CN105936943B - A kind of Marburg virus RT-LAMP detection method of rapid sensitive - Google Patents
A kind of Marburg virus RT-LAMP detection method of rapid sensitive Download PDFInfo
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Abstract
The present invention relates to a kind of Marburg virus RT-LAMP detection methods of rapid sensitive.Detection method of the invention is by design specific primer, isothermal duplication (LAMP) technology mediated by ring, using the visual reagent of addition fluorescence, can be simple and quick detect amplified production；Detection method includes: that sample RNA is extracted, RT-LAMP amplification, as a result interpretation.The present invention can carry out the detection of rapid sensitive, instrument and equipment and easy to operate, detection time section to Marburg virus.The detection efficiency of first-line inspection and quarantine personnel at import and export ports can be increased substantially through the invention, not only workload can have been reduced but also can have solved the problems, such as traditional detection method positive missing inspection that may be present to the maximum extent, to prevent the incoming of Marburg virus to the maximum extent.
Description
Technical field
The present invention relates to technical field of in vitro diagnostic reagents, and in particular to a kind of Marburg virus RT- of rapid sensitive
LAMP detection method.
Background technique
With global economic integration and liberalization of trade, external Medical Vectors by the advanced vehicles and
Infectious disease can be transmitted to the whole world from a country rapidly, cause international propagation by international trade.In recent years, new hair
Viral infectious brings human health and seriously threatens in the frequent outbreak of epidemic in countries in the world, causes tremendous economic to society
Loss.Such as 2003 serious acute respiratory syndrome (SARS) Countries and area prevalence, China in 2004 and
The bird flu of surrounding countries is popular, and global Influenza A H1N1 epidemic situation in 2009, African Ebola's epidemic situation in 2014 is to society
The life and health of stable, economic construction and people bring great influence.These neopathy toxoinfection diseases take place frequently to me
Beaten alarm bell, it is vital for reinforcing research to it and monitoring with prevention and control early.
Marburg hemorrhagic fever is a kind of deadly infectious disease that lethality is high as caused by Marburg virus.Marburg virus
Filamentous virus category is collectively formed with Ebola virus, but belongs to two different germlines.Earliest in relation to marburg hemorrhagic thermal explosion
Record is in Marburg, Germany in 1967.It is reported that having the morbidity of 31 people's virus infections at that time, wherein 7 people are dead.This 31 infection
It is pathogenic due to contacting the monkey of infection Marburg virus for having 25 people in the people of virus.Some Africa are often betided later
Country has occurred and that 12 epidemic situations, an epidemic situation of most serious betided Angola in 2004 to 2005 years so far, adds up
Reported cases 374, wherein dead 329.MARV is mainly propagated by close contact, and case fatality rate is 23%~90%, at present
There are no commercialized vaccines.
Marburg virus full-length genome about 19kb is sub-thread minus-stranded rna virus.7 kinds of virus proteins of genome encoding, packet
Include nucleoprotein (NP), stromatin VP24 and VP40, structural proteins VP30 and VP35, dependenc RNA RNA polymerase (L albumen),
Glycoprotein 7 (gp7).
The detection method of Marburg virus mainly has viral Isolation and identification, serum neutralization test, enzyme-linked exempts from present
Epidemic disease absorption detection (ELISA), detection of nucleic acids etc..For viral disease, virus purification is most reliable with culture identification
Detection means.But above-mentioned virus purification need to carry out under 4 grades of bio-safety (P4) laboratory conditions, and required time is longer,
Laboratory and experimenter are required it is high, be not suitable for yet be widely applied and frontier port rapid screening.In serum and try
Test be virus serology detection main method, but also need to carry out in P4 grades of laboratories.Simultaneously in epidemic situation in 2005
Find that the serology such as ELISA, IgM or immunologic detection method sensitivity are lower in detection, false negative rate is high, only core
The means sensitivity of acid detection has obtained consistent approval, but conventional PCR is complicated for operation, and equipment is huge, skill
Art difficulty is relatively high, is unfavorable for port field diagnostic and epidemic-stricken area on-site test.Therefore a kind of novel fast there is an urgent need to research and develop
Fast, simple nucleic acid detection method.The invention patent is by studying a kind of reverse transcription loop-mediated isothermal amplified reaction (RT-LAMP)
Method detects 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
A section designs 4 kinds or 6 kinds of primers, is then reacted at a certain temperature using strand replacement reaction.2 in 4 kinds of primers
Its 3 ' end of kind of inner primer and 5 ' ends can identify 2 different zones in target nucleic acid sequence respectively, and be designed to that its 5 ' terminal sequence can be with
The complementary strand region annealed combination as synthesized by the extension of 3 ' end startings.Inner primer can form loop-stem structure, and carry out certainly
My extension then has new inner primer annealed combination to carry out strand displacement synthetic reaction in cyclic structure position.The two is continuous
Circulating repetition realizes amplified reaction.Therefore, even if only constant-temperature amplification also may be implemented in a kind of enzyme, ring mediated isothermal amplification method.
When using RNA as template, reverse transcriptase need to only be added in advance, so that it may which a step completes the synthesis of cDNA and amplification process (reverses
Record loop-mediated isothermal amplification).
Ring mediated isothermal amplification gene amplification method is characterized in also generating a large amount of by-products while synthesizing a large amount of nucleic acid
Object pyrophosphate ion.Calcein contained in fluorescence visual observation agent is initially in fluorescent quenching shape due in conjunction with manganese ion
State.But with the progress of loop-mediated isothermal amplification, because being seized the manganese ion of combination by byproduct of reaction pyrophosphate ion,
Calcein restores free to issue fluorescence.And then combined with the magnesium ion in reaction solution, enhance fluorescence.According to
This principle can easily judge whether that ring mediated isothermal amplification has occurred by fluorescence visual detection.
