CN105936943A - Rapid and sensitive method for detection of Marburg virus RT-LAMP - Google Patents
Rapid and sensitive method for detection of Marburg virus RT-LAMP Download PDFInfo
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Abstract
The invention relates to a rapid and sensitive method for detection of Marburg virus RT-LAMP. The detection method of the invention employs specific primers design, loop mediated isothermal amplification (LAMP) technique, and fluorescent visual reagent for simple and fast detection of amplification products. The method includes: specimen RNA extraction, RT-LAMP amplification, and interpretation of results. The method can conduct fast and sensitive detection of Marburg virus, uses simple equipment and operations and has less testing time. The invention can greatly improve the detection efficiency of the inspection and quarantine officers in the front line of import and export ports, and can reduce the workload, and solve the positive leak detection in the traditional detection method by the maximum, so as to maximally prevent the introduction of Marburg virus.
Description
Technical field
The present invention relates to external diagnosis reagent technical field, be specifically related to the Marburg virus RT-of a kind of rapid sensitive
LAMP detection method.
Background technology
Along with global economic integration and liberalization of trade, external Medical Vectors by the advanced vehicles and
International trade, can be transmitted to the whole world infectious disease from a country rapidly, cause international propagation.In recent years, newly send out
Viral infectious is frequent outbreak of epidemic in countries in the world, and human health is brought serious threat, causes tremendous economic to society
Loss.The severe acute respiratory syndrome (SARS) of such as 2003 is popular Countries and area, China in 2004 and
The bird flu of surrounding countries is popular, global influenza A H1N1 epidemic situation in 2009, and Ebola's epidemic situation in Africa in 2014 gives society
The life and health of stable, economic construction and people brings great impact.Sick the taking place frequently to me of these neopathy toxoinfections
Beaten alarm bell, strengthen the research to it and monitoring with prevention and control early it is critical that.
Marburg hemorrhagic fever is the deadly infectious disease that a kind of fatality rate caused by Marburg virus is high.Marburg virus
Collectively form filamentous virus with Ebola virus to belong to, but belong to two different germlines.The earliest about marburg hemorrhagic thermal explosion
Record is at Marburg, Germany in 1967.It is reported and have 31 people to infect virus morbidity at that time, wherein 7 people are dead.These 31 infection
Having 25 people in the people of virus is to infect the monkey of Marburg virus and pathogenic owing to contacting.The most often betide some Africa
Country, has occurred and that 12 epidemic situations, a most serious epidemic situation betided Angola in 2004 to 2005 years up to now, accumulative
Reported cases 374 example, the most dead 329 examples.MARV is mainly propagated by close contact, and case fatality rate is 23%～90%, at present
Also there is no commercialized vaccine.
Marburg virus full-length genome about 19kb, for sub-thread minus-stranded rna virus.7 kinds of virus proteins of genome encoding, bag
Include nucleoprotein (NP), stromatin VP24 and VP40, structural protein VP30 and VP35, the RNA polymerase (L albumen) of dependenc RNA,
Glycoprotein 7 (gp7).
The detection method of Marburg virus mainly has viral Isolation and identification, serum neutralization test, enzyme connection to exempt from present
Epidemic disease absorption detection (ELISA), detection of nucleic acids etc..For viral disease, virus purification and culture identification are most reliable
Detection means.But above-mentioned virus purification need to be carried out under 4 grades of (P4) laboratory conditions of bio-safety, and required time is longer,
Laboratory and experimenter are required high, is not also suitable for extensively applying and the rapid screening of frontier port.In serum and examination
Testing is the main method of virus serology detection, but needs also exist for carrying out in P4 level laboratory.Simultaneously the epidemic situation of 2005
Finding in detection that the serology such as ELISA, IgM or immunologic detection method sensitivity are relatively low, false negative rate is high, only core
The means sensitivity of acid detection has obtained consistent accreditation, but the PCR of routine operation is complicated, and equipment is huge, skill
Art difficulty is higher, is unfavorable for port field diagnostic and epidemic-stricken area Site Detection.Therefore a kind of novel fast in the urgent need to research and development
Nucleic acid detection method fast, simple.Patent of the present invention is by a kind of reverse transcription loop-mediated isothermal amplified reaction (RT-LAMP) of research
Method detection Marburg virus.
