CN105759028A - Immunochromatographic detection method of Norovirus Raman microprobe labeling - Google Patents
Immunochromatographic detection method of Norovirus Raman microprobe labeling Download PDFInfo
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- CN105759028A CN105759028A CN201511005062.2A CN201511005062A CN105759028A CN 105759028 A CN105759028 A CN 105759028A CN 201511005062 A CN201511005062 A CN 201511005062A CN 105759028 A CN105759028 A CN 105759028A
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- 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
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- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/10—Detection of antigens from microorganism in sample from host
Abstract
The invention relates to an immunochromatographic detection method of Norovirus Raman microprobe labeling.A principle of the detection method includes: using nitrocellulose membrane (NC) as an SERS substrate, labeling an immunoprobe by Raman labeling, constructing novel surface enhanced Raman spectroscopy and immunochromatographic assay (SERS-ICA) based on colloidal gold ICA, and detecting Norovirus by using the method.Detection includes sample fixation, colloidal gold penetration, antigen antibody reaction, detection and the like, detection results are used to read peaks under the band of 1074 cm<-1> through software on a detection instrument, some peaks are positive, and no peaks are negative.The method is suitable for quickly and sensitively detecting Norovirus and is simple to perform, it is possible to greatly improve detection efficiency for inspection and quarantine staff on the front line of entry and exit ports through the method, and the method is significant to controlling diarrhea due to Norovirus.
Description
Technical field
The present invention relates to external diagnosis reagent technical field, be specifically related to the immunochromatography detection method of a kind of norovirus Raman microprobe labelling.
Background technology
Norovirus infectious diarrhea is to be belonged to, by norovirus, the diarrhoea that virus causes, and has the features such as morbidity is anxious, spread speed is fast, coverage is wide, is the Etiological causing Non-bacterial diarrhea to break out.The U.S. is every year in all of Non-bacterial diarrhea breaks out, and 60-90% is to be caused by norovirus.Also there are similar results in the developed countries such as Holland, Britain, Japan, Australia.In developing country, norovirus infectious diarrhea generally exists, and the most often causes outbreak of epidemic.In less than 5 years old diarrhea children of China, norovirus recall rate is about 15%, and antibody level of serum investigation shows that in population of China, the infection of norovirus is the most universal.Nineteen ninety-five, China reports the first norovirus and infects, and the area such as Shanxi, Beijing, Anhui, Foochow, Wuhan, Guangzhou successively occurs a lot of norovirus infectious diarrhea epidemic outbreaks afterwards.December in 2012 13 days is to December during 16 days, and Japan various places occur a series of collective's Food poisoning cases caused because of norovirus in succession.
Norovirus is single strand plus RNA virus, without peplos.Being structured small round virus under Electronic Speculum, diameter 26～35nm, in icosahedral symmetry, 90 dimers that shell is made up of 180 same coat protein are constituted.Norovirus contains the Strain that kind more than 40 is different, according to its gene expression characteristics, is broadly divided into 5 genomes.Five genomes are GI, G II, G III, G IV and GV.II and G IV 3 genome main infection mankind of GI, G, and G III and GV infected cattle and Mus respectively.Norovirus restructuring often betides G II .2, G II .3, G II between .4 and G II .12.Nineteen ninety is to cause the major gene group of Infection outbreak in world wide for species specific norovirus strain in mid-term one (G II .4 strain).Over 20 years, G II .4 is always the popular topmost genotype of norovirus in world wide.There are some researches show that G II .4 group's Strain is the popular advantage strain that China has been reported.
