detection technology is: the LAMP amplified product in step ④ is subjected to 2.0% agarose gel electrophoresis. If there is a brightly diffused ladder-like nucleic acid band in the gel, it is a positive reaction, indicating a sample Contains LNYV; if there is no nucleic acid band in the gel, it is a negative reaction, which means that the sample does not contain LNYV.
本发明将免疫捕获IC与LAMP技术有机结合，成功建立了IC-RT-LAMP技术特异检测莴苣坏死黄化病毒LNYV的方法。本发明的检测方法，其检测对象是完整的病毒粒子，通过固相化的特异抗体捕获特定的病原物抗原，再利用病原物基因组序列特异引物进行扩增，通过对扩增产物的检测和分析达到对完整病原物的检测。IC-RT-LAMP检测方法将血清学方法和分子生物学方法有机地结合起来，特异性强，灵敏度达到了3.5pg/mL，比普通PCR高100倍，且无需提取RNA，也不需要专业的仪器设备，简化了操作过程，降低了检测难度，提高了检测效率。The invention organically combines the immune capture IC and the LAMP technology, and successfully establishes a method for specifically detecting the lettuce necrotic yellowing virus LNYV with the IC-RT-LAMP technology. In the detection method of the present invention, the detection object is a complete virus particle, the specific pathogen antigen is captured by a solid-phased specific antibody, and then the pathogen genome sequence specific primer is used for amplification, and the amplified product is detected and analyzed Achieve the detection of intact pathogens. The IC-RT-LAMP detection method organically combines serological methods and molecular biology methods with strong specificity and a sensitivity of 3.5 pg/mL, which is 100 times higher than ordinary PCR. It does not require RNA extraction or professional expertise. The equipment simplifies the operation process, reduces the detection difficulty, and improves the detection efficiency.
与现有技术相比，本发明具有以下优点和积极效果：Compared with the prior art, the present invention has the following advantages and positive effects:
(1)操作方便：本发明提供的特异性检测莴苣坏死黄化病毒的IC-RT-LAMP方法克服了现有技术中莴苣坏死黄化病毒的检测方法特异性差、灵敏度低、提取RNA难度 高、试剂昂贵以及需要热循环仪器等问题。本发明将血清学和LAMP扩增技术有机地结合，充分发挥了两种检测方法的优越性，直接以捕获的LNYV病毒粒子作为检测对象进行RT-LAMP扩增，避免了RNA抽提，降低了实验难度。(1) Convenient operation: The IC-RT-LAMP method for specific detection of lettuce necrotic yellowing virus provided by the present invention overcomes the poor specificity, low sensitivity, and high difficulty of extracting RNA in the detection method of lettuce necrotic yellowing virus in the prior art. Problems such as expensive reagents and the need for thermal cycling equipment. The present invention organically combines serology and LAMP amplification technology to give full play to the advantages of the two detection methods. The captured LNYV virus particles are directly used as the detection object for RT-LAMP amplification, which avoids RNA extraction and reduces Experiment difficulty.
(2)准确性高：本发明利用高质量的LNYV抗体IgG捕获待测样品中的LNYV粒子，提高了检测的特异性，减少了假阳性。LAMP反应通过6条引物(F3、B3、FIP、BIP、LF、LB)特异性识别靶基因(LNYV蛋白N)的8个序列，特异性强，灵敏度达到了3.5pg/mL，比普通PCR高100倍。(2) High accuracy: The present invention uses high-quality LNYV antibody IgG to capture LNYV particles in the sample to be tested, which improves the specificity of detection and reduces false positives. The LAMP reaction specifically recognizes 8 sequences of the target gene (LNYV protein N) through 6 primers (F3, B3, FIP, BIP, LF, LB), with strong specificity, and the sensitivity reaches 3.5 pg/mL, which is higher than ordinary PCR 100 times.
(3)适用性好：本发明摆脱了对热循环仪器的依赖，LAMP反应只需在恒温水浴中就可以发生，极大地扩展了该方法的使用范围。反应结束后可通过颜色变化用肉眼可以直接判断结果，使检测结果更直观，从而增加了检测的应用价值。(3) Good applicability: The present invention gets rid of the dependence on thermal cycling equipment, and the LAMP reaction only needs to occur in a constant temperature water bath, which greatly expands the application range of the method. After the reaction is over, the result can be directly judged by the naked eye through the color change, which makes the detection result more intuitive, thereby increasing the application value of the detection.
(4)检测成本低，检测结果直观：该方法不需要PCR仪等分子生物学仪器设备，在恒温水浴中即能完成扩增反应，检测结果可用肉眼直接观测，非常适合生菜等蔬菜病毒的检测，能为LNYV的有效防治提供可靠的技术依据。(4) The detection cost is low, and the detection results are intuitive: this method does not require molecular biology instruments such as PCR machines, and can complete the amplification reaction in a constant temperature water bath. The detection results can be directly observed with the naked eye, which is very suitable for the detection of vegetable viruses such as lettuce , Can provide a reliable technical basis for the effective prevention and treatment of LNYV.
(5)本发明为生菜病毒病检测提供了新的技术平台，可用于莴苣坏死黄化病毒LNYV的快速检测，也能够用于监控LNYV病毒的发生、扩散和流行，非常适合在基层推广应用。(5) The present invention provides a new technology platform for lettuce virus disease detection, which can be used for rapid detection of lettuce necrotic yellowing virus LNYV, and can also be used to monitor the occurrence, spread and prevalence of LNYV virus, and is very suitable for popularization and application at the grassroots level.
