CN110042173A - Nano-gold biosensor and detection method a kind of while that detect four kinds of hemorrhagic fever viruses - Google Patents

Nano-gold biosensor and detection method a kind of while that detect four kinds of hemorrhagic fever viruses Download PDF

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Publication number
CN110042173A
CN110042173A CN201910148421.1A CN201910148421A CN110042173A CN 110042173 A CN110042173 A CN 110042173A CN 201910148421 A CN201910148421 A CN 201910148421A CN 110042173 A CN110042173 A CN 110042173A
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pad
gold
dna probe
nano
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曾令文
赵锦
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Guangzhou Institute of Biomedicine and Health of CAS
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Guangzhou Institute of Biomedicine and Health of CAS
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6825—Nucleic acid detection involving sensors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes

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Abstract

The invention discloses a kind of nano-gold biosensors and detection method for detecting four kinds of hemorrhagic fever viruses simultaneously.The sensor includes substrate, sample pad, absorption pad, nitrocellulose membrane and bonding pad, and the sample pad and absorption pad are fixed on the both ends of the substrate, and the nitrocellulose membrane is fixed on the middle part of the substrate；There is the overlapping of 2-3mm between the absorption pad and the nitrocellulose membrane, has the overlapping of 2-3mm between the nitrocellulose membrane and the bonding pad, there is the overlapping of 2-3mm between the bonding pad and the sample pad；There is gold nanoparticle DNA probe on the bonding pad.The present invention has high sensitivity, specificity height, detection time short, and the advantage detected, testing cost is low can be completed in 60 minutes.