Entry and exit food safety is the international significant problem being concerned about, norovirus the diarrhoea poisoning caused not only directly affects port safe and sanitary, and affects the foreign trade of China.And the traditional sensing techniques workflow length of norovirus pathogen, automatization level are low, it is difficult to adapt to the actual demand of quickly detection, other conventional detection methods can not meet again the requirement of multivariate detection.Raman spectroscopy is a kind of quick, non-destructive photon scattering technology, and it is provided that the information about analyte molecule and its characteristic spectrum are vibrated, is a kind of analytical technology and instrument [1] developed based on Raman scattering principle.Surface enhanced raman spectroscopy (Surface Enhanced Raman Scattering, it is called for short SERS) refer to that its Raman scattering intensities can increase by 10 when some molecules are adsorbed on some coarse metal (such as silver, copper, gold etc.) surface4～106Times.In SERS technology for detection pathogenic bacterium field, SERS has shown the prominent superiority relative to traditional detection method, particularly strengthening in the substrate combination with antibacterial, technical and various enhancing preparation means unique for SERS is successfully to realize SERS hypersensitive, quickly detection pathogenic bacterium to lay a solid foundation.Application SERS technical research microorganism in recent years it has been reported that but, experiments involved in these reports are higher to instrument requirements, and experimentation is complicated, is not suitable for carrying out on-line checking.
The present invention is with nitrocellulose filter (Nitrocellulose membrane, NC) it is SERS substrate, Raman labels immunological probe labelling based on colloidal gold immunity chromatography (Immunochromatographic Assay, ICA) construct novel surface enhanced raman spectroscopy immunochromatography technique (SERS-ICA), and use the method that norovirus is detected.The present invention is simple to operate, time-consuming, highly sensitive, and specificity is good.Site Detection for norovirus provides quickly, easy, sensitive Raman spectrum immunochromatography detection method.
Summary of the invention
The technical problem to be solved is to provide the immunochromatography detection method of a kind of quick, easy, sensitive norovirus Raman microprobe labelling.The immunochromatography detection method of norovirus Raman microprobe labelling of the present invention, including following detecting step:
1. the preparation of Raman labels immune colloid gold-antibody complex, Raman labels immune colloid gold-antibody complex includes gold colloidal, norovirus VP1 protein antibodies, probe molecule；
2. prepared by sample；
3. prepared by colloidal gold strip: first Raman labels immune colloid gold-antibody complex is sprayed to colloidal gold pad, again sample pad, colloidal gold pad, nitrocellulose filter and absorbent paper are assembled in successively on polyethylene PVC base plate, after cutting into 6X2cm specification, become a colloidal gold immunochromatographitest test strip.
4. with a stroke film instrument, the sample prepared is drawn at colloidal gold strip T line.
5. colloidal gold chromatographic: the well at colloidal gold strip drips buffer, stands 15min.
6. testing result reads: use Raman spectrometer surveying card T line position detection SERS signal, and according to detection signal judged result.Its power 20mW, the signals collecting time is 10s, chooses equally distributed 10 points along T line position and carries out signals collecting, and by 1074cm-1The meansigma methods of the peak height at displacement is considered as representing the valid data of true horizon.
Gold colloidal described in step 1 is 1.2*10-4Gold chloride and 0.7*10-4Trisodium citrate aqueous solution interact formed；Described probe molecule is 4-mercaptobenzoic acid.
The preparation of the Raman labels immune colloid gold-antibody complex described in step 1 comprises the following steps: to add the antibody of 20 μ g in 1mL colloidal gold solution, add the 4-mercaptobenzoic acid solution of 1mM, vortex mixes, 1mL is added containing 1% caseic PBS solution after agitator reaction 20min, 1h is reacted after vortex mixing, 5000r/min collects bottom precipitation about 100 μ L after being centrifuged 10min, and 4 DEG C of Refrigerator stores are stand-by.
Sample preparation procedure described in step 2 is: in addition 1ml sample diluting liquid to the little centrifuge tube of 1.5ml, then, add 0.1g solid manure specimen or 0.1ml liquid manure specimen, it is placed in whirlpool oscillator fully to mix, room temperature stands 10min, under room temperature >=5000rpm is centrifuged 5min, take supernatant and detect.
The sample diluting liquid in sample preparation procedure described in step 2 is the sodium chloride albumen buffer containing 0.1%Kathon CG.
