41results about How to "Fast amplification" patented technology

The invention relates to a preparation method and application of an autologous CAR (chimeric antigen receptor)-T cell. An established CD28-CD137-CD19-CD3 full-length gene is guided into a T-cell of a patient by a CRISPR / Cas9 technology to prepare the CAR-T cell, and the CAR-T cell is subjected to expansion in vitro and then returns in the body of the patient to perform anti-tumor treatment. Compared with the traditional tumor treatment method, the method has the advantages that the method is cell targeted therapy and small in side effect; the gene modified T cell can stably express an antigen binding domain on the surface and identify a target antigen, and does not have MHC limit; and the tumor treatment effect is improved.

The invention discloses a kit for mesenchymal stem cell culture and its application. The kit provided by the invention includes a cell culture solution A and a cell culture solution B. The cell culture solution A is a Knockout-DMEM culture medium containing 80-120ng / ml cytokine LIF and 80-120ng / ml cytokine bFGF. The cell culture solution B is fetal bovine serum. The kit provided by the invention can separate umbilical cord mesenchymal stem cells from an in-vitro umbilical cord and culture the umbilical cord mesenchymal stem cells, and has the advantages of rapid cell expansion speed and high expansion efficiency. After multiple subculturing, cell totipotency can still be maintained. In the invention, the problems of umbilical cord mesenchymal stem cell separation purity, quantity and primary culture time are solved, and a lot of umbilical cord mesenchymal stem cells can be obtained. Thus, the kit provided in the invention provides abundant sources for making use of umbilical cord mesenchymal stem cells to conduct further experimental research.

The invention relates to an effector cell combination for preventing and treating tumors. The effector cell combination comprises CIK (cytokine induced killer) cells and MSC (Mesenchymal Stem Cells) which are sequentially injected into a human body in the form of a cell suspension and are extracted and prepared from peripheral blood of exogenous healthy people in non-tumor patients. The invention also discloses a preparation method of the effector cell combination, and the method comprises the following steps of: blood mononuclear cell collection, CIK cell culture and MSC culture. According to the scheme disclosed by the invention, the CIK cells and the MSC are extracted and prepared from the peripheral blood of the exogenous healthy people in the non-tumor patients, thereby breaking the routine treatment of auto-CIK cells, improving the clinical effects of tumor biotherapy and especially the clinical effects of CIK cell therapy, and solving the problems in graft-versus-host reaction (GVHD).

The invention relates to a novel method for preparing menstrual blood mesenchymal stem cells. The method comprises the following steps: a, collecting menstrual blood in vitro, and storing in menstrual blood collecting liquid to obtain menstrual blood mixed liquid; b, separating single karyocyte from the menstrual blood mixed liquid; C, culturing the single karyocyte in an adherent manner, proliferating cells, and then through adherent purification, obtaining purified cells, namely the menstrual blood mesenchymal stem cells. The novel method has the advantages of being nondestructive in the body, strong in uniformity and high in cell activity which is reflected in the characteristics of high cell proliferation speed, high passage times and the like.

The invention discloses an in vitro induction amplification, freeze preservation and anabiosis method of immune cells. The method comprises the following steps: using a CD16 antibody enveloped culture container to obtain an enveloped culture container; using an immune cell activated culture medium to put single karyocytes into the enveloped culture container to carry out first induction and amplification culture so as to obtain primarily induced and amplified immune cells; using an immune cell amplification culture medium to carry out second induction and amplification culture on the primarily induced and amplified immune cells so as to obtain differentiated immune cells; using an immune cell large-scale amplification culture medium to carry out third induction and amplification culture on the differentiated immune cells so as to obtain large-scale function activated immune cells; using an immune cell freeze preservation liquid to freeze and preserve the immune cells so as to obtain frozen and preserved immune cells; water-bathing the frozen and preserved immune cells for melting, mixing with a freezing anabiosis liquid of the immune cells so as to obtain an anabiosis cell mixed liquid; carrying out centrifugal treatment on the anabiosis cell mixed liquid so as to obtain anabiosis immune cells.

