36 results about "Genus Echinococcus" patented technology

The invention discloses a method for detecting Echinococcus multilocularis pathogen from fox excrement. The method includes: placing the collected Tibetan fox excrement sample into 70% ethanol, and performing cryopreservation at minus 80 DEG C for more than three weeks; using double-layer sterilizing gauze to enrich excrement sample Echinococcus multilocularis eggs, using a tissue crusher to crush egg embryonic membranes in a mechanical oscillation manner, using an excrement DNA extracting kit to extract the DNA of the Echinococcus multilocularis egg in the excrement sample, and using nest PCR amplification to detect the Echinococcus multilocularis eggs. The method is simple to operate, capable of fast detecting whether Echinococcus multilocularis infection exists in the fox excrement or not, high in sensitivity and high in specificity.

The invention discloses a kit for detecting echinococcus granulosus, echinococcus multilocularis and echinococcus shiquicus. Three pairs of specific amplification primers aiming at three pathogens are respectively arranged in the detection kit. By utilizing the three pairs of primers in the kit, a composite polymerase chain reaction (PCR) method can be used for detecting independent or mixed infection of the three pathogens, and whether the detected sample is infected with echinococcus can be determined according to specific strips occurring in electrophoresis of amplification products.

The invention discloses a multiple PCR method for simultaneously detecting and identifying three human tissue echinococcosis pathogens, including echinococcus granulosus G1-G3, echinococcus multilocularis and echinococcus granulosus G6-G10. According to the invention, 3 pairs of specific primers are designed for the mitochondrial genes of 3 pathogens; the specific primers are added into a same PCR reaction system; the specific primers are complementarily combined with the target genes of the corresponding parasite species; through amplified reaction, different parasite species can generate target stripes in different sizes; gel electrophoresis is adopted for separating and detecting. The three pairs of primers according to the invention are free from mutual interference and have no cross reaction with 8 tapeworms in close genetic relationship or do not interfere with the result judgment. The method is high in specificity and sensitivity, can effectively and accurately realize the synchronous detection for 3 pathogens, can greatly save the detection time and cost, can effectively increase the working efficiency and can be applied to the research on the parting identification for human echinococcosis pathogens and echinococcosis molecular epidemiology.

The invention discloses a kit of dog echinococcus granulosus and echinococcus multilocularis based on POCKIT Micro fluorescent PCR platform, and application of the kit. The kit comprises fluorescent quantitative PCR reaction fluid, positive comparison product and negative comparison product; the fluorescent quantitative PCR reaction fluid comprises upstream general primers of echinococcus granulosus and echinococcus multilocularis, downstream general primers of echinococcus granulosus and echinococcus multilocularis, general probes of echinococcus granulosus and echinococcus multilocularis, Taq enzyme; premixing buffer and deionized water. The nucleotide sequence of the upstream general primers of echinococcus granulosus and echinococcus multilocularis and the nucleotide sequence of the downstream general primers of echinococcus granulosus and echinococcus multilocularis are shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3. The kit design is specific to the general primers and the probes of two hydatids; the result can be determined by detection once, thus the time is greatly saved, and detection cost is reduced.

The invention provides an Echinococcus multilocularis circulating antigen dot immunogold filtration kit and a preparation method, relating to an Echinococcus multilocularis circulating antigen detection reagent. The kit is provided with a detection plate, a gold-marked antibody for resisting an Echinococcus multilocularis circulating antigen, and a washing solution; and the detection plate is provided with a carrier medium, a micro-pore filtration membrane, a water absorbing medium, an Echinococcus multilocularis circulating antigen detection point and a contrasting point. The preparation method comprises the following steps of: preparing the Echinococcus multilocularis circulating antigen; preparing the antibody for resisting the Echinococcus multilocularis circulating antigen; preparing colloidal gold; marking the colloidal gold and the antibody for resisting the Echinococcus multilocularis circulating antigen; and preparing the dot immunogold filtration kit. The Echinococcus multilocularis circulating antigen dot immunogold filtration kit can be used for detecting the Echinococcus multilocularis circulating antigen in samples including whole blood, blood serum, blood plasma and the like. When the Echinococcus multilocularis circulating antigen dot immunogold filtration kit is used for detecting, the needed sample amount is extremely small, a special instrument is not needed and a result can be directly judged and read by eyes; and the Echinococcus multilocularis circulating antigen dot immunogold filtration kit has the advantages of simplicity and rapidness in detection, strong specificity, high flexibility, accuracy and reliability, low cost and wide application.

