CN102268489A - Non-diagnostic method for detecting chikungunya virus nucleic acid through nanogold fluorescence quantitative PCR (Polymerase Chain Reaction) - Google Patents
Non-diagnostic method for detecting chikungunya virus nucleic acid through nanogold fluorescence quantitative PCR (Polymerase Chain Reaction) Download PDFInfo
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- CN102268489A CN102268489A CN2011102422860A CN201110242286A CN102268489A CN 102268489 A CN102268489 A CN 102268489A CN 2011102422860 A CN2011102422860 A CN 2011102422860A CN 201110242286 A CN201110242286 A CN 201110242286A CN 102268489 A CN102268489 A CN 102268489A
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Abstract
The invention discloses a non-diagnostic method for detecting chikungunya virus nucleic acid through nanogold fluorescence quantitative PCR (Polymerase Chain Reaction). The method comprises the following steps of: determining concentration of nanogold added into a reaction system; comparing capacities of detecting the chikungunya virus nucleic acid before and after the addition of the nanogold; and guaranteeing that cross reaction is not performed between the nanogold and other viruses.
Description
Technical field
The present invention relates to a kind of non-diagnostic methods of utilizing nanometer gold fluorescence quantitative PCR detection Chikungunya virus nucleic acid.
Background technology
Along with nanotechnology is constantly used in life science, the nano material that more and more has peculiar property be it is found that gradually.Nanometer gold (AuNPs) is widely used a kind of small gold grain in the nanotechnology, and its diameter has high electron density, dielectric characteristics and katalysis at 1～100nm, can combine with multiple biomacromolecule, and not influence its biological activity.Because having in fields such as bioelectrochemistry, materialogy and biomedicines very widely, this special physics, chemical property and biocompatibility, gelationus golden nanometer particle use.
In recent years, there is the scholar to find that nanometer gold can improve the amplification efficiency of PCR system.The Taiwan success LiM of university etc. has reported the amplification efficiency that nanometer gold can improve the PCR system, a series of subsequently research application and the very fast expansion of Exploration on mechanism of optimizing PCR about nanometer gold; (2009) such as Lee Hai Kuo utilize the genomic low copy gene template of nano Au particle amplifying human, have realized the amplification to low copy gene in the complex system, have improved the productive rate of specific amplification; Shen Hebai etc. (2005) are connected the nano Au particle surface with primer, and research is based on the PCR of nano Au particle, with the PCR technological expansion in the homogeneous system to nanoparticle surface.Simultaneously, Li etc. (2005) utilizes the nano Au particle of 10 nm to significantly improve the specificity of PCR and improves non-specific amplification.
Nanometer gold is applied in the quantitative fluorescent PCR reaction preliminary research has been arranged also.Huang etc. (2008) add the nano Au particle of 13 nm in the real-time quantitative reverse transcription PCR reaction system of japanese encephalitis virus RNA after, not only can improve reverse transcription PCR output, reduce proliferation time, and improved the detection sensitivity of low copy viral RNA gene.
Summary of the invention
The object of the present invention is to provide a kind of raising amplification efficiency, increase detection sensitivity, the non-diagnostic methods of the nanometer gold fluorescence quantitative PCR detection Chikungunya virus nucleic acid of technical support is provided for the accurate rapid detection of virus disease.
For achieving the above object, the non-diagnostic methods of nanometer gold fluorescence quantitative PCR detection Chikungunya virus nucleic acid of the present invention adds an amount of nanometer gold in reaction system, come the real-time fluorescence quantitative PCR reaction system is carried out catalysis and optimization.
Further, described nanometer gold is the 10nm nanometer gold.
Further, the concrete steps of described method comprise: the nucleic acid that 1) extracts virus; 2) determine nano-Au solution concentration in the reaction system, set up nanometer gold quantitative fluorescent PCR reaction system; 3) by detecting unknown sample, detection yellow fever virus, dengue fever virus, japanese encephalitis virus are verified the validity of this reaction system.
Further, described real-time fluorescence quantitative PCR reaction system amplification is specially the 25 μ L PCR reaction systems that concentration of component is set in preparation.
Further, in described real-time fluorescence quantitative PCR reaction system, the fluorophor that connects on its probe comprises FAM-BHQ, FAM-TAMARA.