附图说明Description of the drawings
图1为本发明实施例IC-RT-LAMP检测莴苣坏死黄化病毒LNYV的特异性显色图；图中：1：阴性对照；2-7分别对应：LSV感病组织；CMV感病组织；LMoV感病组织；ArMV感病组织；当归JHMV感病组织；7：生菜LNYV感病组织；Figure 1 is an example of the present invention IC-RT-LAMP detection of lettuce necrotic yellow virus LNYV specific color diagram; Figure: 1: negative control; 2-7 respectively correspond to: LSV diseased tissue; CMV diseased tissue; LMoV susceptible tissue; ArMV susceptible tissue; Angelica JHMV susceptible tissue; 7: Lettuce LNYV susceptible tissue;
图2为本发明实施例电泳分析IC-RT-LAMP检测莴苣坏死黄化病毒LNYV的特异性；图中：M：600bp Marker；1：阴性对照；2-7分别对应：LSV感病组织；CMV感病组织；LMoV感病组织；ArMV感病组织；JHMV感病组织；7：生菜LNYV感病组织；Figure 2 shows the specificity of IC-RT-LAMP for detection of lettuce necrotic yellowing virus LNYV by electrophoresis analysis according to the embodiment of the present invention; Figure: M: 600bp Marker; 1: negative control; 2-7 respectively correspond to: LSV susceptible tissue; CMV Affected tissue; LMoV susceptible tissue; ArMV susceptible tissue; JHMV susceptible tissue; 7: Lettuce LNYV susceptible tissue;
图3为本发明实施例莴苣坏死黄化病毒IC-RT-LAMP反应温度优化试验结果的电泳检测图；图中：M：600bp Marker；1：阴性对照；2-8分别对应：56℃；58℃；60℃；62℃；65℃；68℃；70℃；Figure 3 is an electrophoresis detection diagram of the results of the IC-RT-LAMP reaction temperature optimization test for lettuce necrotic yellowing virus in the embodiment of the present invention; Figure: M: 600bp Marker; 1: negative control; 2-8 respectively correspond to: 56°C; 58 ℃; 60℃; 62℃; 65℃; 68℃; 70℃;
图4为本发明实施例莴苣坏死黄化病毒IC-RT-LAMP反应时间优化试验结果的电泳检测图；图中：M：600bp Marker；1：阴性对照；2-6分别对应：20min；40min；60min；80min；100min；Figure 4 is an electrophoresis detection diagram of the results of the lettuce necrotic yellowing virus IC-RT-LAMP reaction time optimization test in the embodiment of the present invention; Figure: M: 600bp Marker; 1: negative control; 2-6 respectively correspond to: 20min; 40min; 60min; 80min; 100min;
图5为本发明实施例IC-RT-LAMP检测莴苣坏死黄化病毒LNYV的灵敏性显色图；图中：1：阴性对照；2-7分别对应LNYV蛋白N基因标准品浓度：10 -1(3.5×10 4ng/mL)；10 -3(3.5×10 2ng/mL)；10 -5(3.5×10 0ng/mL)；10 -7(3.5×10 -2ng/mL)；10 -8(3.5×10 -3ng/mL)；10 -9(3.5×10 -4ng/mL)； Figure 5 shows the sensitivity and color development diagram of IC-RT-LAMP for detection of lettuce necrotic yellowing virus LNYV; Figure: 1: negative control; 2-7 respectively correspond to the concentration of LNYV protein N gene standard product: 10 -1 (3.5×10 4 ng/mL); 10 -3 (3.5×10 2 ng/mL); 10 -5 (3.5×10 0 ng/mL); 10 -7 (3.5×10 -2 ng/mL); 10 -8 (3.5×10 -3 ng/mL); 10 -9 (3.5×10 -4 ng/mL);
图6为本发明实施例电泳分析IC-RT-LAMP检测莴苣坏死黄化病毒LNYV的灵敏性；图中：M：600bp Marker；1：阴性对照；2-7分别对应LNYV蛋白N基因标准品浓度：10 -1(3.5×10 4ng/mL)；10 -3(3.5×10 2ng/mL)；10 -5(3.5×10 0ng/mL)；10 -7(3.5×10 -2ng/mL)；10 -8(3.5×10 -3ng/mL)；10 -9(3.5×10 -4ng/mL)； Figure 6 shows the sensitivity of IC-RT-LAMP to detect lettuce necrotic yellowing virus LNYV by electrophoresis analysis according to the embodiment of the present invention; Figure: M: 600bp Marker; 1: negative control; 2-7 respectively correspond to the concentration of LNYV protein N gene standard product ：10 -1 (3.5×10 4 ng/mL); 10 -3 (3.5×10 2 ng/mL); 10 -5 (3.5×10 0 ng/mL); 10 -7 (3.5×10 -2 ng /mL); 10 -8 (3.5×10 -3 ng/mL); 10 -9 (3.5×10 -4 ng/mL);
图7为本发明实施例电泳分析RT-PCR检测莴苣坏死黄化病毒LNYV的灵敏性；图中：M：600bp Marker；1：阴性对照；2-7分别对应2-7分别对应LNYV蛋白N基因标准品浓度：原倍(3.5×10 5ng/mL)；10 -1(3.5×10 4ng/mL)；10 -3(3.5×10 2ng/mL)；10 -5(3.5×10 0ng/mL)；10 -6(3.5×10 -1ng/mL)；10 -7(3.5×10 -2ng/mL)。 Figure 7 shows the sensitivity of RT-PCR to detect lettuce necrotic yellowing virus LNYV by electrophoresis analysis according to the embodiment of the present invention; Figure: M: 600bp Marker; 1: negative control; 2-7 respectively correspond to 2-7 respectively correspond to LNYV protein N gene Standard concentration: original times (3.5×10 5 ng/mL); 10 -1 (3.5×10 4 ng/mL); 10 -3 (3.5×10 2 ng/mL); 10 -5 (3.5×10 0 ng/mL); 10 -6 (3.5×10 -1 ng/mL); 10 -7 (3.5×10 -2 ng/mL).
具体实施方式detailed description
下面结合具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好的理解本发明并能予以实施，但所举实施例不作为对本发明的限定。The present invention will be further described below in conjunction with specific examples, so that those skilled in the art can better understand and implement the present invention, but the examples cited are not intended to limit the present invention.