Ring mediated isothermal amplification method is a kind of novel constant-temperature nucleic acid amplification method, it is characterized in that for six on target nucleic acid
Individual section design 4 kinds or 6 kinds of primers, then utilize strand replacement reaction to react at a certain temperature.In 4 kinds of primers 2
Its 3 ' end of kind of inner primer and 5 ' ends can identify 2 zoness of different in target nucleic acid sequence respectively, and be designed to its 5 ' terminal sequence can be with
Complementary strand regional annealing extension synthesized by initial by 3 ' ends combines.Inner primer can form loop-stem structure, and carries out certainly
My extension, has new inner primer annealed combination to carry out strand displacement synthetic reaction subsequently in circulus position.The two is constantly
Circulation repeats, it is achieved amplified reaction.Therefore, even if only a kind enzyme, ring mediated isothermal amplification method can also realize constant-temperature amplification.
During with RNA for template, only need to add reverse transcriptase in advance, so that it may a step completes synthesis and the amplification process (reverse of cDNA
Record loop-mediated isothermal amplification).
The feature of ring mediated isothermal amplification gene amplification method is while synthesizing a large amount of nucleic acid, also can generate a large amount of by-product
Thing pyrophosphate ion.Calcein contained in fluorescence visual observation agent is initially in fluorescent quenching shape because being combined with manganese ion
State.But along with the carrying out of loop-mediated isothermal amplification, because seized the manganese ion of combination by byproduct of reaction pyrophosphate ion,
Calcein recovers free thus sends fluorescence.And then combine with the magnesium ion in reactant liquor, make fluorescence be strengthened.According to
This principle, easily can judge whether to there occurs ring mediated isothermal amplification by fluorescence visual detection.
Isothermal duplication (LAMP) method of ring mediation possesses quick, efficient, high specificity, sensitivity advantages of higher.LAMP
Method speed is fast, the highest to requirement of experiment, it is only necessary to a water-bath, it is not necessary to sterile working, the most permissible
Detect a large amount of sample, be suitable to laboratory serum diagnosis, customs quarantine control and extensive epidemic disease generaI investigation.Additionally, LAMP detection method compares
Sensitive, and result is the most reliable.
Molecular Biological Detection for Marburg virus is mainly by round pcr at present, and the present invention is according to Marburg
Specific gene fragment V35 of virus, devises a group-specific primers, and the method using RT-LAMP can be short half
Hour to detecting amplified fragments in one hour, as long as a thermostat water bath in operation, method is simply, highly sensitive, time
Between short.The detection method of the present invention can operate with the detection of Marburg virus rapid sensitive, as the method for port rapid screening,
Improve the accuracy and ageing of detection, the cycle of detection can be greatly reduced.
Summary of the invention
One of the technical problem to be solved is to provide the Marburg virus RT-LAMP detection of a kind of rapid sensitive
Method, the present invention can carry out the detection of rapid sensitive, can increase substantially import and export by the present invention Marburg virus gene
The detection efficiency of port one X-ray inspection X quarantine functionary, not only can reduce workload, but also can solve traditional detection method to greatest extent
Positive missing inspection problem that may be present, thus prevent the generation of epidemic situation and the incoming of external Marburg virus to greatest extent.
The Marburg virus RT-LAMP detection method of rapid sensitive provided by the present invention, comprises the steps:
(1) whole blood sample carries out RNA liquid extraction；
(2) RT-LAMP amplification:
A.RT-PCR reaction system includes: 2 × reaction buffer (RM), sample to be tested gene-specific primer, dd H2O,
Enzymatic solution, RNA extracting solution, the visual reagent of fluorescence；
B. PCR reactant liquor is added PCR pipe, put into thermostatical instrument or the LAMP detector of 63 DEG C, the reaction of isothermal reaction
Condition: 63 DEG C of reaction 60min.；
(3) result interpretation: negative control color is brown, positive sample is green, if with LAMP detector, having bright
Aobvious amplification curve.
RNA described in step (1) extracts and blood sample geneome RNA can be used to extract test kit extraction RNA.Take
100ul whole blood sample, extracts RNA according to the extraction step in test kit description；
PCR reaction system described in step (2) is: 2X reaction buffer 12.5 μ l, specific primer mixed liquor 2.5 μ
L, enzyme liquid 1.0 μ l, dd H2O 3.0 μ l, fluorescence visual observation agent 1.0 μ l, RNA extracting solution 5 μ l.