The invention discloses a real-time quantitative PCR (Polymerase Chain Reaction) method for detecting echinococcus multilocularis and echinococcus granulosus by types based on an MGB (Minor Groove Binder) probe and a detection kit. The real-time quantitative PCR method comprises the following steps: designing primers, designing the MGB probe and selecting PCR amplification conditions; the detection kit contains an outer primer pair sequence of a PCR primer pair and an MGB type detection probe sequence. The method disclosed by the invention can be used for pertinently detecting samples containing the echinococcus multilocularis and / or the echinococcus granulosus by types and the accuracy is high; mitochondrion DNAs (Deoxyribonucleic Acid) of the echinococcus multilocularis and the echinococcus granulosus can be accurately detected by types; the sensitivity is high so that the echinococcus multilocularis and the echinococcus granulosus can be rapidly identified by types in a large batch; the operation is simple and convenient, and trace insect source mitochondrion DNAs in the samples can be amplified and the level of detecting by types is realized.

The invention discloses a liver echinococcus gene segment screening method, an amplification primer and a kit.The screening method comprises the following steps: eliminating an influence of a human genome and a close genetic relationship tapeworm group genome from whole genomes of echinococcus granulosus and echinococcus multilocularis; and screening to obtain a third echinococcus granulosus gene segment, a third echinococcus multilocularis gene segment and a common gene segment, and designing three types of amplification primers by using three types of the gene segments respectively. A primer pair group for detecting echinococcosis of human tissues is obtained by further screening and a kit and a use method of the kit are provided based on the primer pair group. False positive results caused by human genes or close genetic relationship tapeworm genes existing in to-be-detected tissue DNA is avoided from the source, the to-be-detected DNA aiming at the primer during design is a human tissue sample, the false negative results in clinical detection are remarkably reduced, specific primers have higher accuracy and higher specificity, and clinical use effects of the primer pair and the kit are obviously enhanced.

The invention relates to the technical field of AE (Alveolar Echinococcoisis) animal models, and provides a construction method of an AE animal model realizing liver infection with Echinococcus multilocularis through the hepatic portal vein. The method comprises steps as follows: a mouse is anaesthetized and fixed, the abdomen of the mouse is disinfected, the hepatic portal vein is exposed through laparotomy, the mouse is inoculated with the Echinococcus multilocularis through hepatic portal vein puncture, a puncture point is pressed until bleeding is stopped, then the abdomen is closed, the mouse subjected to puncture inoculation and abdomen closure is normally fed after reviving, and the AE animal model realizing liver infection with the Echinococcus multilocularis through the hepatic portal vein is obtained. The AE animal model obtained with the construction method of the AE animal model realizing liver infection with the Echinococcus multilocularis through the hepatic portal vein has the advantages of being close to a natural infection route and high in infection rate; therefore, the good animal model is provided for research of the mouse infection rate and the immune tolerance mechanism in different time periods and under different dosages and a host liver damage mechanism caused by Echinococcus multilocularis infection as well as screening of new drugs for treating AE.

The invention discloses a PCR-RFLP detection kit for authenticating and differentiating the infections of echinococcus multilocularis (Em) and echinococcosis shiquicus (Es). The kit comprises an Em and Es universal primer shown in SEQ ID NO. 1 and 2, restriction endonuclease EcoR I and / or Ssp I, a PCR reagent, and echinococcus multilocularis and echinococcosis shiquicus DNA template reference substances. The purpose of authenticating and differentiating the infections of echinococcus multilocularis and echinococcosis shiquicus can be achieved by extracting the DNA of the single cyst of the sample to be detected as a template, carrying out PCR amplification by using the universal primer, purifying a PCR product, carrying out enzyme digestion by using EcoR I or Ssp I incision enzyme, and then carrying out electrophoresis on an enzyme digestion product. According to the kit disclosed by the invention, the time consumption and money consumption due to sequencing, and the cumbersome process of data processing are omitted, a complex operation system is not required, and infection of echinococcus multilocularis or echinococcosis shiquicus to wild rodents can be rapidly, conveniently and economically differentiated and authenticated.