实施例1：阳性对照品的获得Example 1: Obtaining the positive reference substance
1、总RNA的提取：1. Extraction of total RNA:
将50-100mg兰州地区感染LNYV的生菜叶片在液氮中研磨，用植物总RNA提取试剂盒提取感病组织的总RNA；Grind 50-100 mg of lettuce leaves infected with LNYV in Lanzhou area in liquid nitrogen, and extract total RNA from diseased tissues with a plant total RNA extraction kit;
2、引物设计与合成：2. Primer design and synthesis:
根据GenBank上登录的生菜LNYV蛋白N基因序列(GenBank登录号：AJ746190.1)设计并合成了1对特异性正向(LNYV-F)和反向引物(LNYV-R)；其中上述引物序列如下：A pair of specific forward (LNYV-F) and reverse primers (LNYV-R) were designed and synthesized according to the LNYV protein N gene sequence of lettuce registered on GenBank (GenBank accession number: AJ746190.1); the sequence of the above primers is as follows :
LNYV-F：5’-CTAGGGTCAGGAACACAGCG-3’LNYV-F: 5’-CTAGGGTCAGGAACACAGCG-3’
LNYV-R：5’-ATACCATGCCGCGAATCTGT-3’；LNYV-R: 5’-ATACCATGCCGCGAATCTGT-3’;
3、阳性对照品的制备：3. Preparation of positive reference substance:
1)RT反应1) RT reaction
利用生菜LNYV反向引物LNYV-R和M-MLV反转录酶进行RT反应，合成 cDNA第一链；Use lettuce LNYV reverse primer LNYV-R and M-MLV reverse transcriptase to perform RT reaction to synthesize cDNA first strand;
10μL RT反应体系如下:总RNA 2μL，10μM LNYV特异性反向引物LNYV-R 1μL，DEPC-H 2O 3μL，70℃变性10min，迅速置冰上急冷2min；再加入5×M-MLV buffer 2μL，10mM dNTPs 1μL，30U/μL RNase抑制剂0.34μL，200U/μL M-MLV反转录酶0.35μL和DEPC-H 2O 0.31μL；混合后42℃水浴1h，70℃保温15min，置冰上待用； The 10μL RT reaction system is as follows: total RNA 2μL, 10μM LNYV specific reverse primer LNYV-R 1μL, DEPC-H 2 O 3μL, denatured at 70℃ for 10min, quickly place on ice and chill for 2min; then add 5×M-MLV buffer 2μL , 10mM dNTPs 1μL, 30U/μL RNase inhibitor 0.34μL, 200U/μL M-MLV reverse transcriptase 0.35μL and DEPC-H 2 O 0.31μL; after mixing, 42℃ water bath for 1h, 70℃ for 15min, put on ice stand-by;
2)PCR反应2) PCR reaction
利用上述cDNA第一链为模板，在Ex Taq DNA聚合酶作用下进行LNYV蛋白N基因的PCR扩增；Use the first strand of cDNA as a template to perform PCR amplification of the LNYV protein N gene under the action of ExTaq DNA polymerase;
PCR反应体系为12.5μL，包括：50ng cDNA 0.5μL，5U/μL Ex Taq DNA聚合酶0.1μL，10×PCR buffer 1.25μL，2.5mM dNTPs 1μL，10μM正向引物LNYV-F 0.25μL，10μM反向引物LNYV-R 0.25μL，和ddH 2O 9.15μL； The PCR reaction system is 12.5μL, including: 50ng cDNA 0.5μL, 5U/μL Ex Taq DNA polymerase 0.1μL, 10×PCR buffer 1.25μL, 2.5mM dNTPs 1μL, 10μM forward primer LNYV-F 0.25μL, 10μM reverse Primer LNYV-R 0.25μL, and ddH 2 O 9.15μL;
PCR扩增条件为：94℃预变性3min；94℃变性30s，52℃退火45s，72℃延伸1min，循环扩增35次，最后72℃延伸10min；PCR amplification conditions are: pre-denaturation at 94°C for 3min; denaturation at 94°C for 30s, annealing at 52°C for 45s, extension at 72°C for 1min, 35 cycles of amplification, and finally extension at 72°C for 10min;
PCR产物经2.0％琼脂糖凝胶电泳检测后，回收目的片段，利用克隆载体试剂盒将目的片段连接在pMD18-T载体上，转化DH5α感受态细胞，进行蓝白斑平板筛选；随机挑取3个白斑菌落分别接种在氨苄LB培养基上，37℃摇菌12～16h；使用质粒微量抽提试剂盒提取质粒；分别取2μL质粒，在与上述PCR反应体系相同的条件下进行PCR扩增；将PCR检测得到的阳性重组质粒测序；测序证实序列完全正确的阳性质粒，即为标准品，生菜LNYV蛋白N基因对应的片段长度分别为614bp；用NanoDrop ND-1000核酸/蛋白分析仪测定标准品的OD 260nm和OD 280nm值，根据OD 260nm和OD 280nm值计算质粒浓度； After the PCR product was detected by 2.0% agarose gel electrophoresis, the target fragment was recovered, and the target fragment was ligated to the pMD18-T vector using a cloning vector kit, transformed into DH5α competent cells, and screened by blue-white plate; 3 randomly selected White spot colonies were respectively inoculated on ampicillin LB medium and shaken at 37°C for 12-16 hours; plasmids were extracted with plasmid micro-extraction kit; 2μL of plasmids were taken respectively, and PCR amplification was carried out under the same conditions as the above PCR reaction system; The positive recombinant plasmids obtained by PCR detection were sequenced; the positive plasmids with completely correct sequence confirmed by sequencing were the standard products. The fragment lengths corresponding to the LNYV protein N gene of lettuce were 614bp respectively; the NanoDrop ND-1000 nucleic acid/protein analyzer was used to determine the standard products OD 260 nm and OD 280 nm values, calculate the plasmid concentration based on the OD 260 nm and OD 280 nm values;
4、结果：4. Results:
经测序，上述设计的标准品与预期完全相符，回收的标准品片段序列如下：After sequencing, the above-mentioned designed standard was completely in line with expectations, and the recovered standard fragment sequence is as follows:
生菜LNYV蛋白N基因标准品序列：Lettuce LNYV protein N gene standard sequence:
Figure PCTCN2020071512-appb-000002
Figure PCTCN2020071512-appb-000002
Figure PCTCN2020071512-appb-000003
Figure PCTCN2020071512-appb-000003
实施例2：一种用于特异性检测莴苣坏死黄化病毒LNYV的IC-RT-LAMP检测试剂盒Example 2: An IC-RT-LAMP detection kit for the specific detection of lettuce necrotic yellowing virus LNYV
