CN105527430A - Kit for directly detecting 71 type enterovirus and A16 type coxsackievirus and application thereof - Google Patents
Kit for directly detecting 71 type enterovirus and A16 type coxsackievirus and application thereof Download PDFInfo
- Publication number
- CN105527430A CN105527430A CN201610041545.6A CN201610041545A CN105527430A CN 105527430 A CN105527430 A CN 105527430A CN 201610041545 A CN201610041545 A CN 201610041545A CN 105527430 A CN105527430 A CN 105527430A
- Authority
- CN
- China
- Prior art keywords
- cell
- kit
- antibody
- fluorescein
- type
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/085—Picornaviridae, e.g. coxsackie virus, echovirus, enterovirus
Abstract
The invention provides a kit for directly detecting 71 type enterovirus and A16 type coxsackievirus and an application thereof. The kit comprises an antibody, a cell fixing solution, a PBS (phosphate buffer solution), a PBST (phosphate buffer solution twen-20), 1% Triton-X100, a cell reference sample, a preservative of sodium azide, an antibody stabilizer of BSA (bovine serum albumin), a fluorescent dye of Evans blue, a mounting medium, a slide carrier and a quality control slide, wherein the antibody contains a fluorescence marker. The kit has the advantages that the 71 type enterovirus and A16 type coxsackievirus in the sample cell can be directly detected; the advantages of flow cytometry and direct immunofluorescent technique on automation and high flux are realized, the virus can be quickly, sensitively and efficiently detected, the important significance is realized for the quick and accurate diagnosis of clinics, and different requirements of large hospitals and base hospitals can be met.
Description
Technical field
The invention belongs to pathogen detection technical field, be specifically related to kit and the application thereof of direct-detection enterovirns type 71 and coxsackie virus A 16-type.
Technical background
Hand-foot-and-mouth disease (hand, footandmouthdisease, HFMD) be the infectious disease caused by enterovirus, cause the enterovirus of hand-foot-and-mouth disease and have kind more than 20 (type), wherein with coxsackie virus A 16-type (CA16) and enterovirns type 71 (EV71) the most common.Multiplely be born in less than 5 years old children, there is exanthema vesiculosum or aphtha, the self-healing in about one week of most infant in the positions such as performance stomatalgia, apocleisis, low-heat, hand, foot, oral cavity, minority infant can cause the complication such as myocarditis, pulmonary edema, AME.Indivedual children with serious disease PD is fast, causes death.
Childhood infection EV71 virus after causing severe hand-foot-and-mouth disease each histoorgan viral antigen distribution situation show as, in central nervous system display neurocyte and the spongiocyte positive, be very with brain stem, secondly be brain and cerebellum, prompting neurocyte and microglia are the Principle Target that it is attacked.In respiratory system, larynx, trachea-bronchial epithelial cell and mucous membrane of bronchiole epithelial cell are positive, and some cases alveolar cell and the macrophage positive, prompting airway epithelial cell is also the target cell that EV71 attacks.Intestinal mucosal epithelial cell EV71 antigen detects and is also positive.Target organ and the target cell of EV71 infection are completely unclear yet.It is generally acknowledged, EV71 enters in human body through upper gastrointestinal or respiratory tract, causes viremia virusemia, and arrives each organ of whole body with blood flow, copies, cause a series of pathology in corresponding target organ or tissue.
According to the literature, certified CA16 and EV71 virus specific receptors has palatelet-selectin glycoprotein receptor-1 (PSGLl) and scavenger receptor B2 (SCARB2), PSGLl is the distribution in high density in human central nervous especially brain stem tissue, throat, trachea-bronchial epithelial cell, bronchiole and alveolar epithelial cells is mainly distributed in respiratory tract, in digestive system, mucomembranous epithelial cell and the gland cell of intestinal tissue also have distribution.SCARB2 acceptor is also mainly distributed in nervous centralis, respiratory tract and intestinal tissue.Another certified CA16 virus receptor is Coxsackie-Adenovirus Receptor (CAR).Coxsackie-Adenoviral Receptor is the coreceptor of Coxsackie virus and adenovirus, the infection of the extracellular region of CAR to virus is only had to play a decisive role, namely cross-film district and the intracellular region of removing CAR can not stop viral infection, simultaneously, the extracellular region of viral capsid proteins and CAR interacts can the release of elicit virus RNA, makes it enter cell by endocytosis or other mechanism of action.There is document to have detected the expression of CAR receptor mrna in human tissue, find all have expression in pancreas, brain, heart, small intestine, testis and prostata tissue.Often showing multiple organ for severe hand-foot-and-mouth disease patient organizes infected, and visible CA16 and EV71 virus can infect many histoorgans in human body.
To develop at present and the diagnostic method used comprises:
(1) RT-PCR method detects virus: highly sensitive, can check carrier in time.But instrument and equipment is expensive, experimental site requires high, and operating personnel require high, easily pollute, and easily produces false positive and causes mistaken diagnosis.
(2) diagnosed by clinical symptoms comprehensive evaluation: early stage in disease of this method, because of atypical symptom, and cannot Accurate Diagnosis.In the late period of disease, although can Accurate Diagnosis, delay the treatment to disease.
(3) sero-immunity checks antiviral antibody (indirect immuno fluorescent method or indirect ELISA).IgG antibody produces needs after morbidity at least 14 days, and generally wants superinfection that enough amounts just may be had to be detected, and therefore, this method can not do early diagnosis.IgM antibody produced at virus infections 5-7 days, and was subject to the restriction of serum IgM concentration, caused sensitivity not high, easily occurred false-negative situation.To the window phase sero-immunity that IgM antibody produces, virus infections checks that antiviral antibody is inapplicable, the method therefore detecting serum IgM also has very large restriction.
Above three kinds of methods, in Clinical practice process, have that operation is not easy, spended time are long, diagnose the shortcomings such as inaccurate.
Summary of the invention
For overcoming the shortcoming of prior art with not enough, a kind of direct-detection has been the object of the present invention is to provide to detect kit and the application thereof of enteron aisle germ 71 type and coxsackie virus A 16-type.