Description

A kind of while four kinds of hemorrhagic fever viruses of detection nano-gold biosensors and detection Method
Technical field
The present invention relates to field of biotechnology, and in particular to a kind of nano gold biological for detecting four kinds of hemorrhagic fever viruses simultaneously Sensor and detection method.
Background technique
Fever virus multiplicity, infectivity and harm it is huge, 2014 outburst lethal more than 7000 people of Ebola virus, it is comprehensive Lethality is closed up to 50% or more.The dengue virus infection number of the same year Guangdong Province outburst surpasses 40000 people.It is directed to Hemorrhagic fever both at home and abroad Viral diagnosis is mainly separately cultured, Electronic Speculum observation, the methods of immunofluorescence and RT-PCR, and previous diagnostic method existed The disadvantages of journey complexity, poor repeatability, inadequate and at high cost sensitivity.
Summary of the invention
In view of the deficiencies of the prior art, the present invention is intended to provide a kind of nanogold of four kinds of hemorrhagic fever viruses of detection simultaneously is raw Object sensor and detection method utilize magnetic Nano separation, the isothermal strand displacement amplification and nanometer gold test paper strip of nicking enzyme auxiliary Technology, the diagnosis prevention and control ability that easy can be detected simultaneously four kinds of hemorrhagic fever viruses quickly, sensitive and accurate, improve to virus.
To achieve the goals above, the present invention adopts the following technical scheme:
It is a kind of at the same detect four kinds of hemorrhagic fever viruses nano-gold biosensor, including substrate, sample pad, absorption pad, Nitrocellulose membrane and bonding pad, the sample pad and absorption pad are fixed on the both ends of the substrate, the nitrocellulose membrane It is fixed on the middle part of the substrate；There is the overlapping of 2-3mm between the absorption pad and the nitrocellulose membrane, the nitrification is fine There is the overlapping of 2-3mm between dimension film and the bonding pad, there is the overlapping of 2-3mm between the bonding pad and the sample pad； There is gold nanoparticle DNA probe on the bonding pad；The gold nanoparticle DNA probe includes:
Hantaan virus gold nanoparticle DNA probe: S-n-GGACAACTTCCACTTAGGGGA；
Chikungunya fever virus gold nanoparticle DNA probe: S-n-CTACAGCCCCATGGTACTGG；
Dengue fever virus gold nanoparticle DNA probe: S-n-GCTGAAACGCGAGAGAAACC；
Ebola virus gold nanoparticle DNA probe: S-n-TCCAACAGCTTGGCAATCAGT.
The present invention also provides a kind of detection method of nano-gold biosensor for detecting four kinds of hemorrhagic fever viruses simultaneously, packets Include following steps:
After S1, pathogen cracking, RNA, which is extracted, uses magnetic nanoparticle enriched nucleic acid, the further progress after elution Reverse transcription；
S2, reverse transcription product reuse magnetic nanoparticle and are screened, and the nucleic acid finally eluted is added to It is placed into warm strand displacement amplification reaction system in 37 DEG C of water-baths, after the reaction of 30min, amplified production is as detection sample Product are added drop-wise in sample pad, and detect by an unaided eye result after 5min.
The beneficial effects of the present invention are:
1, high sensitivity of the present invention, up to 0.01pM；
2, present invention specificity is high, can distinguish single base；
3, detection time of the present invention is short, and detection can be completed in 60 minutes；
4, testing cost is low, and as a result naked eyes are as it can be seen that convenient and efficient.
Detailed description of the invention
Fig. 1 is that a kind of structure of nano-gold biosensor for detecting four kinds of hemorrhagic fever viruses simultaneously prepared by the present invention is shown It is intended to；
Fig. 2 is the gel electrophoresis figure of SDA amplified production；
Fig. 3 is a kind of specificity of nano-gold biosensor for detecting four kinds of hemorrhagic fever viruses simultaneously prepared by the present invention Analyze result schematic diagram；
Fig. 4 is a kind of sensitivity of nano-gold biosensor for detecting four kinds of hemorrhagic fever viruses simultaneously prepared by the present invention Analyze result schematic diagram.
Specific embodiment
Below with reference to attached drawing, the invention will be further described, it should be noted that the present embodiment is with this technology side Premised on case, the detailed implementation method and specific operation process are given, but protection scope of the present invention is not limited to this reality Apply example.
Embodiment 1
The present embodiment provides a kind of nano-gold biosensors for detecting four kinds of hemorrhagic fever viruses simultaneously, including substrate 1, sample Product pad 2, absorption pad 3, nitrocellulose membrane 4 and bonding pad 5, the sample pad 2 and absorption pad 3 are fixed on the two of the substrate 1 End, the nitrocellulose membrane 4 are fixed on the middle part of the substrate 1；Between the absorption pad 3 and the nitrocellulose membrane 4 There is the overlapping of 2-3mm, there is the overlapping of 2-3mm, the bonding pad 5 and institute between the nitrocellulose membrane 4 and the bonding pad 5 State the overlapping for having 2-3mm between sample pad 2；There is gold nanoparticle DNA probe on the bonding pad 5；The gold nanoparticle DNA probe includes:
Hantaan virus gold nanoparticle DNA probe: S-n-GGACAACTTCCACTTAGGGGA；
Chikungunya fever virus gold nanoparticle DNA probe: S-n-CTACAGCCCCATGGTACTGG；
Dengue fever virus gold nanoparticle DNA probe: S-n-GCTGAAACGCGAGAGAAACC；
Ebola virus gold nanoparticle DNA probe: S-n-TCCAACAGCTTGGCAATCAGT.
It is viral every particular by searching four kinds on NCBI it should be noted that above-mentioned gold nanoparticle DNA probe Kind of genotype compares what design primer after its conserved sequence obtained:
The primer and probe of 1 Hantaan virus of table design
The primer and probe of 2 chikungunya fever virus of table design
The primer and probe of 3 dengue fever virus of table design
The primer and probe of 4 Ebola virus of table design
Embodiment 2
Described in embodiment 1 while four kinds of hemorrhagic fever viruses of detection nano gold biological biographies are utilized the present embodiment provides a kind of The detection method of sensor, includes the following steps:
After S1, pathogen cracking, RNA, which is extracted, uses magnetic nanoparticle enriched nucleic acid, the further progress after elution Reverse transcription；
S2, reverse transcription product reuse magnetic nanoparticle and are screened, and the nucleic acid finally eluted is added to 37 DEG C of water-baths are placed into warm strand displacement amplification reaction (strand displacement amplification, SDA) system In, after the reaction of 30min, amplified production is added drop-wise in sample pad as test sample, and can detect by an unaided eye knot after 5min Fruit.
Embodiment 3
The present embodiment verifies the performance of nano-gold biosensor provided by embodiment 1 by experiment.
1, testing principle amplified production running gel is verified
It is feasible in order to verify isothermal strand displacement amplification system, four kinds of viral recombinant plasmids are used herein, and plasmid is diluted For 6 concentration gradients 1 × 1012、1×108、1×106、1×104、1×102、1×101Copy/μ L, is added to isothermal strand displacement Amplified production is added on 3% Ago-Gel by amplification reaction system after 30min reacts, and is used after 100V 30min Gel imager observes result (as shown in Figure 2).
Fig. 2 is the gel electrophoresis figure of SDA amplified production.It is from left to right respectively with 1 × 1012、1×108、1×106、1× 104、1×102、1×101Copy/μ L positive plasmid is the gel electrophoresis figure of amplified production that template carries out that SDA is expanded, 0 For blank control (PBS).A is Hantaan virus；B is chikungunya fever virus；C is dengue fever virus；D is Ebola virus.Knot Fruit shows that isothermal strand displacement amplification reacts the detection that can be used for hemorrhagic fever viruse.
2, specificity analysis
In order to analyze nano-gold biosensor described in embodiment 1 hemorrhagic fever viruse detection specificity, respectively to receiving Rice gold biosensor instills the 90 μ L of serum of dengue fever virus and Hantaan virus, Chikungunya fever and Ebola virus positive mark Quasi- product each 1 × 104Each 90 μ L of copy/μ L positive plasmid mixed liquor.As shown in figure 3, nano-gold biosensor detects four The different hemorrhagic fever viruse of kind, is from left to right positive control, Hantaan virus, Chikungunya fever, dengue fever, Ebola respectively； Every kind of viral detection is not interfered mutually as the result is shown, and specificity is high, and positive control and negative control result also demonstrate utilization Nano-gold biosensor described in embodiment 1 can detect four kinds of hemorrhagic fever viruses simultaneously.
3, sensitivity analysis
In order to verify the sensitivity that nano-gold biosensor detects four kinds of different hemorrhagic fever viruses, respectively to embodiment Nano-gold biosensor described in 1 instills 1 × 1012、1×106、1×104、1×102、1×101Copy/μ L positive plasmid with And each 90 μ L of blank control (PBS).As a result as shown in figure 4, the nano-gold biosensor detects four kinds of different Hemorrhagic fevers Virus, A are Hantaan virus；B is chikungunya fever virus；C is dengue fever virus；D is Ebola virus.The biography as the result is shown Copy number is converted into Concentration Testing sensitivity up to 0.01pM by the high sensitivity of sensor.
For those skilled in the art, it can be provided various corresponding according to above technical solution and design Change and modification, and all these change and modification, should be construed as being included within the scope of protection of the claims of the present invention.