Further, described real-time fluorescence quantitative PCR reaction system is applicable to ABI real-time PCR system, BioRad PCR in real time detection system, Stratagene quantitative polumerase chain reaction instrument.
The present invention utilizes the katalysis of nanometer gold to real-time fluorescence quantitative PCR, adds an amount of 10nm nanometer gold in its reaction system, at existing primer and probe, optimizes the PCR reaction system, Virus Sample is carried out real-time quantitative detect.This method improves, improves amplification efficiency compared to general fluorescence quantifying PCR method on sensitivity detects, increase detection sensitivity, for the accurate rapid detection of virus disease provides technical support.
Description of drawings
Fig. 1 is for after adding the nanometer gold of different concns in the quantitative fluorescent PCR reaction, and viral RNA amplification fluorescent intensity is with the graphic representation of reaction cycle number variation;
Fig. 2 is nanometer gold quantitative fluorescent PCR (A represents) and the graphic representation that does not add the detected result of nanometer gold quantitative fluorescent PCR (B represents) sensitivity;
The graphic representation that Fig. 3 detects the RNA of unknown sample for the method set up with the present invention;
Fig. 4 carries out the graphic representation of cross reaction detection to the sample rna of yellow fever virus, dengue fever virus, japanese encephalitis virus for the method for setting up with the present invention.
Embodiment
Below, with reference to the accompanying drawings, the present invention is more fully illustrated, exemplary embodiment of the present invention has been shown in the accompanying drawing.Yet the present invention can be presented as multiple multi-form, and should not be construed as the exemplary embodiment that is confined to narrate here.But, these embodiment are provided, thereby make the present invention, and scope of the present invention is fully conveyed to those of ordinary skill in the art comprehensively with complete.
Basic skills among the present invention: the method for fluorescence quantitative PCR detection Chikungunya virus nucleic acid, corresponding basic method steps and primer, probe are wherein disclosed, still can adopt above-mentioned primer, probe among the present invention.The fluorophor that connects on the described probe comprises FAM-BHQ, FAM-TAMARA etc.
Secondly detection method of the present invention comprises the step that detects Chikungunya virus nucleic acid, and this step is specially:
(1) nucleic acid extraction
Viral RNA extracts and selects for use
QIAamp Mini kit 52906Viral RNA extracts test kit;
(2) determine nano-Au solution concentration in the reaction system
The amplification kit of selecting for use is the AgPath-ID of ABI company
TMOne-step RT-PCR Kit,
Reaction system component and volume thereof see Table 1.
Annotate: X represents the different concns of Radioactive colloidal gold.
Table 1
| Component | Volume (μ L) |
| Upstream primer (40 μ M) | 0.25 |
| Downstream primer (40 μ M) | 0.25 |
| Probe (10 μ M) | 0.30 |
| X (10nm) Radioactive colloidal gold | 1.00 |
| 2×RT-PCR buffer | 12.50 |
| 25×RT-PCRenzymeMix | 0.5 |
| Sample rna | 5.00 |
| Nuclease free water | Complement to 25.00 |
Amplification condition:
For example
1:
Determining of nanometer gold concentration
1) selects Chikungunya virus nutrient solution 1 * 10
2Pfu/mL extracts RNA and detects, and the Radioactive colloidal gold concentration of adding 10nm in the reaction system is respectively 0.095nM, 0.19nM, 0.38nM, 0.76nM, 1.52nM.
2) negative control: normal mosquitoes is organized RNA;
3) reaction system is prepared according to table 1;
Detected result is shown in Fig. 1, table 2.Among Fig. 1, X-coordinate is a cycle index, the corresponding back report fluorescence intensity (Rn) of proofreading and correct of ordinate zou representative.
Table 2
| 10nm Radioactive colloidal gold concentration (nM) | The CT value |
| 0.095nM | 32.25 |
| 0.19nM | 32.32 |
| 0.38nM | 32.61 |
| 0.76nM | 33.10 |
| 1.52nM | 33.02 |
| NTC | — |
Among Fig. 1,5 curves are respectively the amplification curve of the 10nm Radioactive colloidal gold gained that adds 0.095nM, 0.19nM, 0.38nM, 0.76nM, 1.52nM in the nanometer gold quantitative fluorescent PCR reaction system.
