44 results about "Nosema bombycis" patented technology

The invention discloses a group of primers and application thereof in quick detection of nosema bombycis. The primers have the nucleotide sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4. Based on the primers disclosed by the invention, related reagents applied to quick detection of nosema bombycis by an LAMP (Loop-Mediated Isothermal Amplification) method can be prepared by combining a conventional technology in the field, can detect the nosema bombycis by the LAMP method quickly and accurately, are suitable for operation on a small amount of sample and simple to operate, and have important significance in practical application.

The invention relates to a detection method of nosema bombycis (Nb), in particular to a nosema bombycis SYBR Green fluorescence quantitative PCR (Polymerase Chain Reaction) detection method as well as a kit and a specific primer thereof. The nosema bombycis SYBR Green fluorescence quantitative PCR detection method comprises the following steps of: (1) collecting samples; (2) extracting the DNA of nosema bombycis; (3) detecting the samples by using the SYBR Green fluorescence quantitative PCR amplification method; and (4) analyzing the corresponding samples according to the fluorescence intensity of the reaction after the amplification reaction is finished so as to judge whether nosema bombycis exists in the collected samples, and performing accurate quantitation to nosema bombycis so as to realize the real-time quick detection of nosema bombycis. Because the specific amplification primer and the SYBR Green Buffer system are introduced in the method, the sensitivity and the specificity of nosema bombycis detection can be furthest enhanced, and miss detection and mistake detection are avoided.

The invention belongs to the technical field of molecular biotechnologies and particularly discloses an application of an EB1 gene to detection of nosema bombycis. Researches prove that the transcriptional expression of the EB1 gene is related to the cell division of the nosema bombycis and the development of a host. A specific primer is designed by taking the EB1 gene as a target gene, whether silkworm eggs are infected by the nosema bombycis is detected through PCR (Polymerase Chain Reaction) amplification, and the detected result proves that if the EB1 gene is used as the detecting target gene, the interference effects of inhibiting substances in silkworm egg extracts to the effective PCR amplification of pathogenic gene DNA (Deoxyribose Nucleic Acid) are very low, the detected result is more accurate and sensitive, and most importantly, whether the silkworm eggs are infected by the nosema bombycis can be rapidly detected at the early stage that the silkworm eggs are infected. The guarantee is provided for the detection and safe egg production of poisonous silkworm eggs in silkworm egg production.

The invention belongs to the technical field of bioinstrumentation, and relates to a triple-fluorescence PCR (Polymerase Chain Reaction) detection method and kit for Nosema bombycis (Nb), Bombyx mori Nuclear Polyhedrosis Virus (BmNPV) and Bombyx mori Densovirus (BmDNV), as well as primers and probes. According to the invention, the primers and the probes for the Nb, the BmNPV and the BmDNV are respectively designed, and different fluorescence signals are marked on the three probes, so that the Nb, the BmNPV and the BmDNV can be synchronously detected by triple-fluorescence-quantitation PCR under the condition of optimizing a PCR reaction system. According to the invention, the synchronous extraction of DNA (deoxyribonucleic acid) can be realized, and the synchronous detection based on fluorescence PCR can be performed, so that the detection method is simplified greatly; and through a program of florescence compensation, false positive due to intersection of the fluorescence signals iseliminated effectively. The method can be used for quickly, peculiarly and sensitively verifying and detecting the Nb, the BmNPV and the BmDNV, and improving the early detection rate, thereby effectively preventing disease transmission and reducing the economic loss.

The invention provides a method for detecting pebrine-causing Nosema bombycis (N.b) from samples, such as silkworm, silkworm egg and the like by fluorescence quantification PCR technology (a PCR-fluorescence probe method) and a kit thereof. The method comprises the following steps of: (1) collecting the samples; (2) extracting Nosema bombycis DNA; (3) detecting the samples by the fluorescence quantification PCR augmentation method; and (4) analyzing the corresponding samples according to the fluorescence intensity of the reaction after the augmentation reaction is ended so as to judge existence of the Nosema bombycis in the collected samples, accurately quantify the Nosema bombycis and realize real-time quick detection on the Nosema bombycis.

The invention relates to a dyeing liquor used to discriminate the protozoan cell activity, a method to discriminate the protozoan cell activity and the application of the method to discriminate activity of nosema bombycis, which is characterized in that: the dyeing liquor comprises the following components: three acridine oranges with the concentration of 0.05% to 0.3% and one propidium iodide with the concentration of 0.0067% to 0.026%; the acridine oranges and the propidium iodide are prepared by NaCl solution with the concentration of 0.38%; the method to discriminate the protozoan cell activity comprises adequate material choosing, dyeing liquor preparing, dyeing, microscopic examination and protozoan cell activity discriminating based on the color change of microscopic examination results. The dyeing liquor used to discriminate the protozoan cell activity has the advantages that: the spore activity of nosema bombycis can be discriminated quickly, sensitively, accurately and visually; the deficiency of the prior art is overcome.