Sample to be tested gene-specific primer described in step (2) is for Marburg virus peculiar gene VP35 gene to be
Target gene, each three pairs of primers based on loop-mediated isothermal amplification technology design: inner primer FIP/BIP, outer primer F3/B3, ring draw
Thing LF/LB, compares VP35 gene sequence in multiple Marburg virus, and the one section of design choosing homology the highest is drawn
Thing can improve the specificity of Marburg virus detection.Three pairs of specific primers are:
Inner primer-FIP:5 '-GTTTCCTCACTGAGTTCAAGGACCCGGTTAAAAATGCAACAACAG-3 ' (SEQ
ID NO 1)
Inner primer-BIP:5 '-CAAGCCAAACCTCTCAGCCAGGATGTGGAATGGAGTGT-3 ' (SEQ ID NO 2)
Outer primer-F3:5 '-AGCTTGTAGTAAGGGGACTA-3 ' (SEQ ID NO 3)
Outer primer-B3:5 '-GTAAGCAATTTTTGAAAGGACC-3 ' (SEQ ID NO 4)
Ring primer-LF:5 '-ACATCTTGTCGGCTGCAT-3 ' (SEQ ID NO 5)
Ring primer-LB:5 '-TTACCCATCTACCCGGCAA-3 ' (SEQ ID NO 6)
The present invention is directed to the peculiar gene of Marburg virus, design three is to specific primer so that the detection method of the present invention
The accuracy rate of detection Marburg virus gene is higher, can detect Marburg virus sensitive, specifically, with conventional PCR method ratio
Relatively, detection sensitivity and specificity are obviously higher than conventional method.The detection method of the present invention is possible not only to meet daily epidemic situation
The requirement of safety detection, it is also possible to meet the testing requirement of clinical sample.The present invention is compared with existing detection method, and reaction only needs
Can will complete in a small-sized LAMP amplification instrument or water bath with thermostatic control, testing result can be detected by the method for range estimation
Arriving, time and the complexity of detection is greatly reduced, detection equipment is simply easy to carry about with one, and substantially increases current detection energy
Power.And can complete in 1 hour completely total time from sample collecting to detection, it is of value to and detects Marburg fast and accurately
Poison.
Accompanying drawing explanation
Fig. 1 is embodiment 1 Marburg virus RT-LAMP testing result.
Left side figure is the detection signal intensity that detects at LAMP amplification instrument of pipe of the right figure, and right figure is that naked-eye observation is arrived
Color changes, the copies/ul of the digitized representation above right figure.
Fig. 2 is that embodiment 1 Marburg virus RT-LAMP detection method Marburg virus V35 positive sample detects specificity
Result.
A left side is different sample amplification product fluorescence intensities, and the right side is different block diagram sample names.
Fig. 3 is embodiment 1 Marburg virus RT-LAMP detection method sensitivity experiments result.
A left side is the concentration of specimens of each bar diagram, and the right side is the amplified production fluorescence intensity of variable concentrations sample.
Fig. 4 is that embodiment 1 Marburg virus RT-LAMP detection method detects timing result.
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 restriction the scope of the present invention.In addition, it is to be understood that after having read the content that the present invention lectures, people in the art
The present invention can be made various changes or modifications by member, and these equivalent form of values fall within the application appended claims equally and limited
Scope.
The specificity of embodiment 1 Marburg virus RT-LAMP detection method and susceptiveness
One, experimental procedure
1. the external synthesis of Marburg virus, Ebola virus RNA
Utilize and analyze the disclosed Marburg virus Angola pnca gene group sequence (Ang delivered through bioinformatics method
1379c), synthetic Marburg virus VP35 fragment Han specific detection (about 600bp) is cloned in PCR cloning vector
On (TOPO TA cloning kit), by extraction of plasmid DNA kits plasmid DNA, check order after confirming that Insert Fragment is correct
Carry out in vitro transcription: first make linearisation to get rid of downstream RNA product with Not I digested plasmid, then use MAXIscript T7Kit
Carry out in vitro transcription, generate purpose RNA, then carry out DNase process with TurboDNase, remove the plasmid DNA do not transcribed
Template, finally precipitates with ethanol, purifies RNA product, be positive control RNA template, be stored in-70 DEG C of ultra cold storage freezers and treat
With.Same method obtains the RNA fragment of Ebola virus VP35.
2. the collection of blood sample
Taking the whole blood sample 1.0ml of port detection patient, centrifugal treating obtains serum.
