47 results about "Cucumber green mottle mosaic virus" patented technology

The invention discloses an RT-LAMP (loop-mediated isothermal amplification) rapid detection kit for cucumber green mottle mosaic virus (CGMMV) and a detection method. The method comprises the steps of extracting plant viruses RNA, detecting an RT-LAMP reaction and electrophoresis detection or fluorochrome, and determining whether a plant carries the viruses. An RT-LAMP primer is designed according to a coat protein gene conserved region of CGMMV, and an RT-LAMP technology is adopted to establish a detection technology which can rapidly and sensitively detect the cucurbitaceous plant CGMMV such as cucumbers, watermelons and calabashes with high specificity and an application method thereof in diagnosing disease. The detection method provided by the invention can utilize the rapid extraction RNA and conventional RNA extraction as an RNA template for an RT-LAMP experiment. The method is utilized to rapidly identify a CGMMV plant sample, the targeted quarantine inspection protective measure is adopted, and the loss caused by the disease is reduced.

The invention discloses a MP (movement protein)-gene-based PCR (polymerase chain reaction) detection method for performing rapid molecular identification on cucumber green mottle mosaic virus (CGMMV), and a probe and primer combination applied to the method. The sequences of primers and a probe include forward primer MMV-F1, downstream primer MMV-R1 and fluorescence probe MMV-1. The method comprises the following steps of: (1) extracting total RNA (Ribose Nucleic Acid) of plant tissue from a sample to be tested; (2) introducing the downstream primer MMV-R1 for implementing synthesis of a reverse transcription template cDNA with taking the total RNA as a template; (3) performing real-time fluorescence PCR amplified reaction when the forward primer, the downstream primer and the fluorescence probe are arranged in a fluorescence PCR instrument; and (4) comparing a collected fluorescence curve CT value with a standard curve CT value after the reaction is ended, and judging the detection result. The method can be applied to the field of molecular diagnosis on biological species.

The invention provides a monoclonal antibody and a kit for CGMMVs. Hybridism cell strains secreting the CGMMV monoclonal antibody are obtained, the obtained CGMMV monoclonal antibody has the advantages of good sensitivity, high specificity and titer and the like. The monoclonal antibody can be used for identifying whether viruses to be detected are CGMMVs and CGMMV samples to be detected are affected by CGMMVs. The monoclonal antibody can be used for preparing immunological diagnostic reagents for CGMMVs, for example, enzyme-linked diagnostic reagents, immune colloidal gold test strips, etc.

The invention discloses a preparing method and an application of a sample for detecting cucumber green mottle mosaic viruses (CGMMV) carried by cucurbitaceae seeds. The preparing method includes the steps that starting from treating seed samples, shell-kernel separating treatment is carried out on the cucurbitaceae seeds, seed kernels with the low virus content are removed, seed shells with the high virus content are left to serve as to-be-tested samples, the seed samples obtained after treatment are sufficiently grinded through a full-automatic rapid sample grinding instrument, and the CGMMV in the large quantity of cucurbitaceae seeds can be rapidly detected through the sufficiently-grinded samples with a RT-PCR method and can also be rapidly detected through the sufficiently-grinded samples with a dot enzyme-linked immunosorbent assay (dot-ELISA) method. The preparing method and the application are suitable for virus detection of the common cucurbitaceae seeds such as calabash seeds, cucumber seeds, lagenaria sicerariae seeds, pumpkin seeds, long gourd seeds and watermelon seeds. A detecting method is high in sensitivity, good in specificity, short in consumed time and low in cost, technical supports are provided for large-scale detection and scientific prevention and control of the CGMMV carried by the cucurbitaceae seeds, and references are provided for detection of plant viruses transmitted by other seeds.