For achieving the above object, fluorescein-labeled antibody, cell immobile liquid, PBS solution, PBST solution, 1%Triton-X100, cell controls product, antiseptic sodium azide and antibody stabilization agent BSA is contained in kit provided by the invention.
Preferably, described fluorescein-labeled antibody comprises the antibody of the enterovirns type 71 covalently bound with fluorescein and the coxsackie virus A 16-type covalently bound with fluorescein.Described antibody is can the monoclonal antibody covalently bound with fluorescein or polyclonal antibody.
Preferably, described fluorescein is any one in Alexa fluorescent dye (AlexaFluor), phycoerythrin (PE) and derivant thereof, anthocyanin (Cy) and derivant, phycoerythrin-anthocyanin (PE-Cy) and fluorescein-5-isothiocyanates (FITC).
Preferably, described cell controls product comprise positive cell reference substance and negative cells reference substance.Positive cell reference substance is the RD cell containing enterovirns type 71 of freeze-drying and the RD cell containing coxsackie virus A 16-type of freeze-drying, and negative controls is the normal RD cell of freeze-drying.
Preferably, described cell immobile liquid to be mass volume ratio be 4% paraformaldehyde.
Preferably, described kit uses according to the following steps:
(1) cell sample to be measured is abandoned supernatant with described PBS solution is centrifugal, then fix with described cell immobile liquid, it is resuspended finally to add PBST solution, prepares single-cell suspension liquid;
(2), after being mixed with 1%Triton-X100, antiseptic sodium azide and antibody stabilization agent BSA by described fluorescein-labeled antibody, dyeing liquor is obtained;
(3) added respectively by the dyeing liquor in described step (2) in the described single-cell suspension liquid in described step (1) and the cell controls product in described kit, room temperature lucifuge hatches 30min;
(4) with the dyeing liquor of described PBST eluant solution removing in conjunction with cell, and with after PBST re-suspended cell, with the viral antigen be combined with described fluorescein-labeled antibody in cell sample described in flow cytomery;
(5) interpretation of result: if Q2-1, Q2-2, Q2-4 quadrant number percent is greater than critical value, then judge that sample infects positive as corresponding virus or Geminivirus; If when Q2-2 is less than or equal to critical value, Q2-1, Q2-4 are less than or equal to critical value, then judge that sample is negative as corresponding virus infections.
Kit of the present invention also comprises fluorescent dye Yi Wensilan, mountant, microslide and Quality Control slide, wherein, porose on described microslide; Described Quality Control slide has positive hole and negative hole, and positive hole is the RD cell of enterovirns type 71 and the RD cell of coxsackie virus A 16-type of 1:1 mixing, and negative hole is normal RD cell; Described mountant is a kind of damping fluid containing Isosorbide-5-Nitrae-two azo dicyclo [2,2,2]-octane, glycerine and polyvinyl alcohol (PVA).
Preferably, described detection kit uses according to the following steps:
(1) cell sample to be measured is abandoned supernatant with described PBS solution is centrifugal, then fix with described cell immobile liquid, it is resuspended finally to add PBST solution, prepares single-cell suspension liquid;
(2), after described fluorescein-labeled antibody being mixed with described 1%Triton-X100, antiseptic sodium azide and antibody stabilization agent BSA, add Yi Wensilan, obtain Fluorescence Identification reagent;
(3) be added in the hole of microslide by the single-cell suspension drop in described step (1), after volatile dry, drip the Fluorescence Identification reagent obtained in described step (2), 37 DEG C of lucifuges hatch 20min;
(4) on the positive hole and negative hole of described Quality Control slide, respectively drip the Fluorescence Identification reagent in step (2), 37 DEG C of lucifuges hatch 20min;
(5) develop a film with described PBST solution and distilled water, mountant is dripped after sopping up excessive moisture with thieving paper, covered, with the viral antigen be combined with described fluorescein-labeled antibody in cell sample to be measured described in fluorescence microscope, Taking Pictures recording experimental result;
(6) interpretation of result: if find more than 2 green cells and hole inner cell sum is greater than 100 in described microslide hole, then judge that described cell sample EV71 to be measured is positive, otherwise be EV71 feminine gender; If find more than 2 orange fluorescence cells and hole inner cell sum is greater than 100 in described microslide hole, then judge that described cell sample CA16 to be measured is positive, otherwise be CA16 feminine gender.
Utilize kit provided by the invention, can enterovirns type 71 and coxsackie virus A 16-type in direct-detection sample cell.
Kit of the present invention takes full advantage of flow cytometry and direct immunofluorescence art in robotization and high-throughout advantage, can realize quick to virus, sensitive, detect efficiently, significant for clinical quick and precisely diagnosis, the different demands of large hospital and basic hospital can be met.
Accompanying drawing explanation
The two-parameter two-dimentional point diagram of cell fluorescence signal of to be cell door (P1), Fig. 1-B be described cell door correspondence that Fig. 1 is the result figure infected in conjunction with flow cytomery sample 2EV71, CA16 with kit of the present invention, Fig. 1-A;
The two-parameter two-dimentional point diagram of cell fluorescence signal of to be cell door (P1), Fig. 2-B be described cell door correspondence that Fig. 2 is the result figure infected in conjunction with flow cytomery sample 3EV71, CA16 with kit of the present invention, Fig. 2-A;
The two-parameter two-dimentional point diagram of cell fluorescence signal of to be cell door (P1), Fig. 3-B be described cell door correspondence that Fig. 3 is the result figure infected in conjunction with flow cytomery sample 4EV71, CA16 with kit of the present invention, Fig. 3-A;
The two-parameter two-dimentional point diagram of cell fluorescence signal of to be cell door (P1), Fig. 4-B be described cell door correspondence that Fig. 4 is the result figure infected in conjunction with flow cytomery sample 5EV71, CA16 with kit of the present invention, Fig. 4-A;
Fig. 5 is the fluorescence photo adding Fluorescence Identification reagent with kit combined with fluorescent microscopic examination sample 2 of the present invention;
Fig. 6 is the synthesis fluorescence photo adding Fluorescence Identification reagent with kit combined with fluorescent microscopic examination sample 3 of the present invention;
Fig. 7 is the synthesis fluorescence photo adding Fluorescence Identification reagent with kit combined with fluorescent microscopic examination sample 4 of the present invention;
Fig. 8 is the fluorescence photo adding Fluorescence Identification reagent with kit combined with fluorescent microscopic examination sample 5 of the present invention.