3. the extraction of geneome RNA
Use blood sample geneome RNA to extract test kit (centrifugal column type, Qiagen company) and extract dengue fever RNA, Huang
Hot RNA, west nile virus RNA.Take 100uL serum sample, extract RNA according to the extraction step in test kit description.
The mensuration of 4.RNA concentration
Measured the concentration of RNA fragment by Nanodrop 2000c, and calculate the copy of RNA according to the size of RNA fragment
Number.
5. the amplification of target gene
The design of 5.1 primers
Each three designed with Marburg virus peculiar gene VP35 gene as target gene, based on loop-mediated isothermal amplification technology
To primer: inner primer FIP/BIP, outer primer F3/B3, ring primer LF/LB, to VP35 gene sequence in multiple Marburg virus
Row compare, and choose one section of the highest design primer of homology and can improve the specificity of Marburg virus detection.Three pairs special
Property primer is:
Inner primer-FIP:5 '-GTTTCCTCACTGAGTTCAAGGACCCGGTTAAAAATGCAACAACAG-3 ' (SEQ
ID NO 1)
Inner primer-BIP:5 '-CAAGCCAAACCTCTCAGCCAGGATGTGGAATGGAGTGT-3 ' (SEQ ID NO 2)
Outer primer-F3:5 '-AGCTTGTAGTAAGGGGACTA-3 ' (SEQ ID NO 3)
Outer primer-B3:5 '-GTAAGCAATTTTTGAAAGGACC-3 ' (SEQ ID NO 4)
Ring primer-LF:5 '-ACATCTTGTCGGCTGCAT-3 ' (SEQ ID NO 5)
Ring primer-LB:5 '-TTACCCATCTACCCGGCAA-3 ' (SEQ ID NO 6)
5.2 reaction system
Expanding on LAMP amplification instrument, amplification system is: 2X reaction buffer 12.5 μ l, specific primer mixed liquor
2.5 μ l, enzyme liquid 1.0 μ l, dd H2O 3.0 μ l, fluorescence visual observation agent 1.0 μ l, RNA extracting solution 5 μ l.
The reaction condition of isothermal reaction: 63 DEG C of reaction 60min.
6. result interpretation
A, expands on LAMP amplification instrument, is judged by amplification block diagram
B, expands in thermostatical instrument, is judged by the change of amplification system color, and negative control is brown, and the positive is
Green.
7. detection system specificity
According to the mensuration of RNA concentration, obtain two Marburg virus V35 nucleic acid of 10E+6copies/ul Yu 1copies/ul
Fragment concentrations sample, respectively with the Marburg virus VP35 gene RNA of two concentration, Ebola virus V35 genetic fragment, Dengue
Fever virus RNA, yellow fever virus RNA, west nile virus RNA are that template detects according to 5.2 steps, observe its specificity.
8. system susceptiveness detection
Each dilution factor is measured by the method according to 5.2, and calculates the Monitoring lower-cut of detection method.
9. the detection time: according to the curve of detection, determine the detection time
Two, result
1. the power of test of Marburg virus
Being tested by the Marburg RNA sample of external synthesis, result is shown in Fig. 1.It is found through experiments, from 1copy/ul
To 109The sample of copies/ul may detect that, and testing result is anti-with perusal by machine fluorescence intensity
The result of variations answering liquid color is consistent.Absolutely prove that the method is simple to operate, as long as there being a water-bath to can be carried out reaction,
And observed result.Compared with gathering fluorescence intensity with machine, may be accurate not as machine.
2. sample specific test:
As Fig. 2 shows, two Marburg virus V35 nucleic acid fragment 10E+6copies/ul Yu 1copies/ul height two
Concentration all can detect, and other viral RNAs and Ebola's synthetic V35 nucleic acid fragment are all not detected by.
3, sensitivity experiments
Detect 100copies/ul, 10copies/ul, 1copies/ul, 0.1copies/ul, 0.01copies/ respectively
The sample of ul, 0.001copies/ul and 0.0001copies/ul difference diluted concentration gradient, carries out method sensitivity experiments,
As Fig. 3 shows, the sample of 1copies/ul concentration can be detected, sample size is 5ul, Monitoring lower-cut 5copies.
4, detect timing
When 10, the sample of 100copies/ul at 28min time can detect, and 1copy/ul sample can when 42min
To detect, it is specifically shown in Fig. 4.Additionally other data display 10E+6copies/ul sample even can be able to be examined when 16min
Measure, have fabulous ageing.
