CN105603125B - A kind of detection kit and its detection method of quick detection poultry diease virus - Google Patents
A kind of detection kit and its detection method of quick detection poultry diease virus Download PDFInfo
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- CN105603125B CN105603125B CN201610134356.3A CN201610134356A CN105603125B CN 105603125 B CN105603125 B CN 105603125B CN 201610134356 A CN201610134356 A CN 201610134356A CN 105603125 B CN105603125 B CN 105603125B
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Abstract
The invention discloses the detection kits and its detection method of a kind of quickly detection poultry diease virus, belong to spectroscopy and molecular diagnosis field.The present invention is using the silver sol of hydroxylamine hydrochloride complexometry preparation as Raman Contrast agent, silver sol with certain proportion and article to be measured after mixing, drop is on quartz plate, after natural air drying, Raman detection is carried out using SmartRaman Ocean5pro Portable Raman spectrometer, after Raman spectrum obtained is by principal component analysis and linear discriminant analysis, the qualitative detection to target pathogen is realized.The detection kit is easy to operate, high sensitivity, has a good application prospect.
Description
Technical field
The invention belongs to spectroscopy and molecular diagnosis field, in particular to a kind of detection reagent of quickly detection poultry diease virus
Box and its detection method.
Background technique
Newcastle disease virus (Newcastle Disease Virus, NDV), is commonly called as philippine fowl disease, can cause chicken group diarrhea,
Expiratory dyspnea and nervous symptoms.Avian influenza virus (Avian Influenza Virus, AIV) includes multiple hypotypes, and wherein H5 is sub-
Type avian influenza virus can cause a large amount of morbidities of fowl group and death, A class infectious disease is classified as by World Organization for Animal Health, by height
Degree is paid attention to.The propagation of newcastle disease virus and avian influenza virus in fowl group is actively monitored and controlled, aquaculture can not only be reduced
Loss, moreover it is possible to largely reduce avian influenza virus from fowl group and be broadcast to the risk of people, there is important public health peace
Full meaning.
Laboratory testing means mainly have pathogeny detection, cause of disease Serologic detection, immunoassay method and molecule at present
Biological detection method.The separation identification of virus is to make a definite diagnosis that this disease is most definite, method of most public credibility, but experimental implementation step
It is rapid more lengthy and jumbled, take compared with；Enzyme-linked immunosorbent assay technology in immunoassay method, i.e. ELISA are current antigen-antibody inspections
In survey field, the immunological detection method being most widely used, specificity and sensibility with higher, but price is more high
It is expensive；Fluorescence quantitative RT-RCR (Reverse Transcription-Polymerase Chain Reaction) technology has height
The sensibility and specificity of degree, and it is substantially shorter the detection time to virus, it overcomes in conventional serological diagnostic method
The interference and sensitive question of non-specific factors provide sensitive, quick, practical detection side for Rapid&Early diagnosis virus
Method, but step is complex, it is desirable to provide corresponding laboratory equipment.Therefore it is easy to operate urgently to establish one kind, quickly, spirit
Quick, lossless high-throughput detection method.
Surface enhanced Raman spectroscopy scattering (Surface Enhanced Raman Scattering, SERS) refers to when certain
When a little Molecular Adsorptions are to the precious metal surface with nanometer rough overshoot, it will not increase in the Raman scattering signal that these extra coarses obtain
It is up to a million times strong, it is even higher.Thus obtained spectrum is referred to as " Surface enhanced Raman spectroscopy ".For other spectrum,
Such as fluorescence spectrum, infrared spectroscopy etc., Surface enhanced Raman spectroscopy has narrow high sensitivity, bands of a spectrum, anti-light bleaching, is not done by water
The features such as disturbing, and can be realized quick, real-time, non-destructive testing, therefore be highly suitable for biomedical research.
Summary of the invention
To overcome the problems, such as that existing detection technique needs expensive apparatus preparation professional technician, of the invention is primary
It is designed to provide a kind of detection kit of quickly detection poultry diease virus.The detection kit uses surface-enhanced Raman
Spectral technique is provided with a kind of kit of detection virus easy to operate, high sensitivity.
Another object of the present invention is to provide the detection methods of mentioned reagent box.
The purpose of the invention is achieved by the following technical solution: a kind of detection kit of quickly detection poultry diease virus, packet
Include metal-sol liquid, quartz plate and Raman spectrometer.
The metal-sol liquid is aurosol liquid or silver sol liquid.
The silver sol the preparation method is as follows: by 2.5mL 10 under room temperature-2mol/L AgNO3It is added to 75mL distillation
In water, 5mL of 10 is added-2Mol/L thioacetic acid；2.5mL10 is instilled after stirring 10min-2Mol/L NaI stirs 20min
Afterwards, jade-green AgI colloidal sol is obtained, 20mg NaBH is added4Reduction continues stir about 10min, generates yellowish-brown silver sol, obtains
To be the uniform spheroidal particle of shape, average grain diameter about 35nm, concentration is about 120 μ g/mL.