The invention relates to a very important protein gene PTP2 related to propagation and infection of nosema bombycis nageli. A gene order CDS for encoding nosema bombycis nageli polar tube PTP2 is obtained according to 9-fold genome data of the nosema bombycis nageli and proven by a series of biotechnology clonings. The nosema bombycis nageli polar tube PTP2 obtained in the invention plays a very important role in studying a propagation and infection mechanism of microsporidian, thus further playing a role in productive practice and prevention and treatment of the microsporidian disease. Experiments and analyses such as RNAi intervention and the like indicate that after the polar tube protein PTP2 is intervened, the microsporidian loses the capacity of infecting a host, therefore, the polar tube protein PTP2 of the microsporidian and potential regulator genes thereof become new factors for treating microsporidian infection, in addition, the method of modifying genes can be adopted to express the polar tube protein PTP2 in an abundant way, thus killing part of pests such as locust and the like.

The invention provides a detection method of the number level of nosema bombycis in silkworm finished- product eggs. The detection method comprises the following steps of: designing a specific primer according to a DNA (deoxyribonucleic acid) sequence of nosema bombycis of silkworms, and with total DNA in the silkworm eggs as a template, carrying out fluorogenic quantitative polymerase chain reaction (Q-PCR) amplification to obtain a Ct value of a detected sample; meanwhile, with different copy numbers of DNA of recombinant nosema bombycis of the silkworms as a template, carrying out Q-PCR amplification, drawing a standard curve by taking the Ct value as a vertical coordinate and a log value of the copy number of the initial plasmid DNA as a horizontal coordinate, and solving an regression equation; and calculating the initial copy number of the nosema bombycis DNA in the silkworms in the detected sample according to the regression equation and the Ct value of the detected sample, thereby estimating the quantity level of the nosema bombycis of the silkworms in the sample. The detection method can be used for directly judging the number level of the nosema bombycis according to the copy number of the nosema bombycis DNA of the silkworms in the detected sample. The estimation of the occurrence risk of the microparticle disease in subsequent breeding according to the number level of the nosema bombycis in the finished-product eggs is more scientific than the estimation of the diseased egg rate of the microparticle disease of the finished-product eggs.

The invention discloses a processing method for a sample of a nosema bombycis naegeli in a graine by utilizing a PCR (Polymerase Chain Reaction) method. The processing method comprises the steps as follows: adding a graine sample to a pretreatment solution for at least 30min; mashing the graine and adjusting the pH value of the solution to neutral so as to obtain a PCR template, wherein the pretreatment solution is alkaline and contains 1 to 10mol / L of glycine betaine. In addition, the invention further provides a method for detecting the nosema bombycis naegeli in the graine by utilizing the sample processing method. The detecting method provided by the invention has the advantages of simplicity in operation and low in cost, and is capable of shortening the time of the whole detection process by 40%, thereby being suitable for detection of common nosema bombycis naegeli and specifically suitable for quarantine inspection of the nosema bombycis naegeli in the graine (silkworm eggs, silkworm chrysalis and moths) with greater sample capacity.

The invention discloses an LAMP (loop-mediated isothermal amplification) detection primer group for microsporidia in silkworm eggs and an application thereof. The primer group comprises outside primers EB1-F3 / EB1-B3 and inside primers EB1-FIP / EB1-BIP, wherein the sequences of the primers are shown in SEQIDNO:1-4. An LAMP detection method and kit for microsporidia in silkworm eggs are established by utilizing the primers. The kit comprises the primer group, 2*reaction buffer, positive control substances, negative control substances, a developing liquid (or a fluorescence staining liquid), Bst DNA (deoxyribonucleic acid) polymerase, a sealing liquid and sterile water. The results of detection carried out by adopting the method can be visually observed in natural light or be observed through agarose gel electrophoresis or be observed and determined via a real-time fluorescence curve. The method is easy to operate, has the advantages of short detection time, easiness in determination of results and strong specificity, and can be used for detecting the DNA, at a concentration of 5.0*10<-3>ng / mu L, of the eggs laid by the silkworms infected with nosema bombycis.