Embodiment
For making the present invention easier to understand, below in conjunction with specific embodiment, set forth the present invention further.Should be understood that embodiment is only not used in for illustration of kit described in the invention and using method thereof and application to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually conveniently experimental technique or carry out according to the method that manufacturer advises.
Embodiment 1: the preparation of kit
1 cell harvesting and process
1.1 cell harvestings: kit of the present invention is for detecting EV71 and the CA16 viral antigen in sample cell, EV71 and CA16 virus infections coverage is wide, therefore, any cell sample that can be prepared to single cell suspension is all applicable to kit of the present invention in theory.Infect the path of EV71 and CA16 virus from patient and collect the experience of sample, Nasopharyngeal swabs collection easily, hurtless measure and rapidly, substantially free of impurities is gathered after patient gargles, do not need cyclic washing, so, Nasopharyngeal swabs is first elected in cell sample collection, and embodiment is all for Nasopharyngeal swabs.
The preparation of 1.2 single-cell suspension liquid: the sampling pipe containing 3mlPBS or virus transport nutrient culture media put into by detection sample, the centrifugal 8min of 500g, supernatant discarded, notes not siphoning away cellular layer; Then add 3ml cell immobile liquid mass volume ratio be 4% paraformaldehyde and re-suspended cell, screw pipe lid, put 4 DEG C of fixing at least 15min, the centrifugal 8min of 500g, supernatant discarded, adds 100 μ lPBST, blows and beats cellular layer gently with re-suspended cell with liquid-transfering gun, obtain single-cell suspension liquid, 4 DEG C of preservations.
Blood sample is by commercial human peripheral lymphocyte parting liquid, and by specification operates, the resuspended rear formation single-cell suspension liquid of final PBS; The body fluid samples such as skin ulcer and bleb, sputum, ascites by multiple oscillation, centrifugal, add PBS washing and remove resuspended rear formation single cell suspension after impurity.
The preparation of 2 dyeing liquors (Fluorescence Identification reagent)
The preparation of 2.1 antibody: the preparation method of the monoclonal antibody in kit of the present invention is conventional method, namely adopt hybridoma technology through Fusion of Cells, obtain continuing by screening, the hybridoma cell strain of the monoclonal antibody of anti-EV71 and CA16 of stably excreting, and obtain antibody by the secretion of this cell line.And the method that routine prepares polyclonal antibody is by the polypeptide immune animal with purified virus, recombinant protein, carrier protein couplet, and carry out purifying acquisition.Therefore, the process of combined with fluorescent element labelled antibody, can think and in every case can the monoclonal antibody covalently bound with fluorescein and polyclonal antibody all can be applicable in kit of the present invention.
It is emphasized that the monoclonal antibody selectivity of specific recognition EV71 in following examples of the present invention is in conjunction with EV71, be not combined with CA16, other types of enterovirus and other pathogen; The monoclonal antibody of specific recognition CA16, is not only single-mindedly combined with EV71, other types of Coxsackie virus and other pathogen with the combination of CA16.
2.2 fluorescein labelled antibodies: labeling method can change to some extent with the different of the fluorescein of antibody coupling, and concrete labeling process can operate according to fluorescein operation instructions.In theory, the fluorescein of all energy labelled antibodies is all applicable to this method kit.In the embodiment of the present invention 2, adopt the covalently bound EV71 mouse IgG of AlexaFluor488, PE-Cy5 and CA16 mouse IgG respectively, AlexaFluor488 flies generation purchased from Sai Mo, and you are scientific and technological, and PE-Cy5 flies generation purchased from Sai Mo, and you are scientific and technological; In the embodiment of the present invention 3, adopt the covalently bound EV71 mouse IgG of FITC, R-PE and CA16 mouse IgG respectively, FITC is purchased from Beijing Fanbo Biochemicals Co., Ltd., and R-PE flies generation purchased from Sai Mo, and you are scientific and technological.
2.3 will adopt fluorescein-labeled EV71 mouse IgG, CA16 mouse IgG, add the Triton-X100 of 1% respectively, antibody stabilization agent BSA and antiseptic sodium azide, after mixing, obtain dyeing liquor, will keep in Dark Place in 4 DEG C in PBS solution.Dyeing liquor basis is added described fluorescent material Evans blue, obtain Fluorescence Identification reagent.
3 reference material materials prepare
In order to realize validity and the specificity of invention kit, involved material prepares also to comprise:
3.1 positive reference substances and negative controls: positive reference substance has two kinds, be respectively the RD cell containing EV71 of freeze-drying and the RD cell containing CA16 of freeze-drying; Negative controls is the normal RD cell of freeze-drying.
The preparation of 3.2 Quality Control slides: Quality Control slide has 1 Positive control wells and 1 negative control hole.Positive control wells includes the RD cell of mixing EV71 and CA16, and negative control hole includes normal RD cell, and through washing the steps such as fixing after cell chulture, correspondence joins on microslide, sealing after dry.
Embodiment 2: enterovirns type 71 and coxsackie virus A 16-type detection kit (flow cytometry)
The present embodiment provides a kind of kit utilizing flow cytometer direct-detection enterovirns type 71 and coxsackie virus A 16-type.This kit can be used for adjuvant clinical diagnosis hand-foot-and-mouth disease.
1. the composition of kit:
Note: in order to the convenience used, the antibody in kit, antiseptic, antibody stabilization agent and 1% Triton-X100 be all prepared into dyeing liquor by the method for operating in embodiment 1, be contained in a brown bottle; Number percent is mass volume ratio (g/100ml);
2. the preparation of seminal plasma fructose detection kit
2.1PBS and PBS-T composition
Use dH
2o is settled to 1L and is PBS solution, and PBST solution is for add 0.05%tween-20 on this basis.
2.2 cell immobile liquids: 4% paraformaldehyde: 4g paraformaldehyde is dissolved in 100mlPBS solution.