Data shown in the table 2 are respectively the CT value of nanometer gold fluorescence quantitative PCR method adding different concns colloidal gold solution amplification gained among the present invention.The negative contrast of NTC, negative findings is represented with "-".
Can find from the result shown in Fig. 1, the table 2, the colloidal gold solution that adds different concns in the reaction of nanometer gold quantitative fluorescent PCR has certain influence to amplification, when the Radioactive colloidal gold concentration of interpolation 10nm was 0.095 nM in the system, amplification curve is outlet the earliest, and the CT value is also minimum.In view of the above, select to add the colloidal gold solution of 0.095 nM.
:
The nanometer gold quantitative fluorescent PCR with do not add nanometer gold fluorescence quantitative PCR detection remolding sensitivity
1) selects Chikungunya virus nutrient solution 1 * 10
2Pfu/mL extracts RNA and detects, and the Radioactive colloidal gold concentration of adding 10nm in the nanometer gold fluorescence quantitative PCR method reaction system is 0.095 nM.Then do not add colloidal gold solution in the common fluorescence quantitative PCR method.
2) negative control: normal mosquitoes is organized RNA;
3) reaction system is prepared according to table 1;
Detected result is shown in Fig. 2, table 3.
Among Fig. 2, select for use chikungunya disease strain nutrient solution as template, the A curve detects the curve that viral RNA increases for the colloidal gold solution that fluorescence quantitative PCR method among the present invention adds 10nm; The B curve detects the curve of viral RNA amplification for the fluorescence quantitative PCR method that does not add nanometer gold among the present invention.Wherein, X-coordinate is a cycle index, the corresponding back report fluorescence intensity (Rn) of proofreading and correct of ordinate zou representative.
From experimental result as can be seen, the outlet of A curve ratio B curve early, nanometer gold fluorescence quantitative PCR method CT value is 32.25, is 35.39 and do not add nanometer gold fluorescence quantitative PCR method CT value.
Table 3
| Viral RNA | Nanometer gold fluorescence quantitative PCR method CT value | Common fluorescence quantitative PCR |
| 1×10 2pfu/mL | 32.25 | 35.39 |
3: the confirmatory reaction system:
(1): detect unknown sample
1) unknown sample of gathering is detected, the screening Chikungunya virus is monitored of the present invention non-specific simultaneously;
2) positive control: virus-culturing fluid (1 * 10
2Pfu/mL) RNA;
3) reaction system is prepared according to table 1.
Detected result is seen Fig. 3 and table 4.Among Fig. 3, X-axis is represented cycle index, and Y-axis is represented the corresponding back report fluorescence intensity of proofreading and correct.
Only occurred a positive contrast of amplification curve among Fig. 3, all the other sample standard deviations do not have amplification.Reaction result is negative.The positive contrast of PTC.
Data presentation in the table 4, it is all negative to detect the unknown sample result.
Table 4
| The unknown sample numbering | The CT value |
| LW01 | — |
| LW02 | — |
| LW03 | — |
| LW04 | — |
| LW05 | — |
| LW06 | — |
| LW07 | — |
| LW08 | — |
| LW09 | — |
| LW10 | — |
| ML01 | — |
| ML02 | — |
| ML10 | — |
| PTC | 32.25 |
| NTC | — |
(2): detect japanese encephalitis virus, dengue fever virus, yellow fever virus;
1) detected object: be respectively the positive mosquito matchmaker sample of japanese encephalitis virus, dengue fever virus and yellow fever virus detection of nucleic acids;
2) positive control: virus-culturing fluid (1 * 10
2Pfu/mL) RNA;
3) negative control: normal mosquitoes sample rna;
4) reaction system is prepared according to table 1.
Detected result is seen Fig. 4 and table 5.Among Fig. 4, X-axis is represented cycle index, and Y-axis is represented the corresponding back report fluorescence intensity of proofreading and correct.