该试剂盒由LNYV抗体IgG、抗体包被缓冲液(CB)、磷酸缓冲液(PBS)、磷酸洗涤缓冲液(PBST)、RT反向引物LNYV-R、LAMP引物组、第一链cDNA合成试剂、LAMP扩增反应试剂、荧光染料检测液、阴性对照品和阳性对照品组成，其中特异性LAMP引物组包括正向外引物F3、反向外引物B3、正向内引物FIP、反向内引物BIP、正向环引物LF和反向环引物LB，上述特异性RT反向引物LNYV-R以及LAMP引物组的序列如下：The kit consists of LNYV antibody IgG, antibody coating buffer (CB), phosphate buffer (PBS), phosphate washing buffer (PBST), RT reverse primer LNYV-R, LAMP primer set, first strand cDNA synthesis reagent , LAMP amplification reaction reagent, fluorescent dye detection solution, negative control substance and positive control substance. The specific LAMP primer set includes forward outer primer F3, reverse outer primer B3, forward inner primer FIP, and reverse inner primer The sequences of BIP, forward loop primer LF and reverse loop primer LB, the above-mentioned specific RT reverse primer LNYV-R and LAMP primer set are as follows:
LNYV-R：5’-ATACCATGCCGCGAATCTGT-3’；LNYV-R: 5’-ATACCATGCCGCGAATCTGT-3’;
F3：5’-ACCTAAGCCAGCAATGACAT-3’；F3: 5’-ACCTAAGCCAGCAATGACAT-3’;
B3：5’-TGCCCAATCCAGCCTCTT-3’；B3: 5’-TGCCCAATCCAGCCTCTT-3’;
FIP：5’-CTGTTAGATACTCCCGCCTGCGACACCTCCTAACACCTCCTT-3’；FIP: 5’-CTGTTAGATACTCCCGCCTGCGACACCTCCTAACACCTCCTT-3’;
BIP：5’-CTCTGGACGCAACCCGGAAGATTGCTTCGTACCTAGCCCT-3’；BIP: 5’-CTCTGGACGCAACCCGGAAGATTGCTTCGTACCTAGCCCT-3’;
LF：5’-CGAGCCTAGTACCTTGATCTGAAG-3’；LF: 5’-CGAGCCTAGTACCTTGATCTGAAG-3’;
LB：5’-AATTTGTTGACTGAGACTGATGAGG-3’；LB: 5’-AATTTGTTGACTGAGACTGATGAGG-3’;
第一链cDNA合成试剂由10mM dNTPs、5×M-MLV反应缓冲液、30U/μL RNase抑制剂、200U/μL M-MLV反转录酶和DEPC-H 2O组成； The first strand cDNA synthesis reagent is composed of 10mM dNTPs, 5×M-MLV reaction buffer, 30U/μL RNase inhibitor, 200U/μL M-MLV reverse transcriptase and DEPC-H 2 O;
LAMP扩增反应试剂由10mM dNTPs、10×Thermpopol反应缓冲液、100mM MgSO 4、8U/μL Bst DNA polymerase和ddH 2O组成； LAMP amplification reaction reagents consist of 10mM dNTPs, 10×Thermpopol reaction buffer, 100mM MgSO 4 , 8U/μL Bst DNA polymerase and ddH 2 O;
抗体包被缓冲液CB为碳酸盐缓冲液，其浓度为0.05M，PH为9.6；所述磷酸缓冲液(PBS)和磷酸洗涤缓冲液(PBST)的浓度为0.02M，PH值为7.4；The antibody coating buffer CB is a carbonate buffer with a concentration of 0.05M and a pH of 9.6; the concentration of the phosphate buffer (PBS) and phosphate washing buffer (PBST) is 0.02M, and the pH is 7.4;
所述荧光染料检测液为1000×SYBR Green I；所述阴性对照品为ddH 2O；所述阳性对照品为生菜LNYV蛋白N基因标准品。 The fluorescent dye detection solution is 1000×SYBR Green I; the negative control substance is ddH 2 O; the positive control substance is the lettuce LNYV protein N gene standard substance.
实施例3：免疫捕获IC-RT-LAMP检测莴苣坏死黄化病毒LNYV方法的建立Example 3: Establishment of a method for detection of lettuce necrotic yellow virus LNYV by immunocapture IC-RT-LAMP
1、本发明中兔抗LNYV多克隆抗体IgG的制备方法：1. The preparation method of rabbit anti-LNYV polyclonal antibody IgG in the present invention:
1)LNYV蛋白N基因融合蛋白的表达和纯化：从兰州地区感染了LNYV的生菜叶片中提取总RNA进行逆转录聚合酶链式反应(RT－PCR)，扩增LNYV的蛋白N基因片段。通过酶切克隆至pET-28a载体。重组质粒转化入大肠杆菌BL21，37℃培养，IPTG诱导表达，镍柱亲和层析纯化获得大小为22.5kDa的LNYV蛋白N基因融合蛋白；1) Expression and purification of LNYV protein N gene fusion protein: Total RNA was extracted from the leaves of lettuce infected with LNYV in Lanzhou area to perform reverse transcription polymerase chain reaction (RT-PCR) to amplify the protein N gene fragment of LNYV. Cloned into pET-28a vector by restriction enzyme digestion. The recombinant plasmid was transformed into E. coli BL21, cultured at 37°C, IPTG induced expression, and purified by affinity chromatography on a nickel column to obtain a 22.5kDa LNYV protein N gene fusion protein;
2)多克隆抗体IgG的制备：用1mg/mL的上述LNYV蛋白N基因的融合蛋白作为免疫原免疫新西兰大白兔；2) Preparation of polyclonal antibody IgG: 1 mg/mL of the above-mentioned LNYV protein N gene fusion protein was used as an immunogen to immunize New Zealand white rabbits;
初次免疫中，将蛋白抗原与弗氏完全佐剂等体积充分混匀，进行皮下多点注射；两周后进行加强免疫，将蛋白抗原与弗氏不完全佐剂等体积充分混匀，进行皮下多点注射；以后每两周加强免疫一次，在第4次加强免疫后的5～7d颈动脉采血，静至，离心，收集到的血清加入质量百分比浓度0.02％的叠氮钠，-20℃保存；所得抗血清依次通过20％、50％、33％三个饱和度的硫酸铵沉淀粗提后，透析至pH7.8的磷酸缓冲液，然后使用DE52阴离子交换柱进行纯化而获得兔抗LNYV多克隆抗体IgG；In the initial immunization, the protein antigen and Freund’s complete adjuvant are mixed in equal volumes and then injected into multiple points under the skin; two weeks later, booster immunization is performed, and the protein antigen and Freund’s incomplete adjuvant are fully mixed in equal volumes and subcutaneously Multi-point injections; booster immunization every two weeks thereafter, collect blood from the carotid artery 5-7 days after the fourth booster immunization, calm down, centrifuge, and add 0.02% sodium azide to the collected serum at -20℃ Save; the obtained antiserum was crudely extracted by ammonium sulfate precipitation with three saturations of 20%, 50%, and 33%, dialyzed to pH 7.8 phosphate buffer, and then purified with DE52 anion exchange column to obtain rabbit anti-LNYV Polyclonal antibody IgG;