Claims (7)
1. the Marburg virus RT-LAMP detection method of a rapid sensitive, it is characterised in that: comprise the steps:
(1) whole blood sample carries out RNA liquid extraction；
(2) RT-LAMP amplification:
A.RT-PCR reaction system includes: 2 × reaction buffer, three pairs of specific primers, dd H2O, enzymatic solution, and RNA extracts
Liquid, the visual reagent of fluorescence；
B. PCR reactant liquor is added PCR pipe, put into LAMP detector and expand；
(3) result interpretation；
Wherein said specific primer is as target gene, based on loop-mediated isothermal amplification with Marburg virus VP35 genetic fragment
Each three pairs of primers of Technology design: inner primer FIP/BIP, outer primer F3/B3, ring primer LF/LB, three pairs of specific primers are:
Inner primer-FIP:5 '-GTTTCCTCACTGAGTTCAAGGACCCGGTTAAAAATGCAACAACAG-3 ' (SEQ ID NO
1)
Inner primer-BIP:5 '-CAAGCCAAACCTCTCAGCCAGGATGTGGAATGGAGTGT-3 ' (SEQ ID NO 2)
Outer primer-F3:5 '-AGCTTGTAGTAAGGGGACTA-3 ' (SEQ ID NO 3)
Outer primer-B3:5 '-GTAAGCAATTTTTGAAAGGACC-3 ' (SEQ ID NO 4)
Ring primer-LF:5 '-ACATCTTGTCGGCTGCAT-3 ' (SEQ ID NO 5)
Ring primer-LB:5 '-TTACCCATCTACCCGGCAA-3 ' (SEQ ID NO 6).
The Marburg virus RT-LAMP detection method of a kind of rapid sensitive the most according to claim 1, it is characterised in that:
RNA described in step (1) extracts and uses blood sample geneome RNA to extract test kit extraction Marburg virus, and sample size is
100ul whole blood.
The Marburg virus RT-LAMP detection method of a kind of rapid sensitive the most according to claim 1, it is characterised in that:
PCR reaction system described in step (2) is: 2X reaction buffer 12.5 μ l, specific primer mixed liquor 2.5 μ l, enzyme liquid 1.0
μ l, dd H2O 3.0 μ l, fluorescence visual observation agent 1.0 μ l, RNA extracting solution 5 μ l.
The Marburg virus RT-LAMP detection method of a kind of rapid sensitive the most according to claim 1, it is characterised in that:
Described PCR amplification is carried out in thermostatical instrument.
The Marburg virus RT-LAMP detection method of a kind of rapid sensitive the most according to claim 1, it is characterised in that:
Described PCR amplification isothermal reaction condition is, 63 DEG C of reaction 60min.
The Marburg virus RT-LAMP detection method of a kind of rapid sensitive the most according to claim 1, it is characterised in that:
Described detection method Monitoring lower-cut 5copies.
The Marburg virus RT-LAMP detection method of a kind of rapid sensitive the most according to claim 1, it is characterised in that:
10, the sample of 100copies/ul detects when 28min, and 1copy/ul sample detects when 42min.
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Cited By (2)
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| CN106636469A (en) * | 2017-01-12 | 2017-05-10 | 中国人民解放军军事医学科学院微生物流行病研究所 | RPA technology-based marburg virus detection kit and application thereof |
| CN109504808A (en) * | 2019-01-11 | 2019-03-22 | 福建出入境检验检疫局检验检疫技术中心 | Monkey Marburg virus real-time fluorescent RT-PCR method for detecting |
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| WO2006076650A2 (en) * | 2005-01-12 | 2006-07-20 | Applera Corporation | Compositions, methods, and kits for selective amplification of nucleic acids |
| CN102140531A (en) * | 2010-12-24 | 2011-08-03 | 中国检验检疫科学研究院 | Novel Marburg virus fluorescent quantitative PCR (Polymerase Chain Reaction) detection method and Marburg virus PCR detection system |
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| CN106636469A (en) * | 2017-01-12 | 2017-05-10 | 中国人民解放军军事医学科学院微生物流行病研究所 | RPA technology-based marburg virus detection kit and application thereof |
| CN109504808A (en) * | 2019-01-11 | 2019-03-22 | 福建出入境检验检疫局检验检疫技术中心 | Monkey Marburg virus real-time fluorescent RT-PCR method for detecting |
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