The invention relates to selection of a cucumber green mottle mosaic virus low virulent strain line and application of the cucumber green mottle mosaic virus low virulent strain line in cross protection. A wild type CGMMV-JN (GenBank No. KR232571) infectious clone is adopted as a basis, and mutation is introduced in fixed points in a genome of CGMMV through a site-directed mutation technique, so that four CGMMV mutants such as CP89A, CP114A, Rd68A, Rd869A and Rd1069A are obtained, wherein the pathogenicity of the mutants such as CP89A and Rd1069A is significantly reduced. The low virulent strain lines such as CP89A and Rd1069A can protect plants against being infected by CGMMV virulent strains.

The invention discloses an immune colloidal gold test strip for quickly detecting a cucumber green mottle mosaic virus (CGMMV) and a preparation method of the immune colloidal gold test strip. The preparation method comprises the following steps: dripping a purified CGMMV polyclonal antibody solution on a detection line of a nitrocellulose membrane; dripping a goat anti-rabbit polyclonal antibody on a quality control line of the nitrocellulose membrane; sequentially attaching a sample pad made of water-absorbing filter paper, a CGMMV immune colloidal gold pad, the nitrocellulose membrane and water-absorbing paper to a polyvinyl chloride (PVC) adhesive plate with an adhesive sticker so as to prepare the test strip. The test strip can be used for detecting the CGMMV in a cucurbitaceous plant and is applicable to detection and diagnosis of a viral disease caused by the CGMMV. The method is simple and quick. The test strip is low in cost. A result can be obtained within 5 to 10 minutes and can be visually and directly observed by naked eyes. The test strip is applicable to quick and extensive screening and detection of diseases through basic agricultural departments at all levels, farmers and entry-exit inspection departments, facilitates timely and effective control and elimination of an epidemic situation, and has a wide application prospect.

The invention provides a primer and a probe for detecting cucumber green mottle mosaic virus. The nucleotide sequence of the primer is shown as the sequence table SEQ ID NO.1&2, and the nucleotide sequence of the probe is shown as a sequence table SEQ ID NO.3. The invention further provides a method for detecting cucumber green mottle mosaic virus, the method takes the general RNA of samples as templates, conducts real-time fluorescence RT-PCR amplification by employing the primer and probe, each cycle finishes data collection, and the invention determines the result according to the amplification curve after reaction. The invention has primer with perfect specificity, rapid and simple detecting method, high accuracy and sensibility, which ensures import and export security.

The invention builds a new VIGS vector capable of being applied to cucurbit plants based on a CGMMV (cucumber green mottle mosaic virus), and provides a CGMMV recombinant vector carrying PDS (phytoenedesaturase) gene segments. The cucurbit plants such as watermelons, sweet melons, cucumbers and bottle gourds are natural hosts of the CGMMV. The recombinant vector can effectively reduce the expression level of a PDS gene in the cucurbit plants, so that the plant generates a PDS gene silenced light bleaching phenotype. The successful building of the recombinant vector can be used for researchingthe gene function; the foundation is laid for digging a cucurbit plant gene with important agronomic trait, and culturing excellent, disease-resistant and specific varieties.

The invention discloses a kit for rapidly detecting cucumber green mottle mosaic viruses (CGMMV) and application thereof. The specific primer pair provided in the invention comprises a DNA (Deoxyribonucleic Acid) shown as a sequence 1 in a sequence table and a DNA shown as a sequence 2 in the sequence table. When used for detecting CGMMV, the specific primer pair or kit has the advantages of simple and rapid operation, reliable result, strong specificity and high sensitivity and is suitable for field detection at ports. The specific primer pair or kit can be used for port detection of the CGMMV and investigation of field epidemic situations, and is especially suitable for use in each quarantine and inspection station, each port inspection and quarantine office, plant virus research institute and the like of agricultural departments.