2、免疫捕获IC：2. Immune capture IC:
1)取100mg感染LNYV的生菜叶片组织，加1mL PBS研磨后将研磨液转入1.5mL灭菌离心管中，4000rpm离心2min，上清液即感病组织粗提液；1) Take 100mg of lettuce leaf tissue infected with LNYV, add 1mL PBS to grind, transfer the grinding solution into a 1.5mL sterile centrifuge tube, centrifuge at 4000rpm for 2min, the supernatant is the crude extract of the diseased tissue;
2)用0.05M，PH9.6的碳酸盐缓冲液将LNYV多克隆抗体IgG稀释后混合，使LNYV抗体IgG的终浓度为10μg/mL；取100μL上述稀释的LNYV多克隆抗体IgG加入0.2mL PCR管中，37℃孵育2h；2) Dilute LNYV polyclonal antibody IgG with 0.05M, pH9.6 carbonate buffer and mix to make the final concentration of LNYV antibody IgG 10μg/mL; take 100μL of the above-diluted LNYV polyclonal antibody IgG and add 0.2mL Incubate in a PCR tube at 37°C for 2h;
3)弃去包被液，用PBST洗3次，每次3min；加入100μL上述感病组织粗提液，37℃孵育2h；3) Discard the coating solution, wash with PBST 3 times, 3 minutes each time; add 100 μL of the crude extract of the diseased tissues above, and incubate at 37°C for 2 hours;
4)弃去感病组织粗提液，用PBST洗3次，ddH 2O洗1次，短暂离心，吸去残液； 4) Discard the crude extract of the susceptible tissue, wash 3 times with PBST and 1 time with ddH 2 O, centrifuge briefly to remove the remaining liquid;
3、RT反应：3. RT reaction:
1)在上述免疫捕获PCR管中加入浓度为10μM的LNYV特异性反向引物LNYV-R 1μL，DEPC-H 2O 4μL，混合后于70℃保温10min，之后迅速冰浴2min； 1) Add 1μL of LNYV-specific reverse primer LNYV-R and 4μL of DEPC-H 2 O at a concentration of 10μM to the above immunocapture PCR tube, mix and keep at 70℃ for 10min, then quickly ice bath for 2min;
2))向上述PCR管中加入5×M-MLV buffer 2μL、10mM dNTPs 1μL、30U/μL RNase抑制剂0.34μL、200U/μL M-MLV反转录酶0.35μL、DEPC-H 2O补足至10μL，混合均匀后，42℃水浴1h，70℃保温15min，得到cDNA第一链用于后续LAMP扩增； 2)) Add 5×M-MLV buffer 2μL, 10mM dNTPs 1μL, 30U/μL RNase inhibitor 0.34μL, 200U/μL M-MLV reverse transcriptase 0.35μL, DEPC-H 2 O to the above PCR tube to make up to 10μL, mix well, in a water bath at 42°C for 1 hour, and incubate at 70°C for 15 minutes to obtain the first strand of cDNA for subsequent LAMP amplification;
4、LAMP扩增反应：4. LAMP amplification reaction:
取一新的PCR管，按以下体系加入各试剂进行LAMP扩增，反应体系为12.5μL，包括50ng cDNA 0.5μL、10×Thermpopol buffer 1.25μL、100mM MgSO 4 0.75μL、10mM dNTPs 1.75μL、10μM B3和F3Primer各0.25μL、10μM FIP和BIP Primer各2.0μL、10μM LF和LB Primer各0.5μL、8U/μL Bst DNA polymerase 0.5μL、ddH 2O2.25μL；并以ddH 2O作为阴性对照，以生菜LNYV蛋白N基因标准品作为阳性对照； Take a new PCR tube and add the reagents according to the following system for LAMP amplification. The reaction system is 12.5μL, including 50ng cDNA 0.5μL, 10×Thermpopol buffer 1.25μL, 100mM MgSO 4 0.75μL, 10mM dNTPs 1.75μL, 10μM B3 And F3Primer 0.25μL each, 10μM FIP and BIP Primer each 2.0μL, 10μM LF and LB Primer each 0.5μL, 8U/μL Bst DNA polymerase 0.5μL, ddH 2 O2.25μL; and ddH 2 O as a negative control, with lettuce LNYV protein N gene standard as a positive control;
LAMP的反应温度如图3泳道3-8所示，为:58-70℃，LAMP的反应扩增时间如图4泳道4-6所示，为60-100min,随后80℃变性10min，终止反应；The reaction temperature of LAMP is shown in lanes 3-8 in Figure 3, which is 58-70°C, and the amplification time of LAMP is shown in lanes 4-6 in Figure 4, which is 60-100 minutes, followed by denaturation at 80°C for 10 minutes to terminate the reaction ；
5、分析判断反应产物：5. Analyze and judge the reaction product:
LAMP反应结束后，在PCR管内侧加1μL 1000×SYBR Green I荧光染料工作液，盖紧PCR管，上下颠倒混匀，采用肉眼直接观察PCR管内混合液的颜色变化：After the LAMP reaction is over, add 1μL 1000×SYBR Green I fluorescent dye working solution to the inside of the PCR tube, cover the PCR tube tightly, mix upside down, and directly observe the color change of the mixture in the PCR tube:
若混合液颜色变为绿色，说明SYBR Green I染料与双链DNA结合，则为阳性反应，表示样品中含有LNYV；If the color of the mixed solution changes to green, indicating that the SYBR Green I dye binds to double-stranded DNA, it is a positive reaction, indicating that the sample contains LNYV;
若混合液颜色为橙色，则为阴性反应，表示样品中不含LNYV；If the color of the mixed solution is orange, it is a negative reaction, indicating that the sample does not contain LNYV;
或者，结果的观察也可以采用琼脂糖凝胶电泳检测技术：取5μL LAMP反应产物进行2.0％琼脂糖凝胶电泳，在1×TAE缓冲液环境中，100V稳压电泳30min，然后用凝胶成像系统观察并记录结果：Or, the observation of the results can also use agarose gel electrophoresis detection technology: take 5μL of the LAMP reaction product for 2.0% agarose gel electrophoresis, in 1×TAE buffer environment, 100V stabilized electrophoresis for 30 minutes, and then use gel imaging The system observes and records the results:
若凝胶中存在明亮的弥散状核酸泳带，则为阳性反应，表示样品中含有LNYV；If there are bright and diffuse nucleic acid bands in the gel, it is a positive reaction, indicating that the sample contains LNYV;
若凝胶中无核酸泳带，则为阴性反应，表示样品中不含LNYV。If there is no nucleic acid band in the gel, it is a negative reaction, indicating that the sample does not contain LNYV.