The quartz plate is that wave-number range is 700-2000cm-1Base ceramic piece without obvious Raman peaks；
The quartz plate can also have sheet glass replacement.
The Raman spectrometer is SmartRaman Ocean5pro Portable Raman spectrometer.
The Raman spectrometer testing conditions are as follows: optical maser wavelength 785nm, laser power 15-120mW, the time of integration
It is 700-2000cm for 1-10s, wave-number range-1。
The detection method of poultry diease virus is carried out using mentioned reagent box, comprising the following steps:
1) by sample to be tested and metal-sol liquid with the mixing of 1:2~3:1 ratio；
2) on the sample drop quartz plate after taking 2~5 μ L steps 1) to mix, natural air drying；
3) Raman detection, Raman instrument SmartRaman are carried out to the round point shape sample formed on quartz plate
Ocean5pro Portable Raman spectrometer.The parameter of test are as follows: power is 15~120mW, and the time of integration is 1~10s, excitation
Wavelength is 785nm；
4) spectrogram is recorded, modeling of class is carried out to two kinds of viruses by principal component analysis and linear discriminant analysis；
5) judging result.
The detection environment temperature of above-mentioned detection method is at 4~40 DEG C.
Above-mentioned virus is usually that inside has nucleic acid (DNA or RNA), and outside has the albumen of capsid, and outermost layer has capsule
Film or without cyst membrane structure.
The poultry diease virus includes other avian virals such as newcastle disease virus, avian influenza virus.
The present invention has the following advantages and effects with respect to the prior art:
The present invention establishes the method and detection kit of a kind of quickly detection poultry diease virus.This method is easy to operate, specific
By force, time-consuming short, the Raman spectrogram of poultry diease virus can be quickly detected, is counted through the chemistry such as principal component analysis and linear discriminant analysis
Amount method can obviously distinguish different poultry diease virus, provide the foundation of optics spectrogram for quickly detection poultry diease virus, have
Good application prospect.
Detailed description of the invention
Fig. 1 is the Raman spectrum result figure of GM strain and H5N6 strain of the invention.
Fig. 2 is GM strain and H5N6 strain Raman spectrogram PCA-LDA (Principal components of the present invention
Analysis-linear discriminate analysis) two-dimensional process figure.
Fig. 3 is GM strain and H5N6 strain Raman spectrogram PCA-LDA (Principal components of the present invention
Analysis-linear discriminate analysis) three-dimensional process figure.
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
The sample that the present invention uses is avian influenza virus H5N6 strain (A/Oriental Magpie-robin/
Guangdong/SW8/2014 (H5N6), gene pool number: KT454936), newcastle disease virus GM strain (gene pool number:
DQ486859), strain is provided by poultry diease teaching and research room, Agricultural University Of South China, Guangdong Province.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Embodiment 1
The present invention provides a kind of detection kit of quickly detection poultry diease virus, including metal-sol liquid, quartz plate with
Raman spectrometer.
The metal-sol liquid is silver sol liquid.
The silver sol the preparation method is as follows: by 2.5mL 10 under room temperature-2mol/L AgNO3It is added to 75mL distillation
In water, 5mL of 10 is added-2Mol/L thioacetic acid.2.5mL10 is instilled after stirring 10min-2Mol/L NaI stirs 20min
Afterwards, jade-green AgI colloidal sol is obtained, 20mg NaBH is added4Reduction continues stir about 10min, generates yellowish-brown silver sol, obtains
To be the uniform spheroidal particle of shape, average grain diameter about 35nm, concentration is about 120 μ g/mL.
The quartz plate is that wave-number range is 700-2000cm-1Base ceramic piece without obvious Raman peaks.
The Raman spectrometer is SmartRaman Ocean5pro Portable Raman spectrometer.
The Raman spectrometer testing conditions are as follows: optical maser wavelength 785nm, laser power 100mW, the time of integration be
3s, wave-number range are 700-2000cm-1。
The detection method of poultry diease virus is carried out using mentioned reagent box, comprising the following steps:
1) by sample to be tested (respectively including newcastle disease virus, avian influenza virus) and metal-sol liquid (the implementation case with
For silver sol) with the mixing of 1:2~3:1 ratio；
2) on the sample drop quartz plate after taking 2~5 μ L steps 1) to mix, natural air drying；
3) Raman detection, Raman instrument SmartRaman are carried out to the round point shape sample formed on quartz plate
Ocean5pro Portable Raman spectrometer, the parameter of test are as follows: power is 15~120mW, and the time of integration is 1~10s, excitation
Wavelength is 785nm；
4) spectrogram is recorded, modeling of class is carried out to two kinds of viruses by principal component analysis and linear discriminant analysis；
5) judging result.
Testing result is as shown in Figure 1-Figure 3, and Fig. 1 is the Raman spectrum result figure of GM strain and H5N6 strain of the invention.
