CN105603125A - Detection reagent kit for rapidly detecting poultry disease viruses and detection method of detection reagent kit - Google Patents

Detection reagent kit for rapidly detecting poultry disease viruses and detection method of detection reagent kit Download PDF

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CN105603125A
CN105603125A CN201610134356.3A CN201610134356A CN105603125A CN 105603125 A CN105603125 A CN 105603125A CN 201610134356 A CN201610134356 A CN 201610134356A CN 105603125 A CN105603125 A CN 105603125A
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detection
raman
reagent kit
quartz plate
detection reagent
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CN105603125B (en
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任涛
冯敏莎
谭阳通
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South China Agricultural University
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Abstract

The invention discloses a detection reagent kit for rapidly detecting poultry disease viruses and a detection method of the detection reagent kit and belongs to the field of spectroscopy and molecular diagnosis. According to the detection reagent kit and the detection method, silver sol prepared through a hydroxylamine hydrochloride complexation method is adopted as a Raman enhancement reagent; after the silver sol and an object to be detected are mixed evenly at a certain proportion, the mixture is dripped onto a quartz plate; after natural air drying, Raman detection is carried out with a SmartRaman Ocean5pro portable Raman spectrometer; after principal component analysis and linear discriminant analysis are carried out on an obtained Raman spectrum, qualitative detection of the target pathogen is achieved. The detection reagent kit is easy and convenient to operate and high in sensitivity and has broad application prospects.