3. do not provide in kit but equipment necessary in experimentation:
3.1 flow cytometers: this example flow cytometer used is ACEANovoCyteTM2060R;
3.2 hydro-extractors (containing horizontal rotor) being not less than 700g;
3.3 common 96 porocyte culture plates;
3.4 flocking swabs and liquid-transfering gun and rifle head, collection tube, waste fluid container, oscillator.
4. detecting step
4.1 sample collections, removal of impurities and cell are fixed (embodiment 1 is shown in operation);
The preparation (embodiment 1 is shown in operation) of 4.2 single-cell suspension liquid;
4.3 sample dyeings: single-cell suspension liquid is proceeded in 96 orifice plates, a corresponding hole of sample, then use the centrifugal 8min of horizontal centrifuge 500g, carefully suck supernatant with pipettor.Add 25 μ l dyeing liquors again, hatch 30min in room temperature lucifuge.The centrifugal 8min of 500g, carefully sucks supernatant with pipettor afterwards.
4.4 wash-outs: every hole adds 100 μ lPBST again, blows and beats cell with re-suspended cell gently with pipettor.The centrifugal 8min of 500g, carefully sucks supernatant with pipettor again.Add 100 μ lPBST again, with pipettor, cell is blown and beaten gently with re-suspended cell.
4.5 flow cytometer are analyzed.Detect with flow cytometer (Essen Biology, NovoCyte2060R).
4.6 interpretation of result methods: according to forward-scattering signal and lateral scattering light signal, mark cell door.First arrange fluorescence to compensate, again according to described cell door, (X-axis is FITC-H to make two-parameter two-dimentional point diagram, Y-axis Cy5-H, AlexaFluor488 and PE-Cy5 is that 488nm laser instrument excites, AlexaFluor488 passage used is identical with FITC (about 510nm), and PE-Cy5 passage used is identical with Cy5 (about 650nm), can refer to the operation of flow cytometer instructions).Then be set on two-parameter two-dimentional point diagram and set quadrant door (Q2), wherein Q2-1 number percent is CA16 percent positive, Q2-2 number percent is Geminivirus percent positive, and Q2-3 number percent is negative number percent, and Q2-4 number percent is EV71 percent positive.Described percent positive as diagnosis basis, Q2-1, Q2-2, Q2-4 quadrant number percent be greater than critical value be judged to corresponding virus or Geminivirus infects sun; Be less than under critical value equals the prerequisite of critical value at Q2-2, Q2-1, Q2-4 be less than or equal to critical value to be judged to corresponding virus infections negative.The positive door critical value of described quadrant delimited according to a large amount of hand-foot-and-mouth disease sample dyeing result.
5. the present embodiment result and conclusion
In the present embodiment, detect the throat swab sample that sample picks up from 4 clinical hand-foot-and-mouth disease patients.Fig. 1 is the result figure infected in conjunction with flow cytomery sample 2EV71, CA16 with kit of the present invention, Fig. 1-B is the two-parameter two-dimentional point diagram of cell fluorescence signal of described cell door correspondence, as shown in Fig. 1-A, according to forward-scattering signal (FSC-H) and lateral scattering light signal (SSC-H), mark cell door (P1).As shown if figure 1-b, according to described cell door, make and make the two-parameter two-dimentional point diagram of cell fluorescence signal (X-axis is FITC-H, Y-axis Cy5-H).Two-parameter two-dimentional point diagram sets quadrant door (Q2), and wherein Q2-1 number percent is CA16 percent positive, and Q2-2 number percent is Geminivirus percent positive, and Q2-3 number percent is negative number percent, and Q2-4 number percent is EV71 percent positive.Described percent positive is as diagnosis basis, the present embodiment Q2-1, Q2-2 and Q2-4 critical value is set to 0.20%, 0.80% and 0.20% respectively, the sample being greater than critical value is judged to the corresponding virus infections positive or the Geminivirus infection positive, if the sample that Q2-1 and Q2-2 is less than or equal to critical value is judged to CA16 infection feminine gender; If the sample that Q2-4 and Q2-2 is less than or equal to critical value is judged to EV71 infection feminine gender.Show Q2-1, Q2-2 and Q2-4 in figure and be all less than or equal to critical value, illustrate that detected sample 2 is that CA16 and EV71 is double-negative.
Fig. 2 is the result figure infected in conjunction with flow cytomery sample 3EV71, CA16 with kit of the present invention, Fig. 2-A is cell door (P1), Fig. 2-B is the two-parameter two-dimentional point diagram of cell fluorescence signal of described cell door correspondence, show Q2-2 and Q2-4 in figure and be all less than or equal to critical value, illustrate that detected sample 3 is for CA16 is positive and EV71 is negative.Fig. 3 is the result figure infected in conjunction with flow cytomery sample 4EV71, CA16 with kit of the present invention, Fig. 3-A is cell door (P1), Fig. 3-B is the two-parameter two-dimentional point diagram of cell fluorescence signal of described cell door correspondence, show Q2-1 and Q2-2 in figure and be all less than or equal to critical value, illustrate that detected sample 4 is for CA16 is negative and EV71 is positive.Fig. 4 is the result figure infected in conjunction with flow cytomery sample 5EV71, CA16 with kit of the present invention, Fig. 4-A is cell door (P1), Fig. 4-B is the two-parameter two-dimentional point diagram of cell fluorescence signal of described cell door correspondence, show Q2-1, Q2-2 and Q2-4 in figure and be all greater than critical value, illustrate that detected sample 5 is for the two positive of CA16 and EV71.
To sum up, in 4 throat swab samples of described detection, as shown in Figure 1, sample 2 is that CA16 and EV71 is negative, as shown in Figure 2, sample 3 is that the positive EV71 of CA16 is negative, as shown in Figure 3,4 is that the positive CA16 of EV71 is negative, and as shown in Figure 4, sample 5 is that EV71 and CA16 is two positive.
Embodiment 3: enterovirns type 71 and coxsackie virus A 16-type detection kit (direct immunofluorescence)
The present embodiment provides a kind of kit utilizing fluorescent microscope direct-detection enterovirns type 71 and coxsackie virus A 16-type.This kit can be used for adjuvant clinical diagnosis hand-foot-and-mouth disease.