Fig. 1 a is the Raman figure of GM strain, and 1b is the Raman figure of H5N6 strain.The main peak band 1206cm of two figures-1(Tyr),1130cm-1
υ(C-C),1003cm-1(Phe),885cm-1(Gly),635cm-1(Tyr) and 592cm-1(Gly) amino acid, albumen are belonged to
The molecule of matter and nucleic acid.1. 2. 3. 4. dash area is GM strain and H5N6 strain main difference peak position in raman spectrum.
It can be seen that the Raman spectrum of GM strain and H5N6 strain there are apparent difference, can carry out principal component and linear according to difference
Discriminant analysis, to establish the mathematical model for differentiating both viruses.
For principal component analysis combination linear discriminant analysis result as shown in Fig. 2-Fig. 3, Fig. 2 is GM strain and H5N6 poison of the present invention
Strain Raman spectrogram PCA-LDA (Principal components analysis-linear discriminate
Analysis) two-dimensional process figure.Star represents GM strain sample (30) as seen from the figure, and dot represents H5N6 strain sample (24
It is a).The score that each principal component is calculated according to the spectrogram information of all samples, by main component 1 and main component 2 and linear analysis
Method mapping obtains the PCA-LDA curve on two kinds of viral X-Y schemes, and Raman spectrum combination PCA-LDA differentiates correct as seen from the figure
Rate H5N6 reaches 86% (20/24), and GM reaches 100% (30/30).
Fig. 3 is GM strain and H5N6 strain Raman spectrogram PCA-LDA (Principal components of the present invention
Analysis-linear discriminate analysis) three-dimensional process figure.Star represents GM strain sample as seen from the figure
(30), dot represent H5N6 strain sample (24).The score that each principal component is calculated according to the spectrogram information of all samples, by
Main component 1, main component 2 and main component 3 and liner fraction mapping, obtain the PCA-LDA on two kinds of viral three-dimensional figure
Curve, Raman spectrum combination PCA-LDA differentiates accuracy 100% as seen from the figure.
It can be seen that use silver sol as surface Raman enhancement agent, can quickly, accurately distinguish H5N6 strain and GM poison
Strain.For clinical rapid differential diagnosis, both deadly infectious diseases are of great significance.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (4)
1. a kind of application of silver sol in preparation detection poultry diease virus agent box, it is characterised in that: the detection kit packet
Include metal-sol liquid, quartz plate and Raman spectrometer；
The metal-sol liquid is silver sol liquid；
The poultry diease virus is avian influenza virus or newcastle disease virus；The silver sol the preparation method is as follows: under room temperature
By 2.5mL 10-2mol/L AgNO3It is added in 75mL distilled water, 5mL 10 is added-2Mol/L thioacetic acid；After stirring 10min
Instill 2.5mL 10-2Mol/L NaI after stirring 20min, obtains jade-green AgI colloidal sol, 20mg NaBH is added4Reduction, after
Continuous stir about 10min, generates yellowish-brown silver sol, what is obtained is the uniform spheroidal particle of shape；
The kit application the following steps are included:
1) by sample to be tested and metal-sol liquid with the mixing of 1:2~3:1 ratio；
2) on the sample drop quartz plate after taking 2~5 μ L steps 1) to mix, natural air drying；
3) Raman detection carried out to the round point shape sample that is formed on quartz plate, Raman instrument be SmartRaman Ocean5pro just
Take formula Raman spectrometer, the parameter of test are as follows: power is 15~120mW, and the time of integration is 1~10s, excitation wavelength 785nm；
4) spectrogram is recorded, modeling of class is carried out to two kinds of viruses by principal component analysis and linear discriminant analysis；
5) judging result.
2. application according to claim 1, it is characterised in that: the quartz plate is that wave-number range is 700-2000cm-1
Base ceramic piece without obvious Raman peaks.
3. application according to claim 1, it is characterised in that: the quartz plate is sheet glass.
4. application according to claim 1, it is characterised in that: detection environment temperature is at 4~40 DEG C.
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Families Citing this family (6)
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| KR102047989B1 (en) | 2017-02-03 | 2019-11-22 | 사회복지법인 삼성생명공익재단 | Point of care testing system and method for diagnosing acute febrile disease |
| 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 |
| GB2580186B (en) * | 2018-12-24 | 2021-09-15 | Cell Therapy Catapult Ltd | Monitoring viral titre using Raman Spectroscopy |
| 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 |
| CN115184339B (en) * | 2022-09-08 | 2022-12-23 | 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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| CN105092559A (en) * | 2015-08-19 | 2015-11-25 | 华南农业大学 | SERS-based newcastle disease virus detection kit and detection method thereof |
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| US20110037976A1 (en) * | 2009-08-17 | 2011-02-17 | Yiping Zhao | Flexible surface enhanced raman spectroscopy (sers) substrates, methods of making, and methods of use |
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| CN104406954A (en) * | 2014-11-26 | 2015-03-11 | 中国科学院微生物研究所 | Surface enhanced Raman spectra substrate for detecting avian influenza virus, and application thereof |
| CN105092559A (en) * | 2015-08-19 | 2015-11-25 | 华南农业大学 | SERS-based newcastle disease virus detection kit and detection method thereof |
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