The invention discloses a method of extracorporeal induction, proliferation and cryopreservation of immune cells. The method comprises the steps of utilizing a CD16 antibody to cover a culture vessel to obtain the culture vessel after covering; utilizing the immune cells to activate a culture medium, and placing single cell nucleus in the culture vessel after covering to activate the culture medium through the immune cells to obtain preliminarily induced and proliferated immune cells; utilizing a immune cell proliferation culture medium to conduct second induction and proliferation culture on the preliminarily induced and proliferated immune cells to obtain differentiated immune cells; utilizing immune cell scale proliferation culture to conduct third induction proliferation culture on the differentiated immune cells to obtain large-scale immune cells with activated functions; utilizing immune cell cryopreservation liquid to cryopreserve the large-scale immune cells with the activated functions. The method of extracorporeal induction, proliferation and cryopreservation of the immune cells has the advantages of being high in induction efficiency, high in proliferation speed, high in safety, low in cost and the like. Moreover, the cryopreserved large-scale immune cells have long effective storing time and high cell recovery rate.

The invention discloses a Y-STR compound amplification detection kit marked by a novel fluorescent labeling method and a use method thereof. The Y-STR compound amplification detection kit performs compound amplification of 27 Y-STR loca with a compound amplification primer by adopting a polymerase chain reaction; the amplification product is detected by means of a gene sequencing instrument of a single channel or multichannel capillary tube; 27 Y-STR loca comprise 20 conventional low mutation rate loca and 7 fast mutant loca. The novel fluorescent labeling method comprises the steps of: marking a first alkaline group at the 5' end of the primer by a conventional dye; and marking the sixth alkaline group with a short excitation wavelength fluorescent dye to realize fluorescent energy transfer. The signal strength generated by the method is 3-11 times higher than that of the conventional method, and an amplification result which is more stable and balanced and higher in sensitivity can be obtained.

The invention discloses an immune cell culture medium system and applications thereof. The immune cell culture medium system comprises: an immune cell activation culture medium, wherein the immune cell activation culture medium is a serum-free lymphocyte culture medium added with plasma, interleukin-2 and sapylin; an immune cell expansion culture medium, wherein the immune cell expansion culture medium is a serum-free lymphocyte culture medium added with interleukin-2 and sapylin; and an immune cell large-scale expansion culture medium, wherein the immune cell large-scale expansion culture medium is a serum-free lymphocyte culture medium added with interleukin-2. According to the present invention, with the application of the immune cell culture medium system to carry out induction expansion culture on mononuclear cells, the advantages of high induction efficiency, rapid amplification, high safety, low cost, wide source, no requirement of feeder layer cells and the like can be achievedso as to meet the requirement on a large number of nature killer cells in clinical treatment.

The invention belongs to the technical field of cell culture media and particularly relates to a serum-free stem cell culture medium. The serum-free stem cell culture medium comprises a basic culture medium, insulin, transferrin, icariin, glycylalanine, alanyl-L-tyrosine, polylysine, vitamin E, polyvinylpyrrolidone, transforming growth factor-beta, reductive glutathione and sodium bicarbonate. The serum-free stem cell culture medium can promote cell growth and proliferation and can remarkably promote stem cell differentiation and culture the proliferated stem cells to protect a cell retrogression model.

The invention discloses an immune-cell in-vitro-induction-amplification method. The immune-cell in-vitro-induction-amplification method includes the steps that a culturing container is coated with a CD16 antibody to obtain the coated culturing container; media are activated through immune cells, and mononuclear cells are put into the coated culturing container for activating the media through the immune cells to obtain activated immune cells; the media are amplified through the immune cells, the immune cells subjected to primary induction amplification are subjected to secondary induction-amplification culturing to obtain amplified immune cells; the media are amplified in a large-scale mode through the immune cells, differentiated immune cells are subjected to tertiary induction-amplification culturing to obtain large-scale-amplified functional activated immune cells. The mononuclear cells are subjected to induction-amplification culturing with the immune-cell in-vitro-induction-amplification method to obtain the large-scale immune cells, the immune-cell in-vitro-induction-amplification method has the advantages of being high in induction efficiency, amplification speed and safety, low in cost and the like, and therefore the requirements of a large number of the immune cells in clinic treatment are met.