The composition of 1 kit is:
Note: in order to the convenience used, the Triton-X100 of the antibody in kit, antiseptic, antibody stabilization agent, EvansBlue and 1% is all prepared into Fluorescence Identification reagent by the method for operating in embodiment 1, is contained in a brown bottle; Number percent is mass volume ratio (g/100ml);
The preparation of 2 seminal plasma fructose detection kit
2.1PBS
The PBS solution that 1L is is settled to dH2O; PBST solution is for add 0.05%tween-20 on this basis.
2.2 cell immobile liquids: 4g paraformaldehyde is dissolved in 100mlPBS solution to be prepared.
Do not there is provided in 2 kits but equipment necessary in experimentation:
2.1 are equipped with and are suitable for detecting that fluorescence B wave band excites group filter (utilizing emitted light filter disc preferably can filter more than 620nm wavelength) and G-band to excite to organize filter just puts fluorescent microscope, can realize the amplifying power of 100 times to 200 times.
2.2 distilled water: be used for diluting PBS dry powder.
2.3 hydro-extractors, can produce 700g speed;
2.4 cover glasses, glass slide dyeing frame or staining jar, liquid-transfering gun and rifle head, centrifuge tube, tweezers, waste fluid container, 37 DEG C of incubation casees, oscillator, hair-dryer and thieving papers.
3 detecting steps
Collection and the cell of 3.1 samples are fixed (embodiment 1 is shown in operation);
The preparation (embodiment 1 is shown in operation) of 3.2 single cell suspensions;
3.3 sample dyeings: above-mentioned single cell suspension 5 μ l is added drop-wise on 1 hole of microslide.Microslide is put into 37 DEG C of constant temperature oven nature volatile dries.Then correspondingly in this 1 hole of every increment, drip 1 Fluorescence Identification reagent respectively, hatch 20min in 37 DEG C of constant temperature oven lucifuges.
Meanwhile, to positive hole and the negative hole correspondence dropping 1 Fluorescence Identification reagent of Quality Control slide, 20min is hatched in 37 DEG C of constant temperature oven lucifuges, in contrast.
3.4 develop a film: first Quality Control slide and specimen slide PBS are washed one time, then wash one time with distilled water, and washing methods is lift 4 times up and down in a liquid.
3.5 sop up excessive moisture with thieving paper after drip mountant, covered, with fluorescence microscope and Taking Pictures recording result.First debug microscope, with 200 times of observations, EV71 positive cell is bright green fluorescence under B wave band excites group filter, CA16 positive cell is bright orange fluorescence under B wave band excites group filter, because containing EvansBlue in Fluorescence Identification reagent, so all cells all can present red fluorescence under G-band excites group filter, after taking pictures, red fluorescence photo and green fluorescence photo or orange fluorescence photo are merged and just can judge CA16 positive cell in yellowish green EV71 positive cell and yellow significantly.
4 interpretation of result methods: under guarantee Quality Control slide result meets the prerequisite of instructions, if find in hole that more than 2 green cells and hole inner cell sum are greater than 100 and (notice that hole inner cell sum is less than 100, judge that this sample is defective, need resample) time judge that sample EV71 is positive, otherwise be sample EV71 feminine gender.In like manner, if find in hole that more than 2 crocus cells and hole inner cell sum judge that sample CA16 is positive when being greater than 100, otherwise be sample CA16 feminine gender.Notice that operating personnel through professional training, need know the fluorescence signal got rid of acellular and produce, avoid the erroneous judgement of result.
5 the present embodiment result and conclusions
Detection sample in the present embodiment, with embodiment 2, picks up from the throat swab sample of 4 clinical hand-foot-and-mouth disease patients.
5.1 Fig. 5 are that sample 2 adds EV71 and CA16 Fluorescence Identification reagent red fluorescence photo, this sample redgreen fluorescence and orange fluorescence, only having Normocellular red fluorescence (being expressed as grey in black and white picture) in figure, can judgement sample 2 be that CA16 and EV71 is negative thus.
5.2 Fig. 6 is sample 3 add EV71 and CA16 Fluorescence Identification reagent after, the photo (2 times, camera is focused) that positive orange fluorescence photo and red normal cell photo synthesize, orange fluorescence (being expressed as brilliant white in black and white picture) and the Normocellular red fluorescence (being expressed as grey in black and white picture) of the CA16 positive is demonstrated, this sample redgreen fluorescence in figure.Can judgement sample 3 be that the positive EV71 of CA16 is negative thus.
5.3 Fig. 7 is sample 4 add EV71 and CA16 Fluorescence Identification reagent after, the photo that positive green fluorescence photo and red normal cell photo synthesize, green fluorescence (being expressed as brilliant white in black and white picture) and the Normocellular red fluorescence (being expressed as grey in black and white picture) of the EV71 positive is demonstrated in figure, this sample, without orange fluorescence, can judgement sample 4 be that the positive CA16 of EV71 is negative thus.
5.4 Fig. 8 are fluorescence photos that sample 5 adds EV71 and CA16 Fluorescence Identification reagent, the yellow-green fluorescence (being expressed as white in black and white picture) of EV71 positive cell and the orange fluorescence (being expressed as grey in black and white picture) of the CA16 positive can judgement sample 5 be that CA16EV71 is two positive thus.
Embodiment 2 and embodiment 3 contrast, and in visible the present embodiment, direct immunofluorescence kit assay is consistent with above-mentioned flow cytometry kit assay.
The flow cytometry of embodiment 4: hand-foot-and-mouth disease EV71 and CA16, direct immunization detection method, PCR-fluorescence probe method and the comparative experiments of cell chulture isolated viral method
The hand-foot-and-mouth disease EV71 of the present embodiment and the FCM analysis method of CA16, direct immunization detection method utilize kit provided by the invention respectively, and PCR-fluorescence probe method used kit is purchased from the Enterovirus 71 nucleic acid detection kit (PCR-fluorescence probe method) of Da An gene incorporated company and coxsackie virus A 16-type nucleic acid detection kit (PCR-fluorescence probe method).Adopt and carry out comparative experimental research with Da An gene incorporated company kit, clinical verification is carried out to EV71 and CA16 antigen detection kit prepared by our company, with cell culture method be detect goldstandard, compare three kinds of kits detect EV71 and CA16 time sensitivity and specificity.