Claims (2)

1. a kind of nano-gold biosensor for detecting four kinds of hemorrhagic fever viruses simultaneously, which is characterized in that including substrate, sample Pad, absorption pad, nitrocellulose membrane and bonding pad, the sample pad and absorption pad are fixed on the both ends of the substrate, the nitre Change the middle part that fiber membrane is fixed on the substrate；There is the overlapping of 2-3mm between the absorption pad and the nitrocellulose membrane, There is the overlapping of 2-3mm between the nitrocellulose membrane and the bonding pad, has 2- between the bonding pad and the sample pad The overlapping of 3mm；There is gold nanoparticle DNA probe on the bonding pad；The gold nanoparticle DNA probe includes:
Hantaan virus gold nanoparticle DNA probe: S-n-GGACAACTTCCACTTAGGGGA；
Chikungunya fever virus gold nanoparticle DNA probe: S-n-CTACAGCCCCATGGTACTGG；
Dengue fever virus gold nanoparticle DNA probe: S-n-GCTGAAACGCGAGAGAAACC；
Ebola virus gold nanoparticle DNA probe: S-n-TCCAACAGCTTGGCAATCAGT.
2. it is a kind of using described in claim 1 while detection four kinds of hemorrhagic fever viruses nano-gold biosensor detection side Method, which comprises the steps of:
After S1, pathogen cracking, RNA, which is extracted, uses magnetic nanoparticle enriched nucleic acid, and further progress inverts after elution Record；
S2, reverse transcription product reuse magnetic nanoparticle and are screened, and the nucleic acid finally eluted is added to isothermal chain It is placed into 37 DEG C of water-baths in displacement amplification reaction system, after the reaction of 30min, amplified production is dripped as test sample It is added in sample pad, detect by an unaided eye result after 5min.
CN201910148421.1A 2019-02-28 2019-02-28 Nano-gold biosensor and detection method a kind of while that detect four kinds of hemorrhagic fever viruses Pending CN110042173A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110628955A (en) * 2019-11-04 2019-12-31 中国人民解放军军事科学院军事医学研究院 CrRNA target and CRISPR-Cas13a system for detecting Ebola virus