Description

A kind of detection kit and detection method thereof of fast detecting poultry diease virus
Technical field
The invention belongs to spectroscopy and molecular diagnosis field, particularly a kind of inspection of fast detecting poultry diease virusTest agent box and detection method thereof.
Background technology
NDV (NewcastleDiseaseVirus, NDV), is commonly called as philippine fowl disease, can cause chicken groupDiarrhoea, expiratory dyspnea and nervous symptoms. Avian influenza virus (AvianInfluenzaVirus, AIV) comprises multiple AsiasType, wherein H5 subtype avian influenza virus can cause a large amount of morbidities of fowl group and dead, by world's animal health groupKnit and classify category-A infectious disease as, paid much attention to. Actively monitoring and control NDV and avian influenza virusPropagation in fowl group, not only can reduce the loss of aquaculture, can also reduce to a great extent bird fluVirus is propagated the risk to people from fowl group, has important public health security meaning.
Laboratory detection means mainly contains aetology detection, the detection of cause of disease serology, immune analysis method at presentAnd molecular biology for detection. The separation qualification of virus be make a definite diagnosis this disease the most definitely, the side of tool public credibilityMethod, but experimental implementation step is comparatively lengthy and jumbled, takes; EUSA in immune analysis methodTechnology, i.e. ELISA, is in current antigen and antibody field, the immunology detection side being most widely usedMethod, has higher specificity and sensitiveness, but price is comparatively expensive; Fluorescence quantitative RT-RCR (ReverseTranscription-PolymeraseChainReaction) technology has sensitiveness and the specificity of height, andCan greatly shorten viral detection time, overcome non-specific factor in conventional serological diagnostic methodDisturb and sensitive question, for Rapid&Early diagnosis virus provides responsive, quick, practical detection method,But step is comparatively complicated, and corresponding laboratory equipment need to be provided. Therefore urgently set up one simple to operate,Fast, sensitive, harmless high-throughout detection method.
SERS scattering (SurfaceEnhancedRamanScattering, SERS) refers to works asSome Molecular Adsorption when thering is the precious metal surface of nanometer extra coarse degree, the Raman scattering obtaining in these extra coarsesSignal will not strengthen up to a million times, even higher. The spectrum obtaining is thus called as " surface-enhanced RamanSpectrum ". Compare other spectrum, as fluorescence spectrum, infrared spectrum etc., SERS hasHighly sensitive, bands of a spectrum are narrow, anti-light bleaching, be not subject to the feature such as interference of water, and can realize fast, realTime, Non-Destructive Testing, be therefore highly suitable for biomedical research.
Summary of the invention
Need expensive apparatus preparation professional and technical personnel's problem for overcoming existing detection technique, the present inventionPrimary and foremost purpose be to provide a kind of detection kit of fast detecting poultry diease virus. Described detection kitAdopt SERS technology, provide a kind of examination of easy and simple to handle, highly sensitive detection virusAgent box.
Another object of the present invention is to provide the detection method of mentioned reagent box.
Object of the present invention is achieved through the following technical solutions: a kind of detection reagent of fast detecting poultry diease virusBox, comprises metal-sol liquid, quartz plate and Raman spectrometer.
Described metal-sol liquid is aurosol liquid or silver sol liquid.
The preparation method of described silver sol is as follows: under normal temperature by 2.5mL10-2mol/LAgNO3JoinIn 75mL distilled water, add 5mLof10-2Mol/L TGA; After stirring 10min, splash into 2.5mL10-2Mol/LNaI, stirs after 20min, obtains jade-green AgI colloidal sol, adds 20mgNaBH4Reduction,Continue stir about 10min, generate yellowish-brown silver sol, what obtain is the spheroidal particle of shape homogeneous, average particleThe about 35nm in footpath, concentration is about 120 μ g/mL.
Described quartz plate is that wave-number range is 700-2000cm-1Without the substrate quartz plate of obvious Raman peaks;
Described quartz plate also can have sheet glass to replace.
Described Raman spectrometer is SmartRamanOcean5pro Portable Raman spectrometer.
Described Raman spectrometer testing conditions is: optical maser wavelength 785nm, laser power be 15-120mW,Be that 1-10s, wave-number range are 700-2000cm the time of integration-1。
Application mentioned reagent box carries out the detection method of poultry diease virus, comprises the following steps:
1) testing sample and metal-sol liquid are mixed with 1:2～3:1 ratio;
2) get 2～5 μ L steps 1) on sample drop quartz plate after mixing, natural air drying;
3) the round point shape sample forming on quartz plate is carried out to Raman detection, Raman instrument is SmartRamanOcean5pro Portable Raman spectrometer. The parameter of test is: power is 15～120mW, the time of integrationBe 1～10s, excitation wavelength is 785nm;
4) record spectrogram, by principal component analysis and linear discriminant analysis, two-strain is carried out to modeling classification;
5) judged result.
The testing environment temperature of above-mentioned detection method is at 4～40 DEG C.
Above-mentioned virus is generally that inside has nucleic acid (DNA or RNA), and outside has the albumen of capsid,Skin has cyst membrane or without cyst membrane structure.
Described poultry diease virus comprises other bird viruses such as NDV, avian influenza virus.
The present invention has following advantage and effect with respect to prior art:
The present invention sets up a kind of method and detection kit of fast detecting poultry diease virus. The method is easy to operate,High specificity, consuming time short, energy fast detecting goes out the Raman spectrogram of poultry diease virus, through principal component analysis and lineThe stoechiometric process such as property discriminant analysis can obviously be distinguished different poultry diease virus, are fast detecting poultry diease diseasePoison provides the foundation of optics spectrogram, has a good application prospect.