1. samples sources and process
Oropharyngeal swab specimen 140 parts, takes from the doubtful hand-foot-and-mouth disease patient of the First Affiliated Hospital of Guangzhou Medical University.Sample is suspended in 3mlDMEM nutrient culture media, breaks up mucus with vortex mixer, the centrifugal 8min of 500g, respectively get 100ul supernatant and detect sample as cell culture method.
2. operation steps
2.1 cell culture methods carry out according to the following steps: get above-mentioned detection sample 100ul, be inoculated in 70-80% saturated monolayer Vero cell, change maintain base and cultivate, observation of cell pathology situation.
2.2PCR-fluorescence probe method (nucleic acid method) operates according to its instructions.
2.3 FCM analysis methods (FCM), direct immunization detection method difference (DFA) are by kit description operation of the present invention.
3 result statistics and conclusions
Detect 140 increment product by above-mentioned 4 kinds of methods, the results are shown in Table 1, table 1 is clinical verification testing result.
Table 1
Note :+be positive ,-represent negative
By finding the statistics of experimental result, in 140 increment product, cell culture method has recorded 35 parts of CA16 positives, 62 parts of EV71 positives, and 1 part of EV71 and CA16 is two positive, and 42 parts is negative; The testing result of FCM with DFA is identical, and the sample that they conform to cell culture method testing result has 133 (91+42) part.Wherein, what CA16 positive sample was consistent has 32 parts, and what EV71 positive sample was consistent has 58 parts, and what the two positive sample of EV71 with CA16 was consistent has 1 part, and what negative sample conformed to has 42 parts; What result did not conform to has 7 parts, can be verified as false negative with cell culture method.The sample that nucleic acid method conforms to cell culture method testing result has 137 (96+41) part, what wherein CA16 positive sample was consistent has 34 parts, what EV71 positive sample was consistent has 60 parts, and what the two positive sample of EV71 with CA16 was consistent has 1 part, and what negative sample conformed to has 41 parts; What result did not conform to has 4 parts, and with cell culture method checking, wherein 3 parts are verified as false positive, and 1 part is verified as false negative.
4 sensitivity (positive coincidence rate):
4.1CA16 positive coincidence rate
Kit of the present invention (FCM and DFA): sensitivity=(32+1)/(35+1) * 100%=91.4%
Nucleic acid method: sensitivity=(34+1)/(35+1) * 100%=97.2%
4.2EV71 positive coincidence rate
Kit of the present invention (FCM and DFA): sensitivity=(58+1)/(62+1) * 100%=93.7%
Nucleic acid method: sensitivity=(60+1)/(62+1) * 100%=96.8%
5 specificitys (negative match-rate):
Kit of the present invention (FCM and DFA): sensitivity=42/42*100%=100%
Nucleic acid method: sensitivity=41/42*100%=97.6%
6 conclusions: can be found out by above result, kit of the present invention (FCM and DFA) detects CA16 and EV71, and in sensitivity (being respectively 91.4%, 93.7%), outline is lower than nucleic acid detection method (being respectively 97.2%, 96.8%), but specificity (100%) is then higher than nucleic acid detection method (96.6%).Compared with nucleic acid detection method, kit of the present invention (FCM and DFA) is fast easy to operate, requires low to experimental apparatus, experimental site.Basic hospital hand-foot-and-mouth disease patient is less, this method lower to equipment requirement of applicable direct immunofluorescence; FCM has the feature of high flux and automatic analysis, but flow cytometer costly, is applicable to the large hospital that patient is more.
Finally should be noted that; above embodiment is only in order to illustrate technical scheme of the present invention but not restriction to invention patent protection scope; although with reference to preferred embodiment to invention has been detailed description; those of ordinary skill in the art is to be understood that; under the prerequisite of the essence and scope that do not depart from technical solution of the present invention; can also make amendment or equivalent replacement to technical scheme of the present invention, these all belong to protection scope of the present invention.
Claims (10)
1. the kit of a direct-detection enterovirns type 71 and coxsackie virus A 16-type, it is characterized in that, described kit comprises fluorescein-labeled antibody, cell immobile liquid, PBS solution, PBST solution, 1%Triton-X100, cell controls product, antiseptic sodium azide and antibody stabilization agent BSA.
2. kit according to claim 1, it is characterized in that, described fluorescein-labeled antibody comprises the antibody of the enterovirns type 71 covalently bound with fluorescein and the antibody of the coxsackie virus A 16-type covalently bound with fluorescein, and described antibody is monoclonal antibody or polyclonal antibody.
3. kit according to claim 1, is characterized in that, described fluorescein is any one in Alexa fluorescent dye, phycoerythrin and derivant thereof, anthocyanin and derivant, phycoerythrin-anthocyanin and fluorescein-5-isothiocyanates.
4. kit according to claim 1, it is characterized in that, described cell controls product comprise positive cell reference substance and negative cells reference substance, described positive cell reference substance is the RD cell containing enterovirns type 71 of freeze-drying and the RD cell containing coxsackie virus A 16-type of freeze-drying, and described negative controls is the normal RD cell of freeze-drying; Described cell immobile liquid to be mass volume ratio be 4% paraformaldehyde.
5. kit according to claim 1, is characterized in that, described kit uses according to the following steps:
(1) cell sample to be measured is abandoned supernatant with described PBS solution is centrifugal, then fix with described cell immobile liquid, it is resuspended finally to add described PBST solution, prepares single-cell suspension liquid;
(2), after being mixed with described 1%Triton-X100, described antiseptic sodium azide and described antibody stabilization agent BSA by described fluorescein-labeled antibody, dyeing liquor is obtained;
(3) added respectively by the dyeing liquor obtained in described step (2) in the described single-cell suspension liquid in described step (1) and the cell controls product in described kit, room temperature lucifuge hatches 30min;
(4) with the dyeing liquor of described PBST eluant solution removing in conjunction with cell, and with after PBST re-suspended cell, with the viral antigen be combined with described fluorescein-labeled antibody in cell sample described in flow cytomery;
(5) interpretation of result: if Q2-1, Q2-2, Q2-4 quadrant number percent is greater than critical value, then judge that described cell sample to be measured infects positive as corresponding virus or Geminivirus; If when Q2-2 is less than or equal to critical value, Q2-1, Q2-4 are less than or equal to critical value, then judge that described cell sample to be measured is negative as corresponding virus infections.