Sample size described in step 4 is 100ul.
Colloidal gold chromatographic buffer described in step 5 is PBST buffer.
The present invention be first by Raman labels probe application in the detection of viral pathogens, by building novel surface enhanced raman spectroscopy immunochromatography technique, Raman immunological probe is fixed in colloidal gold pad, buffer dissolves gold colloidal through chromatography, and chromatograph to T line, mix with the detected sample at T line, use Raman spectrometer scanning, at 1074cm-1Place has peak value then for positive, is then negative without peak value.The colloidal gold technique that the remolding sensitivity of the present invention is traditional wants height, and detection equipment is simple, and speed is fast, is suitable for the quick detection at scene.The detection efficiency of import-export ports one X-ray inspection X quarantine functionary can be increased substantially by the present invention, not only can reduce workload, but also traditional detection method positive missing inspection problem that may be present can have been solved to greatest extent, thus prevent the generation of the diarrhoea infectious disease that norovirus causes to greatest extent.
The immunochromatography detection method result of embodiment 1 norovirus Raman microprobe labelling.
Surveying testing result display Detection of antigen in the present embodiment and the detection of norovirus the infected's feces is illustrated as the positive, normal person's fecal sample is shown as negative.
Accompanying drawing explanation
Fig. 1 is the immunochromatography detection method sensitivity experiments result of embodiment 1 norovirus Raman microprobe labelling.
Left figure is Raman spectrogram, and right figure is the standard curve of detection.
Fig. 2 is the immunochromatography detection method specificity experiments result of embodiment 1 norovirus Raman microprobe labelling
Left figure is that Raman spectrum machine detects collection of illustrative plates, and a is norovirus positive sample, and b is negative control, and c is rotavirus sample, and d is intestinal Ev71 sample；Right figure is perusal band color change results.
Fig. 3 is the immunochromatography detection method repeatability testing result of embodiment 1 norovirus Raman microprobe labelling.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is expanded on further.Should be understood that these embodiments are merely to illustrate the present invention rather than limit the scope of the present invention.In addition, it is to be understood that after having read the content that the present invention lectures, the present invention can be made various changes or modifications by those skilled in the art, and these equivalent form of values fall within the application appended claims limited range equally.
The immunochromatography detection method performance evaluation of embodiment 1 norovirus Raman microprobe labelling
One, experimental procedure
1. sample prepares
In the present embodiment, positive sample is the feces of norovirus the infected, and negative control sample is the feces of Healthy People.
2. pattern detection flow process:
(1) preparation of Raman labels immune colloid gold-antibody complex: Raman labels immune colloid gold-antibody complex, including gold colloidal (1.2*10-4Gold chloride, 0.7*10-4Trisodium citrate aqueous solution), norovirus VP1 protein antibodies, probe molecule 4-mercaptobenzoic acid (4-MBA).Preparation method is the antibody to add 20 μ g in 1mL colloidal gold solution, add the 4-MBA solution of 1mM, vortex mixes, 1mL is added containing 1% caseic PBS solution after agitator reaction 20min, 1h is reacted after vortex mixing, 5000r/min collects bottom precipitation about 100 μ L after being centrifuged 10min, and 4 DEG C of Refrigerator stores are stand-by.
(2) prepared by sample: in addition 1ml sample diluting liquid to the little centrifuge tube of 1.5ml, then, add 0.1g solid manure specimen or 0.1ml liquid manure specimen, it is placed in whirlpool oscillator fully to mix, room temperature stands 10min, under room temperature >=and 5000rpm is centrifuged 5min, take supernatant and detect.
(3) prepared by colloidal gold strip: Raman labels immune colloid gold-antibody complex has been sprayed to colloidal gold pad.Sample pad, colloidal gold pad, nitrocellulose filter and absorbent paper are assembled in successively on PVC (polyethylene) base plate, after cutting into 6X2cm specification, become a colloidal gold immunochromatographitest test strip.