The invention relates to a very important protein gene PTP3 related to propagation and infection of nosema bombycis nageli. A gene order CDS for encoding nosema bombycis nageli polar tube PTP3 is obtained according to 9-fold genome data of the nosema bombycis nageli and proven by a series of biotechnology clonings. The polar tube PTP3 obtained in the invention plays a very important role in studying a propagation and infection mechanism of microsporidian, thus playing a role in productive practice and prevention and treatment of the microsporidian disease. Experiments and analyses such as RNAi intervention and the like indicate that after the polar tube protein PTP3 is intervened, the microsporidian loses the capacity of infecting a host, therefore, the polar tube protein PTP3 of the microsporidian and potential regulator genes thereof become new factors for treating microsporidian infection, in addition, the method of modifying genes can be adopted to express the polar tube protein PTP3 in an abundant way, thus killing part of pests such as locust and the like.

The invention provides a method for rapidly obtaining large amount of spore shells of nosema bombycis. The method comprises the following steps of adding 10<9>-10<20> 400muL of nosema bombycis into a solution, mixing the nosema bombycis suspension with 212-300mu m of glass beads according to the proportion of 1mu L:1mg to obtain a mixture, putting the mixture into a micro burette mixer and oscillating for 8-12 hours, carrying out gradient density centrifugation on the spore suspension, and taking sediment. According to the method, the spore shell extracting and purification processes are simplified, the operation is simple and fast, the requirement on equipment is low, the repeatability is high, a large amount of spores can be rapidly broken in one day so as to obtain the spore shells, the yield of the spore shell can reach 70-90% or even 100%, and the acquisition of the spore shell is not affected by the weakened vitality of the spores.

The invention discloses a method for constructing transgenic Nosema bombycis, characterized by injecting a transgenic vector wrapped by liposome in body cavity of the Bombyx mori infected with Nosema bombycis, after 2-4 days, adding silkworm hemolymph in Bombyx mori BmN culture cells to cultivate; when microsporidian presents green fluorescent light, using an insect medium to extremely dilute and adding into a cell culture plate, selecting a culture pore where only one culture cell presents green fluorescent light, adding normal culture cells to culture for 2-4 days to obtain infected culture cells; repeating for at least three times, taking the obtained infected culture cells, conducting homogenate and crushing the cells, conducting differential centrifugation, purifying microsporidian, infecting the Bombyx mori, after the Bombyx mori falls ill, purifying the infected tissues to obtain the transgenic Nosema bombycis. According to the invention, exogenous genes can be integrated into the Nosema bombycis genome, the modification of Nosema bombycis by gene engineering is realized, the adjustment of expression of the microsporidian genes is realized, and a method for microsporidian gene function research is provided.

This invention publishes a kind of method of detecting pebrine eggs in the domestic silkworm finished eggs. Firstly, the whole DNA of the silkworm finished eggs to be checked be taken out. Secondly we design and composite a pair of the specific primer according to the specific sequence of the gene of the silkworm mini sporozoon. The distance between the two primer is 0.5-2.0 kb. Thirdly we set the whole DNA as the form board to make PCR amplification and then we do the electrophoretic detection for the amplified product. When we detect out the specific gene fragment, we can determine the egg is sick. As we check the silkworm finished eggs in large groups, we can get the sick eggs' ratio. To sum up, this invention can directly check the silkworms' eggs and the eggs don't need to be catalyzed to incubation. As a result, the checking time is short and the sensitivity is high, so it can satisfy the need of the departments.

The invention relates to a vector and method for eukaryotic soluble expression of polar-tube glycoprotein of Nosema bombycis. The method comprises the following concrete steps: constructing a recombinant vector containing the NbPTP1 gene of Nosema bombycis, and then transfecting the sf9 cell of Spodoptera frugiperda for expression, wherein the expression of the recombinant vector containing the NbPTP1 gene of Nosema bombycis is regulated by an IE2 promoter. The method of the invention successfully realizes the soluble expression of the polar-tube glycoprotein of Nosema bombycis, and has a glycosylation modification, so a theoretical foundation is laid for further understanding of the functions of the polar-tube protein of Nosema bombycis and explication of the infection mechanism of Nosemabombycis.

The invention relates to a separating and enriching method of nosema bombycis sporoplasm. The separating and enriching method comprises the following specific steps of (1) treating nosema bombycis spores with a 0.1M KOH aqueous solution, performing centrifugation, removing supernatant, adding TC100 insect culture medium suspension spore precipitate, and performing incubation to obtain culture medium suspension spore samples; (2) absorbing the spore samples obtained in the step (1), performing cell nucleus dyeing, observing the situation of sporoplasm ejected from a polar tube, and counting thesprouting rate of the spores; (3) when the sprouting rate of the spores reaches 60%, adding the sprouted spore samples into a percoll solution having two density gradients of 30% and 60%, and performing density gradient centrifugal separation to obtain sporoplasm; and (4) collecting the percoll solution at 30% density layer of an upper layer, and performing cleaning and centrifugation. The separating and enriching method disclosed by the invention is convenient to operate, besides, the sporoplasm can be efficiently separated and enriched, and the separating and enriching method has far-reaching significance on deep researching of the infestation mechanism and the life history of the nosema bombycis.