The invention discloses a kit for assaying echinococcus in lesion tissue or dog feces by multiplex recombinase-aid amplification (RAA) and multiple PCR. Meanwhile, the kit is also used for assaying echinococcus granulosus G1 and echinococcus multilocularis. The kit comprises primers as follows: echinococcus granulosus specific primers shown in SEQ ID NO.1 and SEQ ID NO.2 and echinococcus multilocularis specific primers shown in SEQ ID NO.3 and SEQ ID NO.4. In addition, the invention further discloses a multiplex RAA and multiplex PCR assay method for the echinococcus granulosus G1 and echinococcus multilocularis. Various verifications prove that the kit has characteristics of rapidness, sensitivity and high specificity, complex steps for repeated assay are eliminated, two genes can be assayed simultaneously in one reaction, functions of pathogens are made clear, the workload is reduced greatly, the assay time is shortened, the assay cost is reduced, and the kit has a greater development trend.

The invention discloses a primer and a kit for simultaneously detecting two types of echinococcosis. The primer is arranged in the kit in the form of freeze-dried powder or dissolved in a buffer solution. The primer comprises three pairs of specific primer pairs respectively comprising: an echinococcus granulosus specific primer pair, an echinococcus canadensis specific primer pair and an echinococcus multilocularis specific primer pair. By utilizing the three pairs of primers in the kit, individual or mixed infection of the three pathogens can be detected by one round of PRC amplification, and it can be determined whether samples to be detected are infected and infected with the species or not according to the specific bands present in the electrophoresis of the amplification products. Byadopting the kit, not only is the operation fast and convenient, but also the hydatidosis caused by three echinococcus infections can be accurately diagnosed and distinguished.

The invention relates to a design and preparation method and an application of an echinococcus multilocularis subunit vaccine LTB-Emy162. The active component of the echinococcus multilocularis subunit vaccine LTB-Emy162 is a polypeptide, which is mainly composed of an echinococcus multilocularis antigen protein Emy162 amino acid sequence and a mucosal immunoadjuvant E. coli heat-labile enterotoxin B subunit (LTB) amino acid sequence. In the invention, the gene sequence of the echinococcus multilocularis subunit vaccine LTB-Emy162 is synthesized through gene synthesis technology, the gene sequence is then linked to an expression vector through dual enzyme-cut, the expression vector then is converted into Arctic Express to perform expression of fusion protein, after purification of the protein, the echinococcus multilocularis subunit vaccine LTB-Emy162 is produced. The echinococcus multilocularis subunit vaccine can induce body to generate T-cell and B-cell immunologic response of the echinococcus multilocularis and humoral immune response of a high-titer specific antibody, and can be applied in prevention and therapy of echinococcus multilocularis infection related diseases.

The invention relates to a design, preparing method and applications of an echinococcus multilocularis multi-epitope vaccine LTB-AE. An active component of the multi-epitope vaccine LTB-AE is a polypeptide. The polypeptide mainly comprises an echinococcus multilocularis multi-epitope peptide AE, and a mucosal immune adjuvant that is an escherichia coli heat-labile enterotoxin B subunit (LTB). A gene sequence of the multi-epitope vaccine LTB-AE is synthesized mainly by a gene synthesis technique, and is connected to an expression vector through double digestion, then the expression vector is converted into arctic express to perform expression of a fusion protein, and after the protein is purified, the multi-epitope vaccine LTB-AE is obtained. The multi-epitope vaccine can induce a body to generate immune responses and high-titer specific antibody humoral immune responses to echinococcus multilocularis T cells and B cells, and can be used for preventing and treating echinococcus multilocularis infection related disease.