The invention discloses an RT-LAMP (reverse transcription loop-mediated isothermal amplification) primer set and kit for detecting porcine epidemic diarrhea virus and application thereof. The primer set is composed of a pair of outer primers, a pair of inner primers and a pair of loop primers. Sequences of the paired outer primers are shown in SEQ ID No.1 and SEQ ID No.2. Sequences of the paired inner primers are shown in SEQ ID No.3 and SEQ ID No.4. Sequences of the paired loop primers are shown in SEQ ID No.5 and SEQ ID No.6. The primer set is effective in detecting the porcine epidemic diarrhea virus. The kit comprises the afore-mentioned primers and is applicable to detecting the porcine epidemic diarrhea virus. The invention further provides a method of detecting the porcine epidemic diarrhea virus with the kit; detection results of the method may be visually detected according to changes in the color of reaction liquid, quickly and accurately. The primer set, the kit and the method are highly specific, highly sensitive and quick and accurate in detecting the porcine epidemic diarrhea virus and are particularly applicable to quick field detection.

The invention provides a CHO-K1 suspension acclimatization culture medium and an acclimatization method thereof. The culture medium at least comprises CD CHO Medium, IGF-1, poloxamer 188 and conditionmedium. When the culture medium is adopted to acclimatize CHO-K1 cells, the cell culture time is short (less than 2 months), the multiplication speed is high, the cells cannot be clustered, and the survival rate is high.

The invention provides a method for preparing high-purity menstrual-blood-derived stem cells. Menstrual blood is collected by means of a menstrual blood collection sleeve; middle layer mononuclear cells are collected; a magnetic bead buffering solution is added, an FcR reagent is added after being fully and evenly mixed, then a cross-linking anti-human-antibody micro magnetic bead reagent is added after incubation, and incubation is conducted after uniform mixing; then a magnetic bead buffering solution is added, and after being washed and centrifuged, the mixture is resuspended in the magnetic bead buffering solution; the mixture is added into a separation column placed on a magnetic separation frame in advance, and then the column is washed by means of the magnetic bead buffering solution; the separation column is taken out, 1.5-2 ml of magnetic bead buffering solution is added, and cell suspension is collected; the obtained suspension is cultured by means of a menstrual blood stem cell primary culture medium, and the half of suspension is replaced every 2-3 days; after culture is conducted for 5-7 days, adherent cells are digested and collected, and multiplication culture continues by means of a menstrual blood stem cell subculture medium; the obtained cells are digested, and a cryopreservation solution is added for cryopreservation. The menstrual-blood-derived stem cells obtained through the method are high in purity, short in growth period, high in self-renewal capacity and low in contamination rate.

The invention discloses an amplification method for a whole genome aiming at different enterovirus serotypes. The method comprises the following steps: extracting RNA of the viruses, carrying out RT-PCR, sequencing and comparison on the VP1 area of the RNA of the viruses by adopting the universal primers 222-224 of the enteroviruses, and determining the serotypes of virus strains by utilizing a molecular biology experimental technology; and designing corresponding primers, namely, designing the primers by utilizing the conservative property of the enteroviruses 5'UTR and 3'UTR, and carrying out corresponding RT-PCR amplification, sequencing and comparison according to the above paired primers till the nucleotide sequence of the whole genome is obtained. The method provided by the invention has the advantages of high amplification speed, as well as simplicity, convenience and feasibility, the cost can be greatly reduced, and the operation is efficient and rapid.

The invention relates to improvement of an isolated culture technology of umbilical cord mesenchymal stem cells and an application of the umbilical cord mesenchymal stem cells in arthritis. The isolated culture method of the umbilical cord derived mesenchymal stem cells and the application of the umbilical cord mesenchymal stem cells in the arthritis comprise the following steps: 1) obtaining themesenchymal stem cells from a human umbilical cord via an enzyme digestion method; 2) implementing culture of the mesenchymal stem cells via a DMEM / F12 medium which is added with basic fibroblast growth factors and platelet-derived growth factors in the combination with blood platelet enriched plasma; 3) improvement of hyaluronic acid: adding methacrylic anhydride to the hyaluronic acid, so that a3D bio-stent is formed, and subsequently, the mesenchymal stem cells can be immobilized to an osteoarticular injury site for conducting fixed-point repair; and 4) injecting the cultivated mesenchymalstem cells, together with PRP and the improved hyaluronic acid, into a bone joint of a volunteer, so as to treat osteoarthritis.