Brief description of the drawings
Fig. 1 is the Raman spectrum result figure of GM strain of the present invention and H5N6 strain.
Fig. 2 is GM strain of the present invention and H5N6 strain Raman spectrogram PCA-LDA (PrincipalComponentsanalysis-lineardiscriminateanalysis) two-dimensional process figure.
Fig. 3 is GM strain of the present invention and H5N6 strain Raman spectrogram PCA-LDA (PrincipalComponentsanalysis-lineardiscriminateanalysis) three-dimensional process figure.
Detailed description of the invention
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present inventionBe not limited to this.
The sample that the present invention uses is avian influenza virus H5N6 strain (A/OrientalMagpie-robin/ Guangdong/SW8/2014 (H5N6), gene pool number: KT454936), NDV GM strain (baseBecause of storehouse number: DQ486859), strain is provided by poultry diease teaching and research room of Agricultural University Of South China of Guangdong Province.
In following embodiment, material, reagent etc. used if no special instructions, all can obtain from commercial channels.
Embodiment 1
The detection kit that the invention provides a kind of fast detecting poultry diease virus, comprises metal-sol liquid, stoneEnglish sheet and Raman spectrometer.
Described metal-sol liquid is silver sol liquid.
The preparation method of described silver sol is as follows: under normal temperature by 2.5mL10-2mol/LAgNO3JoinIn 75mL distilled water, add 5mLof10-2Mol/L TGA. After stirring 10min, splash into 2.5mL10-2Mol/LNaI, stirs after 20min, obtains jade-green AgI colloidal sol, adds 20mgNaBH4Reduction,Continue stir about 10min, generate yellowish-brown silver sol, what obtain is the spheroidal particle of shape homogeneous, average particleThe about 35nm in footpath, concentration is about 120 μ g/mL.
Described quartz plate is that wave-number range is 700-2000cm-1Without the substrate quartz plate of obvious Raman peaks.
Described Raman spectrometer is SmartRamanOcean5pro Portable Raman spectrometer.
Described Raman spectrometer testing conditions is: optical maser wavelength 785nm, laser power is 100mW, long-pendingDividing the time is that 3s, wave-number range are 700-2000cm-1。
Application mentioned reagent box carries out the detection method of poultry diease virus, comprises the following steps:
1) by testing sample (comprising respectively NDV, avian influenza virus) and (this enforcement of metal-sol liquidCase is taking silver sol as example) mix with 1:2～3:1 ratio;
2) get 2～5 μ L steps 1) on sample drop quartz plate after mixing, natural air drying;
3) the round point shape sample forming on quartz plate is carried out to Raman detection, Raman instrument is SmartRamanOcean5pro Portable Raman spectrometer, the parameter of test is: power is 15～120mW, be the time of integration1～10s, excitation wavelength is 785nm;
4) record spectrogram, by principal component analysis and linear discriminant analysis, two-strain is carried out to modeling classification;
5) judged result.
As shown in Figure 1-Figure 3, Fig. 1 is the Raman of GM strain of the present invention and H5N6 strain to testing resultSpectral results figure. Fig. 1 a is the Raman figure of GM strain, and 1b is the Raman figure of H5N6 strain. Two figure are mainPeak band 1206cm-1(Tyr),1130cm-1υ(C-C),1003cm-1(Phe),885cm-1(Gly),635cm-1(Tyr)And 592cm-1(Gly) all belong to the molecule of amino acid, protein and nucleic acid. 4. 3. 2. 1. dash area beGM strain and H5N6 strain main difference peak position in Raman spectrogram. As can be seen here, GM strain withThere is obvious difference in the Raman spectrum of H5N6 strain, can carry out principal component and linear discriminant divides according to differenceAnalyse, to set up the Mathematical Modeling of differentiating this two-strain.
Principal component analysis is in conjunction with linear discriminant analysis result as shown in Fig. 2-Fig. 3, and Fig. 2 is GM poison of the present inventionStrain and H5N6 strain Raman spectrogram PCA-LDA (Principalcomponentsanalysis-linearDiscriminateanalysis) two-dimensional process figure. Star represents GM strain sample (30) as seen from the figure, circlePoint represents H5N6 strain sample (24). Calculate the score of each principal component according to the spectrogram information of all samples,Mapped by main component 1 and main component 2 and linear analysis method, obtain on two-strain X-Y schemePCA-LDA curve, Raman spectrum reaches 86% in conjunction with PCA-LDA differentiation accuracy H5N6 as seen from the figure(20/24), GM reaches 100% (30/30).
Fig. 3 is GM strain of the present invention and H5N6 strain Raman spectrogram PCA-LDA (PrincipalComponentsanalysis-lineardiscriminateanalysis) three-dimensional process figure. Star representative as seen from the figureGM strain sample (30), round dot represents H5N6 strain sample (24). According to the spectrum of all samplesFigure information is calculated the score of each principal component, by main component 1, main component 2 and main component 3 and linear dividingAnalyse method mapping, obtain the PCA-LDA curve on two-strain graphics, Raman spectrum combination as seen from the figurePCA-LDA differentiates accuracy 100%.
As can be seen here, use silver sol as surface Raman enhancement agent, can fast, accurately distinguish H5N6Strain and GM strain. Be significant for these two kinds of deadly infectious diseases of clinical rapid differential diagnosis.
Above-described embodiment is preferably embodiment of the present invention, but embodiments of the present invention are not subject to above-mentioned enforcementThe restriction of example, other any do not deviate from change, the modification done under Spirit Essence of the present invention and principle, replacesGeneration, combination, simplification, all should be equivalent substitute mode, within being included in protection scope of the present invention.