实施例4：免疫捕获IC-RT-LAMP检测莴苣坏死黄化病毒LNYV的特异性Example 4: Detection of the specificity of lettuce necrotic yellowing virus LNYV by immunocapture IC-RT-LAMP
为了分析免疫捕获IC-RT-LAMP检测莴苣坏死黄化病毒LNYV的特异性，以感染百合、当归和生菜的6个主要病毒(百合隐症病毒-LSV、百合黄瓜花叶病毒-CMV、百合斑驳病毒-LMoV、南芥菜花叶病毒-ArMV、日本金鱼藻花叶病毒-JHMV和莴苣坏死黄化病毒-LNYV)的感病叶片为样品，分别用LNYV多克隆抗体IgG捕获病毒粒子，用上述实施例中的IC-RT-LAMP检测体系进行反应。以健康生菜叶片为阴性对照，实验重复3次；In order to analyze the specificity of immunocapture IC-RT-LAMP to detect the lettuce necrotic yellowing virus LNYV, the 6 main viruses that infect lily, angelica and lettuce (lily recessive virus-LSV, lily cucumber mosaic virus-CMV, lily mottle) Virus-LMoV, Arabidopsis Mosaic Virus-ArMV, Japanese Ceratophyllum Mosaic Virus-JHMV and Lettuce Necrotic Yellowing Virus-LNYV) were samples of infected leaves. The virus particles were captured with LNYV polyclonal antibody IgG, and the above implementation The IC-RT-LAMP detection system in the example reacts. With healthy lettuce leaves as a negative control, the experiment was repeated 3 times;
SYBR Green I荧光染料肉眼观察法结果显示如图1所示，只有7号EP管中感染LNYV的生菜叶片的扩增产物出现绿色，其余感病叶片的扩增产物与健康生菜叶片的扩增产物均为橙色；琼脂糖凝胶电泳结果也证实，除了7号感染LNYV的生菜叶片 能够扩增出弥散状核酸泳带外，其余样品均没有扩增出核酸泳带，如图2所示。这表明本发明建立的IC-RT-LAMP方法对莴苣坏死黄化病毒LNYV有很高的特异性。The results of the SYBR Green I fluorescent dye naked eye observation method showed that as shown in Figure 1, only the amplified products of the lettuce leaves infected with LNYV in the No. 7 EP tube appeared green, and the amplified products of the remaining diseased leaves and the amplified products of healthy lettuce leaves All are orange; the results of agarose gel electrophoresis also confirmed that, except for lettuce leaves infected with LNYV on No. 7 that can amplify a diffuse nucleic acid band, the other samples did not amplify a nucleic acid band, as shown in Figure 2. This indicates that the IC-RT-LAMP method established in the present invention has high specificity to the lettuce necrotic yellowing virus LNYV.
实施例5：免疫捕获IC-RT-LAMP检测莴苣坏死黄化病毒LNYV的灵敏性Example 5: Sensitivity of immunocapture IC-RT-LAMP for detection of lettuce necrotic yellowing virus LNYV
为了分析免疫捕获IC-RT-LAMP检测莴苣坏死黄化病毒LNYV的灵敏性，以上述实施例1中生菜LNYV蛋白N基因标准品为样品，经NanoDrop ND-1000核酸/蛋白分析仪测定其标准品浓度(3.5×10 5ng/mL)后，再用DEPC-H 2O对上述标准品进行10倍比稀释，-20℃保存作为模板。分别取10倍比稀释后的各稀释液1.0μL作为模板，加入上述实施例中的LAMP反应试剂进行LAMP扩增，反应程序为65℃扩增100min； In order to analyze the sensitivity of immunocapture IC-RT-LAMP to detect the lettuce necrotic yellowing virus LNYV, the standard LNYV protein N gene of lettuce in Example 1 was used as the sample, and the standard was determined by NanoDrop ND-1000 nucleic acid/protein analyzer After the concentration (3.5×10 5 ng/mL), the above standards were diluted 10-fold with DEPC-H 2 O and stored at -20°C as a template. Take 1.0 μL of each diluted solution diluted by 10 times as a template, and add the LAMP reaction reagents in the above examples to carry out LAMP amplification. The reaction procedure is 65°C for 100 min;
作为对比检测，将上述10倍比稀释后的各稀释液利用上述实施例1中的PCR反应进行PCR扩增。PCR扩增条件为：94℃预变性3min；94℃变性30s，52℃退火45s，72℃延伸1min，循环扩增35次，最后72℃延伸10min；As a comparative test, each dilution after the above 10-fold dilution was subjected to PCR amplification using the PCR reaction in Example 1 above. PCR amplification conditions are: pre-denaturation at 94°C for 3min; denaturation at 94°C for 30s, annealing at 52°C for 45s, extension at 72°C for 1min, cyclic amplification 35 times, and finally extension at 72°C for 10min;
LAMP和PCR反应结束后，分别取5μL扩增产物上样，SYBR Green I荧光染料肉眼观察法和琼脂糖凝胶电泳结果显示LAMP对生菜LNYV蛋白N基因标准品的反应灵敏度为3.5×10 -3ng/mL，即3.5pg/mL，见图5中2-6号EP管和图6。PCR对生菜LNYV蛋白N基因标准品的反应的灵敏度为3.5×10 -1ng/mL，即350pg/mL，见图7。可见免疫捕获IC-RT-LAMP检测JHMV的灵敏性是普通PCR的100倍。 After the LAMP and PCR reactions are completed, 5μL of the amplified products are respectively loaded on the sample. The results of naked eye observation with SYBR Green I fluorescent dye and agarose gel electrophoresis show that the sensitivity of LAMP to the standard LNYV protein N gene of lettuce is 3.5×10 -3 ng/mL, that is, 3.5pg/mL, see Figure 5 in No. 2-6 EP tube and Figure 6. The sensitivity of PCR to the standard LNYV protein N gene of lettuce is 3.5×10 -1 ng/mL, which is 350 pg/mL, as shown in Figure 7. It can be seen that the sensitivity of immune capture IC-RT-LAMP to detect JHMV is 100 times that of ordinary PCR.