The invention discloses an application of Ag nanoparticles to preparation of medicines for restraining nosema bombycis as a main pathogen of bombyx mori. The Ag nanoparticles are spherical, are 3-24nmin dimension and are 0.23nm in crystal lattice distance. Through Ag nanoparticle solution of different concentrations, the toxicity of bombyx mori larvae is tested, and the result shows that the weight, the cocoon shell rate and the oviposition number of the bombyx mori larvae between groups do not have significant differences, which indicates that the Ag nanoparticle solution having the concentration being 100 [mu]g / mL or below is safe to the bombyx mori larvae; a mechanism of Ag nanoparticles for resisting nosema bombycis is analyzed, then the inventor finds that the Ag nanoparticles can seriously destroy the structure of the nosema bombycis, when the Ag nanoparticles act with spore coats, the permeability is increased, so that some substances of DNA, proteins and the like are destroyed, the infestation capacity is greatly reduced, the pathogenic capacity of the nosema bombycis is reduced, and the disinfection effects of the nosema bombycis can be achieved; and the Ag nanoparticleshave important significance in controlling the nosema bombycis of the bombyx mori.

The invention discloses a nosema bombycis Met-AP2 gene and an application thereof in the aspect of nosema detection, as well as a group of universal nosema detection primers and a kit. The universal detection primers include an upstream primer MC-F and a downstream primer MC-R, wherein the nucleotide sequence of the upstream primer MC-F is as shown in SEQ ID NO. 4 and the nucleotide sequence of the downstream primer MC-R is as shown in SEQ ID NO.5. The detection primers take the gene Met-AP2 as a target gene and the detection primers are simultaneously applicable to the detection of various nosemas; the detection primers are reliable in detection result, easy in operation and high in sensitivity, and especially, the detection primers have an important practical application value for the early detection of various nosemas in a sample in practical inspection and quarantine.

The invention belongs to the technical field of bombyx mori transgenosis, and particularly relates to a promoter of a nosema bombycis inducible expression gene BmPUGT2 and application of the promoter.A nucleotide gene of the promoter is shown as SEQ ID NO:6, and the promoter can drive an exogenous gene to be expressed in bombyx mori under induction of nosema bombycis, the promoter is suitable formolecular biology theoretical research such as gene function analysis and the like, meanwhile is suitable for bombyx mori variety improvement by utilizing genetic engineering, particularly suitable for bombyx mori nosema-disease-resistant variety cultivation, and has a good application prospect.

The invention provides an application of a small molecule compound in preparation of a medicine for preventing or treating pebrine. The structure of the small molecule compound is as shown in a general formula (I) aiming at core protein of nosema bombycis, and the three-dimensional structure of the small molecule compound is obtained by utilizing a virtual modeling technology based on a mainstream molecular docking technology. The small molecule compound can be used for preparing a medicine for preventing and treating pebrine, when the final use concentration of the small molecule compound in cells is 0.5 [mu]M or above, replication of nosema bombycis can be inhibited, and the morbidity of pebrine can be remarkably reduced when the small molecule compound is added to silkworms with the concentration of 1 mM or above. The method can be used for research, development and production of medicines for preventing and treating pebrine, and has application and popularization values.

The invention discloses a Nosema bombycis ED2013 for controlling Athetis lepigone, belonging to the field of biological control on crop pests. The Nosema bombycis is collected by China General Microbiological Culture Collection Center on August 7th, 2014, and the collection number is CGMCC No.9557. The invention also discloses a pesticide containing the ED2013 Athetis lepigone and a preparation method thereof. The Nosema bombycis ED2013 has higher pesticidal activity and pesticidal effect for Athetis lepigone larvae, can effectively lower the population quantity of Athetis lepigone, and provides a high-efficiency way for controlling Athetis lepigone. The invention belongs to a biological control method which has no environmental pollution and is harmless to other natural enemies; and the Nosema bombycis ED2013 can substitute the pesticide or lower the pesticide consumption, and is environment-friendly.