The invention discloses an LAMP (Loop-Mediated Isotheral Amplification) test kit for detecting the presence of an echinococcus multilocularis pathogen in dog excrement and application thereof. Thet kit for detecting the echinococcus multilocularis pathogen in dog excrement disclosed by the invention comprises four specific primers, i.e., an upstream external primer SEQ No., a downstream external primer SEQ No.2, an upstream internal primer SEQ No.3 and a downstream internal primer SEQ No.4. The test kit can be used for rapidly detecting the presence of the echinococcus multilocularis pathogeny in dog excrement, and has the characteristics of high sensitivity, easiness and convenience for operating, high specificity, rapidness, and the like.

The invention provides a novel alveolar echinococcosis subunit vaccine. A polypeptide serves as an active ingredient of the alveolar echinococcosis subunit vaccine, which is mainly composed of a surface antigen Emy162 of alveolar echinococcosis and a mucosal immune adjuvant, namely cholera toxin B subunit (CTB). According to the invention, the gene sequence of the alveolar echinococcosis surface antigen Emy162 is synthesized by virtue of a gene synthesis technology, and then the gene is coupled with the gene sequence of the cholera toxin B subunit, so that a fusion gene is formed. The fusion gene is expressed by virtue of an escherichia coli prokaryotic expression system, and protein is purified, so that the alveolar echinococcosis subunit vaccine is obtained. The alveolar echinococcosis subunit vaccine is capable of inducing a body to generate alveolar echinococcosis targeted T cell and B cell immune response and high-titer specific antibody humoral immune response, and the subunit vaccine can be used for preventing and treating alveolar echinococcosis infection related diseases.

The invention provides a primer for detecting echinococcus multilocularis in canine feces, wherein the sequence of an upstream primer is as shown in SEQ ID No. 1; and the sequence of a downstream primer is as shown in SEQ ID No. 2. The invention also provides a probe for detecting the echinococcus multilocularis in the canine feces, wherein the sequence is as shown in SEQ ID No. 3. The invention further provides a kit for detecting the echinococcus multilocularis in the canine feces; the kit comprises the primer for detecting the echinococcus multilocularis in the canine feces and the probe for detecting the echinococcus multilocularis in the canine feces, wherein the sequence of the upstream primer is as shown in SEQ ID No. 1; the sequence of the downstream primer is as shown in SEQ ID No. 2; and the sequence of the probe is as shown in SEQ ID No. 3. In the invention, the fluorescence quantitative PCR reaction of the echinococcus multilocularis can be effectively amplified by utilizing the primer and the probe, and the echinococcus multilocularis infection can be specifically identified and diagnosed.

The invention discloses an echinococcus multilocularis leucine aminopeptidase subunit vaccine LAP, a preparation method and application thereof, and the active ingredient of the subunit vaccine LAP isa polypeptide and is mainly composed of an amino acid sequence of echinococcus multilocularis antigen protein LAP. An echinococcus multilocularis subunit vaccine LAP genetic sequence is synthesized mainly through a gene synthesis technology and is connected to an expression vector through double digestion, and then the expression vector is transformed into Arctic Express for fusion protein expression. After the protein is purified, the echinococcus multilocularis subunit vaccine LAP is obtained. The echinococcus multilocularis subunit vaccine can induce an organism to generate T cell and B cell immune responses and high-titer specific antibody humoral immune responses aiming at echinococcus multilocularis, can effectively prevent mice from infecting echinococcus multilocularis, and can beused for preventing and treating echinococcus multilocularis infection related diseases.

The invention discloses a primer pair and a primer probe set for identifying echinococcus multilocularis and application thereof. The specific primer pair is composed of single-stranded DNA moleculesshown as a sequence 2 and single-stranded DNA molecules shown as a sequence 3. The primer probe set is composed of the specific primer pair and a specific probe, the specific probe is composed of a first segment, tetrahydrofuran and a second segment, the first segment is shown as a sequence 4, and the second nucleotide of 3' terminal is modified by fluorophore; the second segment is shown as a sequence 5, the second nucleotide of 5' terminal is modified by quencher, and C3 Spacer modification is performed on the 3' terminal. The specific primer pair or the primer probe set can be used for identifying echinococcus multilocularis and identifying whether a sample contains echinococcus multilocularis or not. The specific primer pair or the primer probe set is used for identifying echinococcusmultilocularis and is simple, convenient and quick in operation and high in specificity and sensitivity.