实施例6：生菜田间样品LNYV的免疫捕获IC-RT-LAMP检测Example 6: Immune capture IC-RT-LAMP detection of LNYV in lettuce field samples
取田间生菜叶片样品，用LNYV多克隆抗体IgG捕获病毒粒子，再用上述实施例中的IC-RT-LAMP检测体系进行RT反应合成cDNA再进行LAMP扩增；若荧光染料肉眼观察法显示橙色，或琼脂糖凝胶电泳扩增出弥散状核酸泳带，说明样品中含有莴苣坏死黄化病毒；若荧光染料肉眼观察法显示绿色，或琼脂糖凝胶电泳没有扩增出弥散状核酸泳带，说明样品中没有感染莴苣坏死黄化病毒。Take field lettuce leaf samples, use LNYV polyclonal antibody IgG to capture virus particles, and then use the IC-RT-LAMP detection system in the above example to perform RT reaction to synthesize cDNA and then perform LAMP amplification; if the fluorescent dye shows orange by naked eye, Or the agarose gel electrophoresis amplified a diffuse nucleic acid band, indicating that the sample contains lettuce necrotic yellowing virus; if the fluorescent dye shows green by naked eye observation, or the agarose gel electrophoresis does not amplify the diffuse nucleic acid band, It indicated that the sample was not infected with lettuce necrotic yellowing virus.
此外，应当理解，虽然本说明书按照实施方式加以描述，但并非每个实施方式仅包括一个独立的技术方案，说明书的这种叙述方式仅仅是为清楚起见，本领域技术人员应当将说明书作为一个整体，各实施例中的技术方案也可以经适当组合，形成本领域技术人员可以理解的其他实施方式。In addition, it should be understood that although this specification is described in accordance with the implementation manners, not each implementation manner only includes an independent technical solution. This narration in the specification is only for clarity, and those skilled in the art should regard the specification as a whole The technical solutions in each embodiment can also be appropriately combined to form other implementations that can be understood by those skilled in the art.

Claims (8)

  1. 一种用于特异性检测莴苣坏死黄化病毒LNYV的IC-RT-LAMP检测引物组合物，其特征在于：由RT反向引物LNYV-R和LAMP引物组组成，所述LAMP引物组由正向外引物F3、反向外引物B3、正向内引物FIP、反向内引物BIP、正向环引物LF和反向环引物LB组成，各引物序列具体如下：An IC-RT-LAMP detection primer composition for specific detection of lettuce necrotic yellowing virus LNYV, which is characterized in that it is composed of an RT reverse primer LNYV-R and a LAMP primer set, and the LAMP primer set consists of a forward The outer primer F3, the reverse outer primer B3, the forward inner primer FIP, the reverse inner primer BIP, the forward loop primer LF and the reverse loop primer LB are composed. The specific sequence of each primer is as follows:
    LNYV-R：5’-ATACCATGCCGCGAATCTGT-3’；LNYV-R: 5’-ATACCATGCCGCGAATCTGT-3’;
    F3：5’-ACCTAAGCCAGCAATGACAT-3’；F3: 5’-ACCTAAGCCAGCAATGACAT-3’;
    B3：5’-TGCCCAATCCAGCCTCTT-3’；B3: 5’-TGCCCAATCCAGCCTCTT-3’;
    FIP：5’-CTGTTAGATACTCCCGCCTGCGACACCTCCTAACACCTCCTT-3’；FIP: 5’-CTGTTAGATACTCCCGCCTGCGACACCTCCTAACACCTCCTT-3’;
    BIP：5’-CTCTGGACGCAACCCGGAAGATTGCTTCGTACCTAGCCCT-3’；BIP: 5’-CTCTGGACGCAACCCGGAAGATTGCTTCGTACCTAGCCCT-3’;
    LF：5’-CGAGCCTAGTACCTTGATCTGAAG-3’；LF: 5’-CGAGCCTAGTACCTTGATCTGAAG-3’;
    LB：5’-AATTTGTTGACTGAGACTGATGAGG-3’。LB: 5’-AATTTGTTGACTGAGACTGATGAGG-3’.
  2. 权利要求1所述的IC-RT-LAMP检测引物组合物在检测莴苣坏死黄化病毒LNYV中的应用。Application of the IC-RT-LAMP detection primer composition of claim 1 in the detection of lettuce necrotic yellowing virus LNYV.
  3. 一种采用如权利要求1所述的IC-RT-LAMP检测引物组合物特异性检测莴苣坏死黄化病毒LNYV的IC-RT-LAMP试剂盒，所述试剂盒由LNYV抗体IgG、权利要求1所述的IC-RT-LAMP检测引物组合物、第一链cDNA合成试剂和LAMP扩增反应试剂组成；An IC-RT-LAMP kit for specifically detecting lettuce necrotic yellowing virus LNYV using the IC-RT-LAMP detection primer composition as claimed in claim 1, said kit is made up of LNYV antibody IgG, as claimed in claim 1. The IC-RT-LAMP detection primer composition, first-strand cDNA synthesis reagent and LAMP amplification reaction reagent composition;
    其特征在于：所述试剂盒还包括LNYV抗体IgG包被缓冲液(CB)、磷酸缓冲液(PBS)、磷酸洗涤缓冲液(PBST)、SYBR Green I荧光染料检测液、阴性对照品和阳性对照品，所述阴性对照品为ddH 2O；所述阳性对照品为生菜LNYV蛋白N基因标准品；所述阳性对照品序列如下： It is characterized in that: the kit also includes LNYV antibody IgG coating buffer (CB), phosphate buffer (PBS), phosphate washing buffer (PBST), SYBR Green I fluorescent dye detection solution, negative control and positive control The negative control substance is ddH 2 O; the positive control substance is the lettuce LNYV protein N gene standard substance; the sequence of the positive control substance is as follows:
    生菜LNYV蛋白N基因标准品：Lettuce LNYV protein N gene standard:
    CGCCAAAGAAGGATAAAGGGAAGGGACCTGAATCCTCGAACACACAAGATGACAATCAGTTGGCGCAAGCAAGCAAAGACAGAGATATTGATATGGGCACTAGTGGCACATTTGCTGTACCTAGATACAAGATTTTTAAATCCAAATTGAGGTTCCCAATGGTTAGGGGCAGGAAGATTATGAATATGAGCCACCTCGCACAATATAACCCAGAGCAGACAGATCTAGCCAACACAAGAGCAACACAGAATCAGTTTGCTCGATGGTTCGACGGCATCAAAGGAGATTATGGTCTGAATGATGCGGAAATGGACGTCATGTTGAATGGGCTTGTCGTGTGGTGCATTGAAAATGGAACCTCCCCAAATATAAACGGATTGTGGACTATGATGGATGGAGAGGAACAAGTTGAGTATCCAATTAAACCA TTAATAGATCATGCGTCACCCACATTCCGACAA。CGCCAAAGAAGGATAAAGGGAAGGGACCTGAATCCTCGAACACACAAGATGACAATCAGTTGGCGCAAGCAAGCAAAGACAGAGATATTGATATGGGCACTAGTGGCACATTTGCTGTACCTAGATACAAGATTTTTAAATCCAAATTGAGGTTCCCAATGGTTAGGGGCAGGAAGATTATGAATATGAGCCACCTCGCACAATATAACCCAGAGCAGACAGATCTAGCCAACACAAGAGCAACACAGAATCAGTTTGCTCGATGGTTCGACGGCATCAAAGGAGATTATGGTCTGAATGATGCGGAAATGGACGTCATGTTGAATGGGCTTGTCGTGTGGTGCATTGAAAATGGAACCTCCCCAAATATAAACGGATTGTGGACTATGATGGATGGAGAGGAACAAGTTGAGTATCCAATTAAACCA TTAATAGATCATGCGTCACCCACATTCCGACAA.