The invention discloses a method for detecting a cucumber green mottle mosaic virus based on a one-step process RPA technology. The method is characterized in that a primer pair for detecting the cucumber green mottle mosaic virus is provided, wherein the primer pair is composed of a primer CGMMV-RPA-F and a primer CGMMV-RPA-R. The primer pair is used for detecting or detecting whether the cucumber green mottle mosaic virus or a to-be-detected plant sample is infected with the cucumber green mottle mosaic virus or not in an auxiliary mode. The primer pair provided by the invention has the advantages of good specificity and high sensitivity. The detecting method has the advantages of simple and convenient operation, high detecting efficiency and the like, and can meet basic needs of port and field detection.

The invention discloses a primer and a method for detecting cucumber green mottle mosaic virus (CGMMV). The primer is specific in the CGMMV, has the characteristics of being good in specificity and high in rate of coverage on different CGMMV isolators, can be used for effectively detecting the different CGMMV isolators, is more accurate in detection result, and is more convenient since auxiliary primers are not needed in the detection. The detection method disclosed by the invention is applicable to the direct detection on the CGMMV in a great number of seeds of cucurbit plants, and maintains the advantages of rapidness, accuracy, high sensitivity and the like while lowering the detection cost and reducing the work load.

The invention relates to an RNA-RP RT-PCR method capable of fast detecting melon viruses. Leaves of melon plants infected with viruses are homogenized by adopting a PBST plus a 2% PVP solution or a 0.5% Triton X-405 PBS solution, sample virus RNA is extracted in supernate, and the supernate is used for one-step RT-PCR amplification of target segments of the viruses, so that the melon viruses are detected. According to the RNA-RP RT-PCR method capable of fast detecting the melon viruses, only trace amounts of samples are needed for obtaining adequate amounts of RNA templates, the RNA templatesare used for detecting the six viruses of a Cucumber green mottle mosaic virus (CGMMV), a Cucumber mosaic virus (CMV), a Zucchini yellow mosaic virus (ZYMV), a Watermelon mosaic virus (WMV), a papayaring spot virus (PRSV) and a squash mosaic virus (SqMV) in watermelons, muskmelons, cucumbers, zucchini and a model plant nicotiana benthamiana in melon plants, the method is high in sensitivity and accurate in result, operation is simple and fast, and a novel detection method of the melon viruses is provided.

The invention discloses application of a nicotiana benthamiana NbSMG7 gene in regulation and control of plant virus resistance and a transgenic plant cultivation method, the virus is cucumber green mottle mosaic virus, and a transcript sequence of the NbSMG7 gene is shown as SEQ ID NO: 1. The NbSMG7 gene in nicotiana benthamiana is cloned and constructed into a YFP-NbSMG7 vector with a fluorescentlabel, the YFP-NbSMG7 vector is transferred into agrobacterium tumefaciens and then infects tobacco for genetic transformation of the tobacco, and a YFP-NbSMG7 transgenic plant is obtained. The obtained nicotiana benthamiana overexpression YFP-NbSMG7 plant can significantly hinder the infection of cucumber green mottle mosaic viruses.

The invention belongs to the field of agricultural microbiology, and relates to a biocontrol strain HW2 for preventing and treating cucumber green mottle mosaic virus and an application of the biocontrol strain HW2. Through authentication, the biocontrol strain HW2 belongs to stenotrophomonas maltophilia, and is preserved in the China General Microbiological Culture Collection Center (CGMCC) on June 29, 2015, wherein the preservation number of the biocontrol strain HW2 is CGMCC NO.11024. Meanwhile, the greenhouse experimental result shows that the biocontrol strain HW2 can obviously reduce the disease severity of the cucumber green mottle mosaic virus, and has an obvious effect in preventing and treating cucumber green mottle mosaic virus, the control effect is within 53.85%-57.02%, the microbial inoculum has a very good growth promoting effect for cucumber, the biomass increase can achieve 42.76%, and the content of chlorophyll of the cucumber leaf is obviously increased. Therefore, the strain and the microbial inoculum prepared by the strain can be applied to the prevention and treatment of the cucumber green mottle mosaic virus and growth promotion.