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006088478A2 (en) * 2004-06-18 2006-08-24 Novartis Vaccines And Diagnostics Inc. Methods and reagents for diagnosing hantavirus infection
CN101942386A (en) * 2009-07-08 2011-01-12 中国科学院广州生物医药与健康研究院 Nucleic acid nanogold biosensor and preparation method thereof
CN102230032A (en) * 2011-06-27 2011-11-02 广州弗赛生物科技有限公司 Isothermal chain multiple detection card of pathogen nucleic acid
CN103725798A (en) * 2014-01-15 2014-04-16 中国人民解放军南京军区军事医学研究所 Primer, kit and detection method of detecting haemorrhagic fever with renal syndrome virus by RT-LAMP (Reverse Transcription Loop-Mediated Isothermal Amplification) method
CN104313179A (en) * 2014-09-26 2015-01-28 浙江省疾病预防控制中心 Isothermal amplification detection kit and detection method for Zaire type Ebola virus
CN105087824A (en) * 2015-03-04 2015-11-25 浙江省疾病预防控制中心 Preparation and application of hemorrhagic fever associated pathogen identifying gene chip
CN105400904A (en) * 2015-12-18 2016-03-16 中国人民解放军疾病预防控制所 RPA kit used for detecting Ebola virus, and special-purpose primers, probes, and applications of RPA kit

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006088478A2 (en) * 2004-06-18 2006-08-24 Novartis Vaccines And Diagnostics Inc. Methods and reagents for diagnosing hantavirus infection
CN101942386A (en) * 2009-07-08 2011-01-12 中国科学院广州生物医药与健康研究院 Nucleic acid nanogold biosensor and preparation method thereof
CN102230032A (en) * 2011-06-27 2011-11-02 广州弗赛生物科技有限公司 Isothermal chain multiple detection card of pathogen nucleic acid
CN103725798A (en) * 2014-01-15 2014-04-16 中国人民解放军南京军区军事医学研究所 Primer, kit and detection method of detecting haemorrhagic fever with renal syndrome virus by RT-LAMP (Reverse Transcription Loop-Mediated Isothermal Amplification) method
CN104313179A (en) * 2014-09-26 2015-01-28 浙江省疾病预防控制中心 Isothermal amplification detection kit and detection method for Zaire type Ebola virus
CN105087824A (en) * 2015-03-04 2015-11-25 浙江省疾病预防控制中心 Preparation and application of hemorrhagic fever associated pathogen identifying gene chip
CN105400904A (en) * 2015-12-18 2016-03-16 中国人民解放军疾病预防控制所 RPA kit used for detecting Ebola virus, and special-purpose primers, probes, and applications of RPA kit

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
赵锦等: "采用等温链置换扩增结合纳米金测流层析试纸条检测登革热病毒", 《浙江大学学报(医学版)》 *
郑夔等: "应用含内参的多重实时荧光RT-PCR方法快速检测登革病毒和基孔肯雅病毒", 《中国人兽共患病学报》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110628955A (en) * 2019-11-04 2019-12-31 中国人民解放军军事科学院军事医学研究院 CrRNA target and CRISPR-Cas13a system for detecting Ebola virus
CN110628955B (en) * 2019-11-04 2023-04-07 中国人民解放军军事科学院军事医学研究院 CrRNA target and CRISPR-Cas13a system for detecting Ebola virus

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Application publication date: 20190723