  4. 权利要求3所述的特异性检测莴苣坏死黄化病毒LNYV的IC-RT-LAMP试剂盒在检测莴苣坏死黄化病毒中的应用。The application of the IC-RT-LAMP kit for specifically detecting lettuce necrotic yellowing virus LNYV according to claim 3 in detecting lettuce necrotic yellowing virus.
  5. 一种采用如权利要求3所述的IC-RT-LAMP试剂盒特异性检测莴苣坏死黄化病毒LNYV的检测方法，其特征在于包括以下步骤：A detection method for specifically detecting lettuce necrotic yellowing virus LNYV by using the IC-RT-LAMP kit according to claim 3, which is characterized by comprising the following steps:
    ①抗体制备：表达并纯化LNYV蛋白N基因融合蛋白，用其免疫动物，获得特异性的LNYV抗体IgG；① Antibody preparation: express and purify the LNYV protein N gene fusion protein, and use it to immunize animals to obtain specific LNYV antibody IgG;
    ②免疫捕获IC：将步骤①获得的特异性的LNYV抗体IgG包被在反应容器中，捕获待测样品中的LNYV粒子；②Immune capture IC: Coat the specific LNYV antibody IgG obtained in step ① in a reaction container to capture LNYV particles in the sample to be tested;
    ③反转录RT：以步骤②捕获的LNYV粒子为模板，利用LNYV的RT反向引物LNYV-R和第一链cDNA合成试剂进行反转录RT反应获得第一链cDNA；③Reverse transcription RT: Using the LNYV particles captured in step ② as a template, use LNYV's RT reverse primer LNYV-R and the first-strand cDNA synthesis reagent to perform reverse transcription RT to obtain the first-strand cDNA;
    ④LAMP扩增：以步骤③得到的第一链cDNA作为模板，用LAMP特异性引物组和LAMP扩增反应试剂进行LAMP扩增；并以ddH 2O作为阴性对照，以生菜LNYV蛋白N基因标准品作为阳性对照； ④LAMP amplification: use the first strand cDNA obtained in step ③ as a template, use LAMP specific primer set and LAMP amplification reaction reagents for LAMP amplification; use ddH 2 O as a negative control, and use lettuce LNYV protein N gene standard As a positive control;
    ⑤分析判断反应产物：以步骤④中所得LAMP扩增反应产物判断待测样品是否含有莴苣坏死黄化病毒。⑤Analyze and determine the reaction product: use the LAMP amplification reaction product obtained in step ④ to determine whether the test sample contains lettuce necrotic yellowing virus.
  6. 根据权利要求5所述的检测方法，其特征在于，步骤①所述LNYV抗体为多克隆抗体或单克隆抗体。The detection method of claim 5, wherein the LNYV antibody in step ① is a polyclonal antibody or a monoclonal antibody.
  7. 根据权利要求5所述的检测方法，其特征在于，步骤⑤中根据扩增结果判断待测样品是否含有LNYV的具体方法为荧光染料肉眼观察法；所述荧光染料肉眼观察法为：取SYBR Green I荧光染料反应液加入步骤④所述LAMP扩增的反应产物中，采用肉眼直接观察，若扩增产物出现绿色，说明SYBR Green I染料与双链DNA结合，则为阳性反应，表示样品中含有莴苣坏死黄化病毒LNYV；若扩增产物颜色为橙色则为阴性反应，表示样品中不含莴苣坏死黄化病毒LNYV。The detection method according to claim 5, characterized in that, in step ⑤, the specific method for judging whether the sample to be tested contains LNYV according to the amplification result is a fluorescent dye macroscopic observation method; the fluorescent dye macroscopic observation method is: SYBR Green The I fluorescent dye reaction solution is added to the reaction product of the LAMP amplification described in step ④, and the amplified product is directly observed with the naked eye. If the amplified product appears green, it means that the SYBR Green I dye binds to the double-stranded DNA. Lettuce necrotic yellowing virus LNYV; if the color of the amplified product is orange, it is a negative reaction, which means that the sample does not contain lettuce necrotic yellowing virus LNYV.
  8. 根据权利要求5所述的检测方法，其特征在于，步骤⑤中根据扩增结果判断待测样品是否含有LNYV的具体方法也可采用琼脂糖凝胶电泳检测技术；所述琼脂糖凝胶电泳检测技术为：将步骤④所述LAMP扩增的反应产物进行琼脂糖凝胶电泳，若扩增产物呈明亮的弥散状核酸泳带，则为阳性反应，表示样品中含有莴苣坏死黄化病毒LNYV；若扩增产物无核酸泳带，则为阴性反应，表示样品中不含莴苣坏死黄化病毒LNYV。The detection method according to claim 5, characterized in that, in step ⑤, the specific method of judging whether the sample to be tested contains LNYV according to the amplification result can also adopt agarose gel electrophoresis detection technology; the agarose gel electrophoresis detection The technique is: subject the reaction product amplified by the LAMP in step ④ to agarose gel electrophoresis, if the amplified product is a bright diffuse nucleic acid band, it is a positive reaction, indicating that the sample contains lettuce necrotic yellowing virus LNYV; If there is no nucleic acid band in the amplified product, it is a negative reaction, which means that the sample does not contain the lettuce necrotic yellowing virus LNYV.
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