(4) with a stroke film instrument, the sample 100ul prepared is drawn at colloidal gold strip T line.
(5) colloidal gold chromatographic: the well at colloidal gold strip drips 100ul buffer, stands 15min.
(6) testing result reads: use Raman spectrometer surveying card T line position detection SERS signal, and according to detection signal judged result.Its power 20mW, the signals collecting time is 10s, chooses equally distributed 10 points along T line position and carries out signals collecting, and by 1074cm-1The meansigma methods of the peak height at displacement is considered as representing the valid data of true horizon.
3. detection method sensitivity and the foundation of standard curve
Configuration concentration is 0,0.3,3,5,10,30,50,100,150,300,500, the antigen mark liquid of 1000ng/mL carry out the immunochromatography process of standard, question response can determine the content of determinand by the intensity of detection Raman signal after terminating.
The most specific establishment:
For detecting the specific performance of the method, have chosen is the pathogen of diarrhea virus equally, enterovirus EV 71 Virus Sample and rotavirus sample together with norovirus fecal sample according to 2 in program detect.
5. method repeatability:
For detecting the repeated situation of detection method, choose positive sample independent detection 10 times.
Two, experimental result
2.1 susceptiveness detections
The SERS-ICA method of the present invention obtains standard curve when detecting the norovirus VP1 antigen of variable concentrations, from standard curve display linear detection range about between 3-150ng/mL, detection limit is about 0.5ng/ml, use the detection line of the ICA method with a collection of antibody then for 5ng/ml,, i.e. the Lod value of SERS-ICA method reduces about 10 times compared to ICA method.It is specifically shown in Fig. 1.
2.2 specificitys are established
After testing, only norovirus fecal sample detects the positive, and testing result is 1074cm-1The peak value at place, EV71 virus and rotavirus are detected as feminine gender, consistent with the result of perusal band variable color.The detection method specificity of the display present invention is good.It is specifically shown in Fig. 2.
2.3 method repeatability
After testing, positive sample duplicate detection 10 times, test peak value essentially coincides, and CV < 5% has good stability.Result is shown in Fig. 3.
Claims (9)
1. the immunochromatography detection method of a norovirus Raman microprobe labelling is characterized in that: includes following detecting step:
(1) preparation of Raman labels immune colloid gold-antibody complex, Raman labels immune colloid gold-antibody complex includes glue
Body gold, norovirus VP1 protein antibodies, probe molecule；
(2) prepared by sample；
(3) prepared by colloidal gold strip: first Raman labels immune colloid gold-antibody complex is sprayed to colloidal gold pad, then by sample
Product pad, colloidal gold pad, nitrocellulose filter and absorbent paper are assembled on polyethylene PVC base plate successively, cut into 6X2cm rule
After lattice, become a colloidal gold immunochromatographitest test strip；
(4) with a stroke film instrument, the sample prepared is drawn at colloidal gold strip T line；
(5) colloidal gold chromatographic: the well at colloidal gold strip drips buffer, stands 15min；
(6) testing result reads: uses Raman spectrometer surveying card T line position detection SERS signal, and sentences according to detection signal
Disconnected result.
The immunochromatography detection method of norovirus Raman microprobe labelling the most according to claim 1, it is characterised in that: step
Suddenly the gold colloidal described in (1) is 1.2*10-4Gold chloride and 0.7*10-4Trisodium citrate aqueous solution interact formed；
Described probe molecule is 4-mercaptobenzoic acid.
3. according to the immunochromatography detection method of the norovirus Raman microprobe labelling described in claim 1 and 2, it is characterised in that:
The preparation of described Raman labels immune colloid gold-antibody complex comprises the following steps: to add in 1mL colloidal gold solution
The antibody of 20 μ g, adds the 4-mercaptobenzoic acid solution of 1mM, and vortex mixes, and adds 1mL after agitator reaction 20min
Containing 1% caseic PBS solution, react after vortex mixing after 1h, 5000r/min are centrifuged 10min and collect bottom precipitation about
100 μ L, 4 DEG C of Refrigerator stores are stand-by.