The invention discloses a co-immunoprecipitation method suitable for nosema bombycis. The three technical problems in the prior art are solved, wherein as the first problem, spore total protein for a co-immunoprecipitation experiment is difficult to extract, and as the sporoderm of the nosema bombycis is thick, common protein extraction liquid cannot enter a spore, and protein cannot be fully extracted; as the second problem, due to the gravity, immune ball beads for co-immunoprecipitation can be naturally sunk to the bottom in the reacting process, stacked ball beads, protein liquid and antibodies cannot be fully combined, and the experiment result is influenced; as the third problem, most antibodies for the co-immunoprecipitation experiment are self-prepared polyclonal antibodies, the specific effect of the antibodies is poor, and nonspecific bands usually appear; in addition, detection to target protein can also be influenced by heavy chains and light chains of the antibodies. By means of the co-immunoprecipitation method suitable for the nosema bombycis, the specificity of the nosema-bombycis co-immunoprecipitation is improved, stability is high, and repeatability is good.

The invention discloses a staining solution for Nosema bombycis spores in lepidopteran insects, which is a phosphate buffer containing isothiocyanate. Accordingly, the inventor also establishes a method for preparing the staining solution, and a detection method using the staining solution. In the detection method, a sample is fluorescently stained by the above staining solution, and then the spores are observed under a fluorescence microscope. The staining solution is used to fluorescently stain shells of the Nosema bombycis spores in the lepidopteran insects, and slides are easy to manufacture and easy to operate. The fluorescence microscope is used, and the staining solution used for staining the Nosema bombycis spores in the lepidopteran insects makes the Nosema bombycis spores in thelepidopteran insects have fluorescence specificity. The repeatability and accuracy of the detection are ensured by comparing with shape characteristics of a standard. In summary, the method for detecting the Nosema bombycis spores in the lepidopteran insects, the staining solution and the preparation method thereof have the characteristics of simple operation, strong specificity and easy identification, and lay a foundation for mechanical detection and fair detection, and have high practical value and broad market prospect.

The present invention discloses a nosema bombycis DNA2 gene and application thereof. According to the invention, firstly cloning is carried out to acquire the nosema bombycis DNA2 gene with a full-length sequence shown as SEQ ID NO.1, an ORF rame sequence is shown as SEQ ID NO.2, and an amino acid sequence of encoding protein is shown as SEQ ID NO.3. On the basis, the invention also provides a specific detection primer set for specific detection of nosema bombycis, the specific detection primer set has very good specificity and sensitivity on nosema bombycis, and has very good application prospect in specific detection of nosema bombycis. Moreover, as a newly discovered protein gene, the nosema bombycis DNA2 gene has potential functional application. Discovery of the DNA2 gene provided by the invention has certain significance for theoretical verification and production practice of treatment and detection.

The invention discloses a method for constructing transgenic Nosema bombycis, characterized by injecting a transgenic vector wrapped by liposome in body cavity of the Bombyx mori infected with Nosema bombycis, after 2-4 days, adding silkworm hemolymph in Bombyx mori BmN culture cells to cultivate; when microsporidian presents green fluorescent light, using an insect medium to extremely dilute and adding into a cell culture plate, selecting a culture pore where only one culture cell presents green fluorescent light, adding normal culture cells to culture for 2-4 days to obtain infected culture cells; repeating for at least three times, taking the obtained infected culture cells, conducting homogenate and crushing the cells, conducting differential centrifugation, purifying microsporidian, infecting the Bombyx mori, after the Bombyx mori falls ill, purifying the infected tissues to obtain the transgenic Nosema bombycis. According to the invention, exogenous genes can be integrated into the Nosema bombycis genome, the modification of Nosema bombycis by gene engineering is realized, the adjustment of expression of the microsporidian genes is realized, and a method for microsporidian gene function research is provided.

The invention relates to a kit for detecting silkworm eggs by using a PCR-ELISA method and a detecting method of the kit. The kit comprises a nucleic acid molecular marker labeled primary pair for detecting the silkworm eggs and an ELISA reagent which detects a nucleic acid molecular marker; suitable gene segments are amplified by labeled specific primers, then an amplified product is subjected to ELISA, screened primers have species specificity, the sensitivity is high, multi-sample high-flux detection can be realized, and therefore, the kit plays an important role in detection of silkworm eggs in silkworm eggs.

The invention discloses a probe for rapidly detecting nosema bombycis. The nosema bombycis can be rapidly detected through an RAA technology. The invention further discloses a kit for rapidly detecting the nosema bombycis, and the kit is rapid in detection and high in sensitivity. The invention further discloses a detection method of the kit for rapidly detecting the nosema bombycis, the steps of reagent mixing and sample adding are reduced, operation is convenient, and time is saved.