The invention discloses a high-throughput anti-echinococcosis drug screening method based on echinococcus granulosus and echinococcus multilocularis microtubulin as targets. The high-throughput anti-echinococcosis drug screening method includes the following steps that each 14 alpha-microtubulin homologues genes and 11 beta-microtubulin homologues genes of echinococcus granulosus and echinococcusmultilocularis are separately connected to plasmid vectors, and protein-expressing genetically engineered bacteria containing the plasmid vectors are obtained; expression and purification of the microtubulin are recombined; and the in vitro polymerization effect of the microtubulin is completed, the recombinant microtubulin is co-incubated with drugs to be tested to screen new anti-echinococcosisdrugs through the effect of drugs on the recombinant microtubulin in vitro polymerization. According to the high-throughput anti-echinococcosis drug screening method based on the echinococcus granulosus and echinococcus multilocularis as the targets, by evaluating the promotion or inhibition effect of the drug on microtubulin polymerization, the anti-echinococcosis drugs based on the microtubulincan be efficiently, easily and economically screened in a high-throughput mode. A novel idea is provided for the screening of the anti-echinococcosis drugs, and the high-throughput anti-echinococcosisdrug screening method can be used for finding effective new drugs or leading compounds for the treatment of the echinococcosis.

The invention discloses a primer group for detecting and distinguishing Echinococcus granulosus and Echinococcus multilocularis through loop-mediated isothermal amplification. The primer group comprises two pairs of outer primers and two pairs of inner primers, wherein the primers are designed according to an Echinococcus granulosus repeat region sequence and an Echinococcus multilocularis EmTriple83 microsatellite sequence. According to the primer group for detecting and distinguishing Echinococcus granulosus and Echinococcus multilocularis through loop-mediated isothermal amplification, genomic DNA of Echinococcus granulosus or Echinococcus multilocularis contained in a positive sample can be distinguished through two reactions, 1.6 fg of template DNA can be detected to the minimum, thedetection sensitivity is high, and the specificity is high.

The invention discloses a primer construction method for detecting hepatic echinococcosis through ddPCR, an amplification primer and application of the amplification primer. The primer construction comprises the following steps that common gene segments of echinococcus granulosus and echinococcus multilocularis are screened, and a plurality of first primers are designed based on the common gene segments; a plasma cfDNA sample of a first patient group is amplified by adopting the first primers, and the first primers capable of amplifying the plasma cfDNA sample of the first patient group are taken as second primers; and a plasma cfDNA sample of a second patient group is detected by adopting ddPCR of a reaction system comprising the second primers, and the second primers with the positive detection rate greater than a preset value are screened out as a target amplification primer. The amplification primer can amplify DNA fragments of echinococcus which are released into peripheral blood and exist in the form of plasma free DNA, by combining with a ddPCR method, whether human plasma is infected with echinococcosis can be diagnosed by detecting the human plasma, so that the ultra-early diagnosis of echinococcus granulosus and echinococcus multilocularis is realized, and a detection tool which is sensitive enough is provided for research and development of an echinococcosis precise therapy.

The invention discloses a preparation method and application of a monoclonal antibody for resisting echinococcus multilocularis adult pellicle antigen. According to the method, a hybridoma cell strain Anti-XJEmSfAg-4D6 is established by fusing splenocytes of a Balb / C mouse with SP2 / 0 cells through a hybridoma technology. A specific monoclonal antibody is obtained from the cell strain through an in-vitro culture method and an animal in-vivo ascites inducing method, and the echinococcus multilocularis adult immunohistochemical kit is prepared. The specific monoclonal antibody for resisting echinococcus multilocularis adult pellicle antigen is provided for clinical and scientific research institutions, can be used for detecting clinical samples in an ELISA (enzyme-linked immuno sorbent assay) experiment, can also be used for detecting pathological specimens in an immunohistochemical experiment, can also provide a convenient research tool for an echinococcosis research department, and has a wide application prospect. The method has a wide application value in the detection of the echinococcus multilocularis infected by a final host, and provides a technical support for baseline investigation and prevention and control effect evaluation of the alveolar echinococcosis.