The invention belongs to the technical field of detection on melon quarantine diseases, especially relates to a method capable of synchronously and quickly detecting RT-PCR detection primer group of two melon quarantine diseases, i,e., cucumber green mottle mosaic virus (CGMMV) and acidovorax avenae subsp. citrulli (Aac), a kit and application of the method. According to the method provided by the invention, specific primers CGMMV-F / CGMMV-R, BFB-F / BFB-R are designed specifically for cucumber green mottle mosaic virus disease and acidovorax avenae subsp. Citrulli disease, and a RT-PCR detection technique is combined, synchronous detection and identification of the two diseases can be realized. The method provided by the invention is a specific, sensitive, convenient and cost-effective detection method which can synchronously and quickly detect CGMMV and Aac, can ensure the sensitivity and specificity of the detection, meanwhile, detection cost is reduced, time is saved.

The invention belongs to the field of agricultural microorganisms, and relates to a biocontrol strain HW17 for preventing and treating cucumber green mottle mosaic viruses and application of the biocontrol strain HW17. After being identified, the biocontrol strain HW17 belongs to bacillus subtilis, and is preserved in a common microorganism center of the China Committee for Culture Collection of Microorganisms on June 29, 2015 under the preserving number of CGMCC NO. 11025. Meanwhile, a greenhouse test result shows that by the biocontrol strain HW17, disease severity of the cucumber green mottle mosaic viruses can be reduced obviously, an effect of preventing and treating the cucumber green mottle mosaic viruses is remarkable, a preventing effect ranges from 49.63% to 53.76%, bactericide has an excellent growth promotion effect on cucumbers, increasing of biomass can reach 97.54%, and the content of chlorophyll of laminae of the cucumbers is increased obviously. Therefore, the biocontrol strain and the bactericide prepared from the same can be used for preventing and treating the cucumber green mottle mosaic viruses and promoting growth.

The invention relates to the technical field of crop disease resistance and breeding, in particular to an inoculation identification and quantitative detection method of cucumber green mottle mosaic viruses of Lagenaria leucantha. A method of quantitatively evaluating the cucumber green mottle mosaic virus resisting ability of Lagenaria leucantha comprises the following steps: firstly, utilizing bacterial liquid carrying an infectious cloning vector to inoculate Lagenaria leucantha seedlings by a cotyledon pressing contact method, then detecting CGMMV absorption values in a Lagenaria leucantha plant within two time quantum after inoculation by utilizing an ELISA technology, and comprehensively evaluating the ability of resisting CGMMV of different Lagenaria leucantha germ plasm according to the content variation condition of the CGMMV. Therefore, the method replaces phenotypic identification based on disease symptoms and is simple to operate and liable to apply.

The invention discloses a RPA (recombinase polymerase amplification) method for diagnosis and pathogen detection of blood flesh disease of watermelons. An efficient and economic technical system for rapidly detecting a CGMMV (cucumber green mottle mosaic virus) is established with a technological means of modern molecular biology study, the RPA rapid detection method is provided, and the method comprises steps as follows: cDNA of a sample with the virus is taken as a template in a RPA reaction, a pair of detection primers are added, a reaction is performed for 20-30 min at constant temperaturebeing 37-40 DEG C in a water bath kettle, sensitivity reaches 10<-6> and is higher, the CGMMV is detected rapidly and accurately, meanwhile, identification time is shortened, biochemical reagents aresaved, cost is reduced, and theoretical basis and technical support are provided for monitoring, forecasting and control of the CGMMV as a quarantine disease.

The invention discloses a nucleic acid test strip for detecting cucumber green mottle mosaic viruses and an application of the nucleic acid test strip. A specific primer group comprises a primer CGMMV-BF, a primer CGMMV-MBF, a primer CGMMV-DF, a primer CGMMV-CPR and a primer CGMMV-BR which are sequentially shown as a sequence 1 to 5 in a sequence table. The specific primer group is used for identifying the cucumber green mottle mosaic viruses or detecting whether a plant sample to be detected contains the cucumber green mottle mosaic viruses or not. The nucleic acid test strip has the advantages of good specificity, high sensitivity, simplicity and convenience in operation, high detection efficiency and the like, and can meet the basic requirements of port and field detection.