The invention provides a method and a detection method for rapidly detecting viral nucleic acid, belongs to the technical field of virus detection, and can solve the technical problems that a conventional nucleic acid detection method is high in environment requirement, relatively long in detection time, high in operation difficulty, difficult to realize on-site rapid detection and the like. The detection method comprises the steps of wiping and sampling, sample cracking treatment, detection, color development treatment and the like. The detection device comprises a base body, a sample cracking cavity, a conveying device and a detection and color development cavity. The method and the device have the characteristics of simplicity and convenience in operation, high sensitivity, short detection time, capability of realizing on-site quick detection and the like. The method and the device can be applied to the field rapid detection of viral nucleic acid.

The invention discloses an in-vitro culture, induction, activation and cryopreservation method and cell bank establishment of immune cells. The method comprises the following steps of carrying out first-stage proliferation culture on mononuclear cells by using a special proliferation culture medium for the immune cells to obtain primarily proliferated immune cells; carrying out second-stage induction and proliferation culture on the primarily proliferated immune cells by utilizing a special induction culture medium for the immune cells to obtain induced immune cells; carrying out third-stage activation and proliferation culture on the induced immune cells by utilizing a special activation culture medium for the immune cells to obtain a large number of immune cells with activation functions; cryopreserving the immune cells by using a special cryopreservation solution for the immune cells to obtain cryopreserved immune cells; and performing storage according to ABO / RH typing and HLA typing, establishing an immune cell information file for retrieval, and constructing an immune cell bank.

The invention discloses an in vitro induction amplification, freeze preservation and anabiosis method of immune cells. The method comprises the following steps: using a CD16 antibody enveloped culture container to obtain an enveloped culture container; using an immune cell activated culture medium to put single karyocytes into the enveloped culture container to carry out first induction and amplification culture so as to obtain primarily induced and amplified immune cells; using an immune cell amplification culture medium to carry out second induction and amplification culture on the primarily induced and amplified immune cells so as to obtain differentiated immune cells; using an immune cell large-scale amplification culture medium to carry out third induction and amplification culture on the differentiated immune cells so as to obtain large-scale function activated immune cells; using an immune cell freeze preservation liquid to freeze and preserve the immune cells so as to obtain frozen and preserved immune cells; water-bathing the frozen and preserved immune cells for melting, mixing with a freezing anabiosis liquid of the immune cells so as to obtain an anabiosis cell mixed liquid; carrying out centrifugal treatment on the anabiosis cell mixed liquid so as to obtain anabiosis immune cells.

The invention discloses a method of extracorporeal induction, proliferation and cryopreservation of immune cells. The method comprises the steps of utilizing a CD16 antibody to cover a culture vessel to obtain the culture vessel after covering; utilizing the immune cells to activate a culture medium, and placing single cell nucleus in the culture vessel after covering to activate the culture medium through the immune cells to obtain preliminarily induced and proliferated immune cells; utilizing a immune cell proliferation culture medium to conduct second induction and proliferation culture on the preliminarily induced and proliferated immune cells to obtain differentiated immune cells; utilizing immune cell scale proliferation culture to conduct third induction proliferation culture on the differentiated immune cells to obtain large-scale immune cells with activated functions; utilizing immune cell cryopreservation liquid to cryopreserve the large-scale immune cells with the activated functions. The method of extracorporeal induction, proliferation and cryopreservation of the immune cells has the advantages of being high in induction efficiency, high in proliferation speed, high in safety, low in cost and the like. Moreover, the cryopreserved large-scale immune cells have long effective storing time and high cell recovery rate.