The immunochromatography detection method of norovirus Raman microprobe labelling the most according to claim 1, it is characterised in that: step
Suddenly the sample preparation procedure described in (2) is: in addition 1ml sample diluting liquid to the little centrifuge tube of 1.5ml, then, add
0.1g solid manure specimen or 0.1ml liquid manure specimen, be placed in whirlpool oscillator and fully mix, and room temperature stands 10min,
Under room temperature >=5000rpm is centrifuged 5min, take supernatant and detect.
The immunochromatography detection method of norovirus Raman microprobe labelling the most according to claim 1, it is characterised in that: step
Suddenly the sample diluting liquid in the sample preparation procedure described in (2) is the sodium chloride albumen buffer containing 0.1%Kathon CG.
The immunochromatography detection method of norovirus Raman microprobe labelling the most according to claim 1, it is characterised in that: step
Suddenly the sample size described in (4) is 100ul.
The immunochromatography detection method of norovirus Raman microprobe labelling the most according to claim 1, it is characterised in that: step
Suddenly the colloidal gold chromatographic buffer described in (5) is PBST buffer.
The immunochromatography detection method of norovirus Raman microprobe labelling the most according to claim 1, it is characterised in that: step
Suddenly the signals collecting described in (6), its power 20mW, the signals collecting time is 10s, chooses along T line position and uniformly divides
10 points of cloth carry out signals collecting and pass through software on detecting instrument at 1074cm-1Reading peak value during wave band, having peak value is sun
Property, it is negative without peak value.
9. according to the immunochromatography detection method of the norovirus Raman microprobe labelling described in claim 1-8, it is characterised in that:
Detection method linear detection range is 3-150ng/mL, and detection is limited to 0.5ng/ml.
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Cited By (5)
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| CN109932507A (en) * | 2017-12-15 | 2019-06-25 | 广东出入境检验检疫局检验检疫技术中心 | A kind of biosensor and its preparation, application method for norovirus detection |
| CN109957014A (en) * | 2017-12-25 | 2019-07-02 | 中国科学院上海巴斯德研究所 | The preparation and application of anti-norovirus GII.3 source of mouse monoclonal antibody |
| CN111537493A (en) * | 2020-05-07 | 2020-08-14 | 中国人民解放军军事科学院军事医学研究院 | Method for detecting rotavirus by combining surface enhanced Raman scattering with immunochromatography |
| CN113238043A (en) * | 2020-12-14 | 2021-08-10 | 黑龙江大学 | Preparation method and application of furadan test paper based on SERS immunochromatographic technique |
| CN113354716A (en) * | 2021-08-11 | 2021-09-07 | 北京溯本源和生物科技有限公司 | Mouse norovirus recombinant antigen, monoclonal antibody and virus detection test strip |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109932507A (en) * | 2017-12-15 | 2019-06-25 | 广东出入境检验检疫局检验检疫技术中心 | A kind of biosensor and its preparation, application method for norovirus detection |
| CN109957014A (en) * | 2017-12-25 | 2019-07-02 | 中国科学院上海巴斯德研究所 | The preparation and application of anti-norovirus GII.3 source of mouse monoclonal antibody |
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| CN111537493A (en) * | 2020-05-07 | 2020-08-14 | 中国人民解放军军事科学院军事医学研究院 | Method for detecting rotavirus by combining surface enhanced Raman scattering with immunochromatography |
| CN113238043A (en) * | 2020-12-14 | 2021-08-10 | 黑龙江大学 | Preparation method and application of furadan test paper based on SERS immunochromatographic technique |
| CN113354716A (en) * | 2021-08-11 | 2021-09-07 | 北京溯本源和生物科技有限公司 | Mouse norovirus recombinant antigen, monoclonal antibody and virus detection test strip |
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Application publication date: 20160713 |