6. kit according to claim 1, is characterized in that, described kit also comprises fluorescent dye Yi Wensilan, mountant, microslide and Quality Control slide, porose on described microslide.
7. kit according to claim 6, it is characterized in that, described Quality Control slide has positive hole and negative hole, and described positive hole is the RD cell of enterovirns type 71 and the RD cell of coxsackie virus A 16-type of 1:1 mixing, and described negative hole is normal RD cell; Described mountant is a kind of damping fluid containing Isosorbide-5-Nitrae-two azo dicyclo [2,2,2]-octane, glycerine and polyvinyl alcohol (PVA).
8. kit according to claim 7, is characterized in that, described kit uses according to the following steps:
(1) cell sample to be measured is abandoned supernatant with described PBS solution is centrifugal, then fix with described cell immobile liquid, it is resuspended finally to add described PBST solution, prepares single-cell suspension liquid;
(2), after described fluorescein-labeled antibody being mixed with described 1%Triton-X100, described antiseptic sodium azide and described antibody stabilization agent BSA, add described fluorescent dye Yi Wensilan, obtain Fluorescence Identification reagent;
(3) be added in the hole of described microslide by the single-cell suspension drop in described step (1), after volatile dry, drip the Fluorescence Identification reagent obtained in described step (2), 37 DEG C of lucifuges hatch 20min;
(4) the Fluorescence Identification reagent obtained in the described step of each dropping (2) on the positive hole and negative hole of described Quality Control slide, 37 DEG C of lucifuges hatch 20min;
(5) develop a film with described PBST solution and distilled water, described mountant is dripped after sopping up excessive moisture with thieving paper, covered, with the viral antigen be combined with described fluorescein-labeled antibody in cell sample to be measured described in fluorescence microscope, Taking Pictures recording experimental result;
(6) interpretation of result: if find more than 2 green cells and hole inner cell sum is greater than 100 in described microslide hole, then judge that described cell sample EV71 to be measured is positive, otherwise be EV71 feminine gender; If find more than 2 orange fluorescence cells and hole inner cell sum is greater than 100 in described microslide hole, then judge that described cell sample CA16 to be measured is positive, otherwise be CA16 feminine gender.
9. the antibody used in the kit that one of claim 1-8 is described, it is characterized in that, described antibody is the antibody of the enterovirns type 71 covalently bound with fluorescein and the coxsackie virus A 16-type covalently bound with fluorescein, and described fluorescein is any one in Alexa fluorescent dye, phycoerythrin and derivant thereof, anthocyanin and derivant, phycoerythrin-anthocyanin and fluorescein-5-isothiocyanates.
10. the kit that one of claim 1-8 is described is detecting the application in enterovirns type 71 and coxsackie virus A 16-type.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610041545.6A CN105527430A (en) | 2016-01-21 | 2016-01-21 | Kit for directly detecting 71 type enterovirus and A16 type coxsackievirus and application thereof |
| CN201710188451.6A CN107121547A (en) | 2016-01-21 | 2016-01-21 | The kit and its application of direct immuno-fluorescent antibody assay enterovirns type 71 and coxsackie virus A 16-type |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610041545.6A CN105527430A (en) | 2016-01-21 | 2016-01-21 | Kit for directly detecting 71 type enterovirus and A16 type coxsackievirus and application thereof |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710188451.6A Division CN107121547A (en) | 2016-01-21 | 2016-01-21 | The kit and its application of direct immuno-fluorescent antibody assay enterovirns type 71 and coxsackie virus A 16-type |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105527430A true CN105527430A (en) | 2016-04-27 |
Family
ID=55769783
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710188451.6A Pending CN107121547A (en) | 2016-01-21 | 2016-01-21 | The kit and its application of direct immuno-fluorescent antibody assay enterovirns type 71 and coxsackie virus A 16-type |
| CN201610041545.6A Pending CN105527430A (en) | 2016-01-21 | 2016-01-21 | Kit for directly detecting 71 type enterovirus and A16 type coxsackievirus and application thereof |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710188451.6A Pending CN107121547A (en) | 2016-01-21 | 2016-01-21 | The kit and its application of direct immuno-fluorescent antibody assay enterovirns type 71 and coxsackie virus A 16-type |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN107121547A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105842452A (en) * | 2016-05-30 | 2016-08-10 | 吉林大学 | Preparation method of duplicate diagnosis gold-colloidal strip for hand-foot-mouth disease EV71 and CA16 |
| CN110836972A (en) * | 2018-08-16 | 2020-02-25 | 上海益诺思生物技术股份有限公司 | Biomarker genetic toxicity detection method based on gamma-H2AX |
| CN111208295A (en) * | 2020-02-29 | 2020-05-29 | 济南德亨医学科技有限公司 | Gardnerella vaginalis quantum dot immunodetection method |
| CN111208288A (en) * | 2020-02-29 | 2020-05-29 | 济南德亨医学科技有限公司 | Trichomonas vaginalis quantum dot fluorescence immunoassay method |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111024468A (en) * | 2019-12-20 | 2020-04-17 | 天津金域医学检验实验室有限公司 | Method for processing sample in respiratory tract pathogen immunofluorescence detection |
| CN112813202A (en) * | 2021-02-20 | 2021-05-18 | 深圳市赛格诺生物科技有限公司 | Fluorescent RT-PCR (reverse transcription-polymerase chain reaction) detection reagent and method for coxsackievirus A16 and enterovirus 71 |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6225046B1 (en) * | 1995-04-03 | 2001-05-01 | Macquarie Research Ltd. | Method for detecting microorganisms |