The invention discloses a combined cellular preparation and an application thereof, and belongs to the technical field of the stem cells. The combined cellular preparation comprises an umbilical cord mesenchymal stem cell preparation and a NKT cellular preparation. An umbilical cord is used as the tissue source of the mesenchymal stem cells, the operation is simple, and the stem cells are easily obtained. A mononuclear cell in peripheral blood is used for inducing to form the NKT cells, and the preparation has the obvious function of improving the organism immunity, and eliminating the body infection. The wound healing of a diabetic foot patient is promoted, and the combined preparation of two preparations has the synergistic interaction function, and is capable of further promoting the recovery of the patient.

The invention discloses a small multifunctional biochemical analyzer based on a total internal reflection resonance principle. The small multifunctional biochemical analyzer mainly comprises a total internal reflection electromechanical coupling system module, a temperature control system module, a communication system module and a control system module. The total internal reflection electromechanical coupling system is used as a main body, and the automatic adjustment of the optical angle can be realized through the driving of a single motor, so that the incident light and the optical receiving device can change simultaneously within the angle range of 25-80 degrees. The prism coupling type total internal reflection resonance technology is adopted, angle modulation, intensity modulation and wavelength modulation are achieved, and spectrum detection, fluorescence detection and the like are carried out. In addition, technologies such as a micro electro mechanical system (MEMS), fluorescence detection, total internal reflection, surface plasmon resonance and polymerase chain reaction (PCR) are combined, and multifunctional qualitative and quantitative detection integrating amplification and detection is achieved. The volume of the device is reduced, the nucleic acid molecule amplification time is shortened, the detection efficiency is improved, and the biological reaction is observed in real time.

The invention discloses a fluorescent LAMP primer used for detecting a vesicular stomatitis virus and a detecting method. A sequence is shown in the specifications. According to the detecting method, the final concentration of inner primers FIP and BIP is 1.6umol / L, the final concentration of outer primers of F3 and B3 is 0.2umol / L, and the final concentration of annular primers of LF and Lb is 0.8umol / L; the reaction temperature is 63 DEG C. According to the fluorescent LAMP primer used for detecting the vesicular stomatitis virus and the detecting method, fluorochrome is introduced on the basis of an original LAMP technology to establish a fluorescent LAMP detecting technology, due to the fact that an instrument is used to conduct fluorescent collecting, sensitivity of detecting is further improved, detecting is more objective, the operation is simpler and more convenient, the detecting method is easy to be standardized, and the fluorescent LAMP primer used for detecting the vesicular stomatitis virus and the detecting method are easy to be used and popularized.

The invention discloses an RT-LAMP (Reverse Transcription Loop-Mediated Isothermal Amplification) primer group and a kit for detecting avian influenza viruses and applications of the RT-LAMP primer group and the kit. The primer group consists of a pair of outer primers and a pair of inner primers, wherein the sequences of the two outer primers are shown in SEQ ID No.1 and SEQ ID No.2, the sequences of the two inner primers are shown in SEQ ID No.3 and SEQ ID No.4, and the primers can be used for detecting the avian influenza viruses effectively. The kit comprises the primers and can be used for detecting the avian influenza viruses. The invention also provides a method for detecting the avian influenza viruses by adopting the kit. The detection result of the kit can be used for intuitively performing judgment quickly and accurately according to color changes of a reaction solution. The RT-LAMP primer group and the kit disclosed by the invention have the characteristics of strong specificity, high sensitivity, fastness and accurate avian influenza viruse detection, and are particularly suitable for field and basement layer detection.

The invention discloses a Taq DNA (Deoxyribose Nucleic Acid) polymerase mutant which can be used for carrying out rapid PCR (Polymerase Chain Reaction) and is tolerant to SYBR Green I. The Taq 01 DNA polymerase mutant obtained by screening in the invention shows rapid PCR (Polymerase Chain Reaction) activity in PCR (Polymerase Chain Reaction) and qPCR (Quantitative Polymerase Chain Reaction) and is more tolerant to SYBR Green I activity. Based on the advantages, the mutant can be subjected to rapid PCR (Polymerase Chain Reaction) in the presence of inhibitors such as SYBR Green I and the like, so that a result can be obtained more rapidly and sensitively in clinical diagnosis and scientific research.