The invention discloses an application of apatinib serving as a medicine for treating echinococcosis. The apatinib is used in vitro for killing multilocular hydatid cyst protoscoleces, and the apatinib and an optional pharmaceutically acceptable auxiliary material can be prepared into a medicine composition, or the apatinib and other echinococcosis resistant medicines can be prepared into a medicine composition for treating the echinococcosis. The medicine composition can be prepared into an optional pharmaceutical preparation including a tablet, a granule, a capsule, a pill, oral liquid, injection or lipidosome for treating the echinococcosis. Besides, the invention discloses a novel medicine for treating the echinococcosis, and the apatinib serves as an effective component of the novel medicine for treating the echinococcosis. The application of the apatinib for resisting the echinococcosis is widened, experimental research indicate that the apatinib has a remarkable effect of killing the hydatid cyst protoscoleces, growth of hydatid cysts can be effectively inhibited, and obvious cytotoxicity is avoided within effective concentration.

The invention discloses an echinococcus multilocularis glucose transporter polypeptide vaccine GLEP as well as a preparation method and application thereof. The active ingredient of the vaccine GLEP is a polypeptide, and the polypeptide is mainly composed of an echinococcus multilocularis glucose transporter polypeptide sequence. The echinococcus multilocularis vaccine GLEP gene sequence is synthesized through a gene synthesis technology, double digestion is connected into an expression vector, and then the expression vector is converted into Arctic Express for expression of fusion protein. After the protein is purified through Ni-NTA affinity chromatography, the echinococcus multilocularis vaccine GLEP is obtained. The echinococcus multilocularis vaccine GLEP can induce an organism to generate immune response and high-titer specific antibody humoral immune response for echinococcus multilocularis T cells and B cells, therefore, mice are effectively prevented from being infected with echinococcus multilocularis, and the echinococcus multilocularis glucose transporter polypeptide vaccine GLEP can be used for preventing echinococcus multilocularis infection related diseases.

The invention discloses multiple real-time fluorescent quantitative PCR (polymerase chain reaction) primers, probes and a kit for simultaneously detecting three types of echinococcus, and belongs to the technical field of parasitic pathogen detection. According to the multiplex real-time fluorescent quantitative PCR primer and the probe for simultaneously detecting the three kinds of echinococcus, the three kinds of pathogens of echinococcus multilocularis, echinococcus granulosus and echinococcus shigii are simultaneously detected, the detection efficiency is greatly improved, and the detection time and the detection cost are saved. According to the present invention, the specificity is strong, no non-specific amplification is performed on other common related parasite gene samples, and the sensitivity is high and is as high as 10 copies / [mu] l;.

The invention discloses an echinococcus multilocularis natural epidemic origin grading prediction method based on an ecological niche model. The method comprises the steps that S1, echinococcus multilocularis natural epidemic origin monitoring point data and geographical environment data are acquired; s2, performing logistic regression analysis on the geographical environment data, and screening out geographical environment risk factor data; establishing an ecological niche model according to the screened geographical environment risk factors; s3, dividing monitoring point data into a training set and a test set; s4, inputting the training set into an ecological niche model and detecting to obtain the relative importance of each environmental risk factor variable in the natural epidemic origin of echinococcus multilocularis; and S5, analyzing the test set based on the ecological niche model processed in the S4, and outputting the epidemic distribution condition of the natural epidemic source of the echinococcus multilocularis. According to the geographical environment risk factors, the method can perform corresponding grading and qualification on the epidemic area of the natural epidemic origin of the echinococcus multilocularis, and fills the blank in the technical field of prediction of the propagation risk of the echinococcus multilocularis in China.