The invention discloses a rapid detection kit for cucumber green mottle mosaic viruses. The rapid detection kit comprises a kit cover, a kit body and a test paper strip, wherein the box cover is provided with a reaction groove for containing a fluorescent nano particle-cucumber green mottle mosaic virus antibody conjugate; the kit body is internally provided with a test paper strip storing position; the test paper strip is a rapid detection test paper strip for the cucumber green mottle mosaic viruses and is composed of a bottom plate, and a sample cushion, a chromatography film and a water absorption cushion which are attached on the bottom plate and are closely connected in sequence; the chromatography film is provided with a detection region and a quality control region; the detection region is used for fixing a monoclonal antibody or a polyclonal antibody which is subjected to specific reaction with the cucumber green mottle mosaic viruses. The detection kit provided by the invention can be used for finishing the detection of the cucumber green mottle mosaic viruses of plants in very short time, has good specificity and sensitivity and is an idea cucumber green mottle mosaic virus infection detection tool.

The invention discloses a molecular marker developed based on single nucleotide polymorphism (SNP) linked to resistance to a cucumber green mottle mosaic virus disease, and the molecular marker is used for resistance identification of the cucumber green mottle mosaic virus disease; a watermelon is used as a species, and molecular marker primers CGMMVR include an allelic primer 1, an allelic primer2 and a universal primer. The molecular marker can be used for identification of resistance of the germplasm to the cucumber green mottle mosaic virus disease in the watermelon and assistant selective breeding of hybrid or backcross descendants of the germplasm.

The invention belongs to the field of biological detection, and particularly relates to a primer group, a kit and a method for on-site rapid constant-temperature visualization and fluorescence detection of a cucumber green mottle mosaic virus. The primer group comprises an outer primer CGMMV-OF as shown in SEQ ID NO.1, an outer primer CGMMV-OB as shown in SEQ ID NO.2, an inner primer CGMMV-IF as shown in SEQ ID NO.3, an inner primer CGMMV-IB as shown in SEQ ID NO.4, a loop primer CGMMV-LF as shown in SEQ ID NO.5 and a loop primer CGMMV-LB as shown in SEQ ID NO.6. The primer group can be used for identifying the cucumber green mottle mosaic virus or detecting whether the cucumber green mottle mosaic virus exists in samples such as plant leaves, seeds, plants, nursery stocks, soil, field wastes and plant products (such as melons and fruits) or not. The detection method disclosed by the invention has the characteristics of simplicity in operation, rapidness, accuracy, good specificity, high sensitivity and result visualization, and is suitable for field and large-scale popularization and application.

Cucumber plants (Cucumis sativus) exhibiting increased resistance to Cumber Green Mottle Mosaic Virus (CGMMV) are provided, together with methods of producing, identifying, or selecting plants or germplasm with a CGMMV resistance phenotype. Such plants include cucumber plants comprising recombinant chromosomal segments conferring CGMMV resistance. Compositions, including novel polymorphic markers for detecting plants comprising introgressed virus resistance alleles, are further provided.

The invention belongs to the field of agricultural microorganisms, and relates to a biocontrol bacterial strain HW17 for preventing and treating cucumber green mottle mosaic virus disease and an application thereof. The biocontrol strain HW17, which was identified as belonging to the genus Bacillus subtilis, was deposited in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures on June 29, 2015, and the strain preservation number is CGMCC NO.11025. At the same time, the greenhouse test results showed that the biocontrol agent HW17 can significantly reduce the disease severity of cucumber green mottle mosaic virus disease, and has a significant effect on the control of cucumber green mottle mosaic virus disease, with a control effect between 49.63% and 53.76%. It has a very good growth-promoting effect on cucumbers, the biomass can be increased by 87.54%, and the chlorophyll content of cucumber leaves can be significantly increased. Therefore, the bacterial strain and the microbial agent prepared therefrom can be used in the prevention and treatment of cucumber green mottled mosaic virus disease and growth promotion.