The invention discloses a culture medium for stem cell culture. The culture medium comprises a bottom protecting cover, wherein a culture medium body is arranged in the cavity of the bottom protectingcover, a top protecting cover is meshed with the top of the bottom protecting cover, buckling grooves which are in interaction for use are formed in two sides of the top of the bottom protecting cover and the bottom of the top protecting cover, and the bottom protecting cover and the top protecting cover are made from acrylic materials; the culture medium body comprises 15% of FBS, 116.6mg / L anhydrous calcium chloride, 59.05mg / L L-leucine, 0.042mg / L linoleic acid, 0.0013mg / L anhydrous copper sulfate, 91.25mg / L L-lysine hydrochloride, 0.105mg / L lipoic acid, 0.05Lmg / L iron(III) nitrate nonahydrate, 17.24mg / L L-methionine, 8.1mg / L phenol red, 0.417mg / L of ferrous sulfate heptahydrate, 35.48mg / L L-phenylalanine, and 0.081mg / L 1,4-diaminobutane dihydrochloride. The preparation method is simple, few material instruments are used, stem cells can be massively prepared for culture, the amplification speed of the stem cells is high, the stem cell differentiation capacity is high, and the stem cells can be differentiated into multiple functional cells to achieve high scientific research and medical application value.

The invention relates to a Taq Plus polymerase kit and a usage method thereof. The Taq Plus polymerase kit comprises a square box, a foam pad and reagent bottles, wherein the foam pad is arranged in the square box and is provided with four holes, the reagent bottles include a reagent bottle 1, a reagent bottle 2, a reagent bottle 3 and a reagent bottle 4 and are put in the four holes, the reagent bottle 1 contains Taq Plus polymerase, the reagent bottle 2 and the reagent bottle 3 contain a buffer solution, and the reagent bottle 4 contains an enhancer solution. The kit is simple in operation, pollution produced in the operating process is reduced, meanwhile the reagent bottles used in the kit are transparent, bottle caps are different in color and are provided with round marks, the time for selecting reagents is shortened, and the Taq Plus polymerase kit is convenient to use.

The invention discloses a fluorescent label compound amplification kit for simultaneously amplifying human autosome and Y chromosome STR loci and application thereof. The kit can simultaneously detect 18 A-STRs, 20 Y-STRs and gender (Amel) loci, realize the simultaneous construction of two libraries of A-STR and Y-STR, and can be used for individual identification, paternity testing, and family investigation And family tree construction, etc., can be used for rapid investigation of cases at the same time, shortening the detection time and improving the efficiency of excluding and identifying criminal suspects. The 20 Y-STR loci have great geographical and surname orientation for case investigation. Unrelated families can be excluded as much as possible. The invention can detect a small amount of male DNA in the background of a large number of female DNA samples, and is a powerful weapon for detecting mixed spot samples such as rape crimes. And when detecting paternity-child paternity, there is no need to do separate Y-STR experimental analysis because the Y-STR data is already included.

The invention discloses a PCR (polymerase chain reaction) amplification method. In the method, a restriction endonuclease sequence is added to a 5'end of an upstream primer or a downstream primer for amplification, a fluorophore is marked at the 5'end of the primer, a quenching group capable of specifically quenching the fluorophore at the 5'end is marked in the middle of the primer, the restriction endonuclease is added to a PCR amplification system and subjected to a PCR. The method has the advantages that the amplification speed is high, the reaction is sensitive, the specificity is high, and real-time, rapid and multiplex detection and analysis of nucleic acid can be realized.

The invention relates to an effector cell combination for preventing and treating tumors. The effector cell combination comprises CIK (cytokine induced killer) cells and MSC (Mesenchymal Stem Cells) which are sequentially injected into a human body in the form of a cell suspension and are extracted and prepared from peripheral blood of exogenous healthy people in non-tumor patients. The invention also discloses a preparation method of the effector cell combination, and the method comprises the following steps of: blood mononuclear cell collection, CIK cell culture and MSC culture. According to the scheme disclosed by the invention, the CIK cells and the MSC are extracted and prepared from the peripheral blood of the exogenous healthy people in the non-tumor patients, thereby breaking the routine treatment of auto-CIK cells, improving the clinical effects of tumor biotherapy and especially the clinical effects of CIK cell therapy, and solving the problems in graft-versus-host reaction (GVHD).