Table 5 japanese encephalitis virus, dengue fever virus, yellow fever virus detected result
| Pathogenic agent | The CT value |
| Japanese encephalitis virus | — |
| Dengue fever virus | — |
| Yellow fever virus | — |
| PTC | 32.25 |
| NTC | — |
Interpretation of result:
Amplified reaction is finished after 40 circulations, and when the nanometer gold concentration of adding in the reaction system of the present invention was 0.095nM, amplification curve occurred the earliest, has shifted to an earlier date with the amplification efficiency of the reaction that does not add nanometer gold.The present invention has added the nanometer gold reaction in original reaction system sensitivity obviously strengthens; The unknown sample detected result is all negative; With other viral no cross reactions.
The result judges:
1) when the Ct(of sample to be detected threshold value)≤be judged to the positive 36 the time;
2) when sample 36 ﹤ Ct value ﹤ 40 to be detected, need to detect again, still at 36 ﹤ Ct value ﹤ 40, then be judged to the positive as the result, otherwise negative.
Among the present invention, sample to be measured comprises viral powder, goods to be imported and exported etc., also can comprise mosquito, bat, human serum and tissue (brain, liver and lymphoglandula etc.), cell culture fluid, wild primates, domestic animal etc.The non-diagnostic methods of nanometer gold fluorescence quantitative PCR detection Chikungunya virus nucleic acid of the present invention, be applicable to that ABI real-time PCR system, BioRad PCR in real time detection system, Stratagene quantitative polumerase chain reaction instrument finish, reaction conditions is different and change to some extent according to the model of instrument.
Claims (6)
1. the non-diagnostic methods of nanometer gold fluorescence quantitative PCR detection Chikungunya virus nucleic acid is characterized in that, this non-diagnostic methods adds an amount of nanometer gold in reaction system, comes the real-time fluorescence quantitative PCR reaction system is carried out catalysis and optimization.
2. non-diagnostic methods as claimed in claim 1 is characterized in that, described nanometer gold is the 10nm nanometer gold.
3. non-diagnostic methods as claimed in claim 1 is characterized in that, the concrete steps of described method comprise: the nucleic acid that 1) extracts virus; 2) determine nano-Au solution concentration in the reaction system, set up nanometer gold quantitative fluorescent PCR reaction system; 3) by detecting unknown sample, detection yellow fever virus, dengue fever virus, japanese encephalitis virus are verified the validity of this reaction system.
4. non-diagnostic methods as claimed in claim 3 is characterized in that, described real-time fluorescence quantitative PCR reaction system amplification is specially the 25 μ L PCR reaction systems that concentration of component is set in preparation.
5. non-diagnostic methods as claimed in claim 4 is characterized in that, in described real-time fluorescence quantitative PCR reaction system, the fluorophor that connects on its probe comprises FAM-BHQ, FAM-TAMARA.
6. non-diagnostic methods as claimed in claim 1 is characterized in that, described real-time fluorescence quantitative PCR reaction system is applicable to ABI real-time PCR system, BioRad PCR in real time detection system, Stratagene quantitative polumerase chain reaction instrument.
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| CN106834550A (en) * | 2017-04-18 | 2017-06-13 | 福建省农业科学院畜牧兽医研究所 | A kind of avian influenza virus nano PCR detection kit and its application |
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| CN101270394A (en) * | 2008-05-05 | 2008-09-24 | 广东出入境检验检疫局检验检疫技术中心 | Chikungunya virus testing method |
| WO2010097806A1 (en) * | 2009-02-25 | 2010-09-02 | Bigtec Private Limited | Probes and primers for detection of chikungunya |
| CN102140530A (en) * | 2010-12-24 | 2011-08-03 | 中国检验检疫科学研究院 | New chikungunya virus fluorescence quantitative polymerase chain reaction detection method and chikungunya virus polymerase chain reaction detection system |
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| CN101270394A (en) * | 2008-05-05 | 2008-09-24 | 广东出入境检验检疫局检验检疫技术中心 | Chikungunya virus testing method |
| WO2010097806A1 (en) * | 2009-02-25 | 2010-09-02 | Bigtec Private Limited | Probes and primers for detection of chikungunya |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106834550A (en) * | 2017-04-18 | 2017-06-13 | 福建省农业科学院畜牧兽医研究所 | A kind of avian influenza virus nano PCR detection kit and its application |
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Application publication date: 20111207 |