The invention belongs to the technical field of molecular biology, and specifically relates to applications of LsWD taken as a reference gene in lagenaria siceraria var.hispida leaf or fruit qRT-PCR analysis under the infection of a cucumber green mottle mosaic virus. Through the utilization of lagenaria siceraria var.hispida transcriptome data, the expression level and stability of a gene can becompared at the whole genome level; and that the disclosed LsWD gene can be stably expressed in lagenaria siceraria var.hispida leaves and fruits under the infection of a cucumber green mottle mosaicvirus can be determined by using a LsIPT or LsDdRP gene to perform verification, so that the LsWD is a reliable reference gene in the analysis of gene expression of lagenaria siceraria var.hispida under the infection of the cucumber green mottle mosaic virus detected by utilizing a qRT-PCR technology. The LsWD gene is firstly taken as the reference gene to be used for the analysis of gene expression of the lagenaria siceraria var.hispida, so that present situations that existing lagenaria siceraria var.hispida quantitative PCR detection has no reference genes can be solved.

The invention discloses an RNA probe for specific detection of cucumber green mottle mosaic virus. The RNA probe is specifically combined with a positive-sense strand RNA of the cucumber green mottle mosaic virus, and detection is performed in a Northern hybridization mode, so that the detection specificity and sensitivity are remarkably improved. The designed RNA probe is used for detecting the accumulation and the integrity of a viral genome RNA (gRNA), a subgenome RNA (sgRNA), defective interfering (DI) RNA and other viral RNA fragments. Through gel electrophoresis to membrane hybridization by a Northern hybridization detection system, the advantages that gRNA, sgRNA and the like of viruses are quantitatively detected, and meanwhile, whether nucleic acid is degraded or not can be judged.

The invention discloses an RT-LAMP (loop-mediated isothermal amplification) rapid detection kit for cucumber green mottle mosaic virus (CGMMV) and a detection method. The method comprises the steps of extracting plant viruses RNA, detecting an RT-LAMP reaction and electrophoresis detection or fluorochrome, and determining whether a plant carries the viruses. An RT-LAMP primer is designed according to a coat protein gene conserved region of CGMMV, and an RT-LAMP technology is adopted to establish a detection technology which can rapidly and sensitively detect the cucurbitaceous plant CGMMV such as cucumbers, watermelons and calabashes with high specificity and an application method thereof in diagnosing disease. The detection method provided by the invention can utilize the rapid extraction RNA and conventional RNA extraction as an RNA template for an RT-LAMP experiment. The method is utilized to rapidly identify a CGMMV plant sample, the targeted quarantine inspection protective measure is adopted, and the loss caused by the disease is reduced.

The invention belongs to the field of plant protection, and relates to a method for preventing and treating cucumber green mottle mosaic virus, in particular to application of melatonin to prevention and treatment of cucumber green mottle mosaic virus and a method for enhancing the prevention and treatment effect in cooperation with melatonin. The method comprises the implementation steps that a melatonin solution is irrigated to plant seedlings and roots or sprayed to leaf surfaces once a day, and treatment is conducted for three days in total. The invention further discloses a method for enhancing the prevention and treatment effect on the cucumber green mottle mosaic virus in cooperation with melatonin. The prevention and treatment effect of the melatonin on the cucumber green mottle mosaic virus is remarkably enhanced through the silent gene NbCRISP1. According to the application, the relationship between the non-toxic macromolecular melatonin and the CGMMV is taken as a research object, the effect of the melatonin in prevention and treatment of the CGMMV is defined, and the application of the melatonin in prevention and treatment of plant diseases is expanded.