Claims (7)

1. a detection kit for fast detecting poultry diease virus, is characterized in that: comprise metal-sol liquid,Quartz plate and Raman spectrometer;
Described metal-sol liquid is aurosol liquid or silver sol liquid;
The preparation method of described silver sol is as follows: under normal temperature by 2.5mL10-2mol/LAgNO3JoinIn 75mL distilled water, add 5mLof10-2Mol/L TGA; After stirring 10min, splash into 2.5mL10-2Mol/LNaI, stirs after 20min, obtains jade-green AgI colloidal sol, adds 20mgNaBH4Reduction,Continue stir about 10min, generate yellowish-brown silver sol, what obtain is the spheroidal particle of shape homogeneous.
2. the detection kit of fast detecting poultry diease virus according to claim 1, is characterized in that:Described quartz plate is that wave-number range is 700-2000cm-1Without the substrate quartz plate of obvious Raman peaks.
3. the detection kit of fast detecting poultry diease virus according to claim 1, is characterized in that:Described quartz plate is sheet glass.
4. the detection kit of fast detecting poultry diease virus according to claim 1, is characterized in that:Described Raman spectrometer is SmartRamanOcean5pro Portable Raman spectrometer.
5. the detection kit of fast detecting poultry diease virus according to claim 4, is characterized in that:Described Raman spectrometer testing conditions is: optical maser wavelength 785nm, laser power is 15-120mW, integrationTime is that 1-10s, wave-number range are 700-2000cm-1。
6. the kit described in application rights requirement 1～5 any one carries out the detection method of poultry diease virus, its spyLevy and be: comprise the following steps:
1) testing sample and metal-sol liquid are mixed with 1:2～3:1 ratio;
2) get 2～5 μ L steps 1) on sample drop quartz plate after mixing, natural air drying;
3) the round point shape sample forming on quartz plate is carried out to Raman detection, Raman instrument is SmartRamanOcean5pro Portable Raman spectrometer, the parameter of test is: power is 15～120mW, be the time of integration1～10s, excitation wavelength is 785nm;
4) record spectrogram, by principal component analysis and linear discriminant analysis, two-strain is carried out to modeling classification;
5) judged result.
7. kit according to claim 6 carries out the detection method of poultry diease virus, it is characterized in that:Testing environment temperature is at 4～40 DEG C.
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CN111118137A (en) * 2018-11-01 2020-05-08 广东中科康仪生物技术有限公司 Gene detection method and kit for thrombocytasthenia
WO2020136376A1 (en) * 2018-12-24 2020-07-02 Cell Therapy Catapult Limited Methods for determining viral titre using raman spectroscopy
CN112525885A (en) * 2020-12-01 2021-03-19 吉林大学 Detect reagent box of trichina muscle larva fast
WO2022109833A1 (en) * 2020-11-25 2022-06-02 深圳先进技术研究院 Method for constructing model for detecting novel coronavirus nucleic acids on basis of surface-enhanced infrared spectroscopy and principal component analysis
CN115184339A (en) * 2022-09-08 2022-10-14 海澳华(黑龙江)生物医药技术有限公司 Method for rapidly detecting viruses based on portable Raman spectrometer

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Publication number Priority date Publication date Assignee Title
CN110546484A (en) * 2017-02-03 2019-12-06 社会福祉法人三星生命公益财团 On-site instant system and method for diagnosing acute febrile disease
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CN111118137A (en) * 2018-11-01 2020-05-08 广东中科康仪生物技术有限公司 Gene detection method and kit for thrombocytasthenia
WO2020136376A1 (en) * 2018-12-24 2020-07-02 Cell Therapy Catapult Limited Methods for determining viral titre using raman spectroscopy
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GB2580186B (en) * 2018-12-24 2021-09-15 Cell Therapy Catapult Ltd Monitoring viral titre using Raman Spectroscopy
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WO2022109833A1 (en) * 2020-11-25 2022-06-02 深圳先进技术研究院 Method for constructing model for detecting novel coronavirus nucleic acids on basis of surface-enhanced infrared spectroscopy and principal component analysis
CN112525885A (en) * 2020-12-01 2021-03-19 吉林大学 Detect reagent box of trichina muscle larva fast
CN115184339A (en) * 2022-09-08 2022-10-14 海澳华(黑龙江)生物医药技术有限公司 Method for rapidly detecting viruses based on portable Raman spectrometer

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