| WO2005116234A2 (en) * | 2004-04-16 | 2005-12-08 | University Of Massachusetts | Detection and quantification of intracellular pathogens |
| US20060216696A1 (en) * | 2004-08-23 | 2006-09-28 | Goguen Jon D | Rapid plague detection system |
| CN101329230A (en) * | 2008-07-14 | 2008-12-24 | 中国人民解放军第三军医大学 | Improved method for dyeing immunofluorescence cell |
| CN102483412A (en) * | 2009-04-16 | 2012-05-30 | 诊断混合公司 | Direct fluorescene immunoassay for viral antigens |
| CN103421112A (en) * | 2012-05-24 | 2013-12-04 | 中国科学院上海巴斯德研究所 | Binding molecule capable of resisting enterovirus, and applications thereof |
| CN104303059A (en) * | 2012-03-29 | 2015-01-21 | 瓦西利奥斯·齐列瓦可斯 | Method of intracellular infectious agent detection in sperm cells |
| CN104513310A (en) * | 2013-09-27 | 2015-04-15 | 中国科学院上海巴斯德研究所 | Preparation and application of anti-Coxsackie virus A16 monoclonal antibody |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN201892679U (en) * | 2010-09-25 | 2011-07-06 | 天津市宝坻区人民医院 | Influenza A H1N1 immunofluorescence detection reagent box |
| CN102288471B (en) * | 2011-07-27 | 2013-06-12 | 上海交通大学医学院附属仁济医院 | Immunofluorescence staining method for suspension cells |
-
2016
- 2016-01-21 CN CN201710188451.6A patent/CN107121547A/en active Pending
- 2016-01-21 CN CN201610041545.6A patent/CN105527430A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6225046B1 (en) * | 1995-04-03 | 2001-05-01 | Macquarie Research Ltd. | Method for detecting microorganisms |
| WO2005116234A2 (en) * | 2004-04-16 | 2005-12-08 | University Of Massachusetts | Detection and quantification of intracellular pathogens |
| US20060216696A1 (en) * | 2004-08-23 | 2006-09-28 | Goguen Jon D | Rapid plague detection system |
| CN101329230A (en) * | 2008-07-14 | 2008-12-24 | 中国人民解放军第三军医大学 | Improved method for dyeing immunofluorescence cell |
| CN102483412A (en) * | 2009-04-16 | 2012-05-30 | 诊断混合公司 | Direct fluorescene immunoassay for viral antigens |
| CN104303059A (en) * | 2012-03-29 | 2015-01-21 | 瓦西利奥斯·齐列瓦可斯 | Method of intracellular infectious agent detection in sperm cells |
| CN103421112A (en) * | 2012-05-24 | 2013-12-04 | 中国科学院上海巴斯德研究所 | Binding molecule capable of resisting enterovirus, and applications thereof |
| CN104513310A (en) * | 2013-09-27 | 2015-04-15 | 中国科学院上海巴斯德研究所 | Preparation and application of anti-Coxsackie virus A16 monoclonal antibody |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105842452A (en) * | 2016-05-30 | 2016-08-10 | 吉林大学 | Preparation method of duplicate diagnosis gold-colloidal strip for hand-foot-mouth disease EV71 and CA16 |
| CN110836972A (en) * | 2018-08-16 | 2020-02-25 | 上海益诺思生物技术股份有限公司 | Biomarker genetic toxicity detection method based on gamma-H2AX |
| CN111208295A (en) * | 2020-02-29 | 2020-05-29 | 济南德亨医学科技有限公司 | Gardnerella vaginalis quantum dot immunodetection method |
| CN111208288A (en) * | 2020-02-29 | 2020-05-29 | 济南德亨医学科技有限公司 | Trichomonas vaginalis quantum dot fluorescence immunoassay method |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107121547A (en) | 2017-09-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105527430A (en) | Kit for directly detecting 71 type enterovirus and A16 type coxsackievirus and application thereof | |
| Drew | Diagnosis of cytomegalovirus infection | |
| CN102066931B (en) | Human papilloma virus early stage and the immunoassay tests of later infections | |
| CN101587043B (en) | Integrated method for enriching and detecting rare cell in biological fluid sample | |
| AU2010236270B9 (en) | Direct fluorescene immunoassay for viral antigens | |
| CN106370867B (en) | A kind of joint-detection assesses the kit of liver Transplantation for Hepatocellular Carcinoma recurrence and metastasis after resection risk | |
| Nath et al. | Diagnosis of herpes simplex virus: laboratory and point-of-care techniques | |
| CN105308458A (en) | Automated immunoanalyzer system for performing diagnostic assays for allergies and autoimmune diseases | |
| Dilnessa et al. | Cell culture, cytopathic effect and immunofluorescence diagnosis of viral infection | |
| CN105137072B (en) | A kind of mycobacterium tuberculosis LAM detection kit, preparation method and using method | |
| CN111007257A (en) | Porcine pseudorabies virus gE protein antibody detection immune blocking chromatography kit and application thereof | |
| CN106771185A (en) | A kind of nasopharyngeal carcinoma circulating tumor cell detection kit | |
| US7067258B2 (en) | Human papillomavirus multiplexed assay | |
| CN101644709A (en) | Method for rapidly detecting neutralizing antibody of virus and kit therefor | |
| CN111334478A (en) | Hybridoma cell strain for detecting canine distemper virus and canine parvovirus and double detection test paper card thereof | |
| CN109799351A (en) | Porcine circovirus 2 type double-antibody sandwich elisa kit and its application | |
| CN104990905A (en) | Kit for diagnosis of hepatocellular carcinoma metastasis based on solid-phase enzyme immunoassay fluorescence spots | |
| CN106771121A (en) | A kind of foot and mouth disease virus colloidal gold strip, cause of disease quick detection kit and preparation method thereof | |
| RU2456617C2 (en) | Panel of serums containins antibodies to hcv antigens of various subtypes | |
| CN104807993A (en) | Mycobacterium tuberculosis ESAT-6 protein detection kit, as well as preparation method and use method | |
| Stirk et al. | Comparative sensitivity of three methods for the diagnosis of cytomegalovirus lung infection | |
| CN101693887A (en) | Fast cultivation and identification method of high throughput virus and applications thereof | |
| Schutzbank et al. | Immunofluorescence | |
| CN105717084A (en) | Non-enveloped virus quantum dot marking method and application | |
| Bosch et al. | Detection of infectious rotaviruses by flow cytometry |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20160427 |
|
| WD01 | Invention patent application deemed withdrawn after publication |