Isothermal duplication (LAMP) method that ring mediates has the advantages that quick, efficient, high specificity, high sensitivity.LAMP
Method speed is fast, not high to requirement of experiment, it is only necessary to which a water-bath does not need sterile working, in a short time can be with
A large amount of samples are detected, laboratory serodiagnosis, customs quarantine control and the generaI investigation of extensive epidemic disease are suitable for.In addition, LAMP detection method compares
It is sensitive, and result is also more reliable.
Round pcr mainly is passed through for the molecular Biological Detection of Marburg virus at present, the present invention is according to Marburg
The specific gene segment V35 of virus, devises a group-specific primers, can be short half using the method for RT-LAMP
Amplified fragments are detected in hour to one hour, as long as a thermostat water bath in operation, method is simple, high sensitivity, when
Between it is short.Detection method of the invention can operate with the detection of Marburg virus rapid sensitive, as the method for port rapid screening,
The accuracy and timeliness for improving detection, can greatly reduce the period of detection.
Summary of the invention
The first technical problem to be solved by the present invention is to provide a kind of Marburg virus RT-LAMP detection of rapid sensitive
Method, the present invention can carry out the detection of rapid sensitive to Marburg disease virus gene, can increase substantially inlet and outlet through the invention
The detection efficiency of port frontline inspection and quarantine personnel can not only reduce workload but also can solve traditional detection method to the maximum extent
Positive missing inspection problem that may be present, to prevent the generation of epidemic situation and being passed to for external Marburg virus to the maximum extent.
The Marburg virus RT-LAMP detection method of rapid sensitive provided by the present invention, includes the following steps:
(1) whole blood sample carries out the extraction of RNA liquid；
(2) RT-LAMP is expanded:
A.RT-PCR reaction system includes: 2 × reaction buffer (RM), gene specificity of sample to be tested primer, dd H2O,
Enzyme solutions, RNA extracting solution, the visual reagent of fluorescence；
B. PCR pipe is added in PCR reaction solution, is put into 63 DEG C of thermostatical instrument or LAMP detector, the reaction of isothermal reaction
Condition: 63 DEG C of reaction 60min.；
(3) result interpretation: negative control color is brown, and positive sample is green, is had if with LAMP detector bright
Aobvious amplification curve.
RNA described in step (1), which is extracted, blood sample geneome RNA extracts kit can be used to extract RNA.It takes
100ul whole blood sample extracts RNA according to the extraction step in kit specification；
PCR reaction system described in step (2) are as follows: 2X reaction buffer 12.5 μ l, 2.5 μ of specific primer mixed liquor
L, 1.0 μ l, dd H2O of enzyme solution 3.0 μ l, 1.0 μ l, RNA extracting solution of fluorescence visual observation agent, 5 μ l.
Gene specificity of sample to be tested primer described in step (2) is to be with the peculiar gene VP35 gene of Marburg virus
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
Object LF/LB is compared sequence of the VP35 gene in a variety of Marburg virus, chooses the highest one section of design of homology and draws
The specificity of Marburg virus detection can be improved in object.Three pairs of specific primers are as follows:
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, three pairs of specific primers is designed, so that detection method of the invention
Detect Marburg disease virus gene accuracy rate it is higher, can it is sensitive, specifically detect Marburg virus, with conventional PCR method ratio
Compared with detection sensitivity and specificity are obviously higher than conventional method.Detection method of the invention not only can satisfy daily epidemic situation
The requirement of safety detection also can satisfy the testing requirements of clinical sample.Compared with the existing detection method, reaction only needs the present invention
It can be completed in a small-sized LAMP amplification instrument or water bath with thermostatic control, testing result can be detected by the method for range estimation
It arrives, time and the complexity of detection is greatly reduced, detection device is simply easy to carry about with one, and substantially increases current detection energy
Power.And collecting detection from sample can complete total time in 1 hour completely, be beneficial to fast and accurately detect Marburg disease
Poison.
Detailed description of the invention
Fig. 1 is 1 Marburg virus RT-LAMP testing result of embodiment.
Left side figure is the signal strength that the detection pipe of the right figure is detected in LAMP amplification instrument, and right figure is what naked-eye observation was arrived
Color change, the copies/ul that the number above right figure represents.
Fig. 2 is 1 Marburg virus RT-LAMP detection method Marburg virus V35 positive sample of embodiment detection specificity
As a result.
A left side is different sample amplification product fluorescence intensities, and the right side is different histogram sample names.
Fig. 3 is 1 Marburg virus RT-LAMP detection method sensitivity experiments result of embodiment.
A left side is the concentration of specimens of each column diagram, and the right side is the amplified production fluorescence intensity of various concentration sample.
Fig. 4 is 1 Marburg virus RT-LAMP detection method detection time measurement result of embodiment.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In addition, it should also be understood that, after reading the content taught by the present invention, those skilled in the art
Member can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited
Range.
The specificity and sensitivity of 1 Marburg virus RT-LAMP detection method of embodiment
One, experimental procedure
1. the external synthesis of Marburg virus, Ebola virus RNA
The Marburg virus Angola pnca gene group sequence (Ang published is analyzed using through bioinformatics method
1379c), the artificial synthesized VP35 of Marburg virus containing specific detection segment (about 600bp) is cloned in PCR cloning vector
On (TOPO TA cloning kit), with extraction of plasmid DNA kits Plasmid DNA, it is sequenced after confirming that Insert Fragment is correct
It is transcribed in vitro: first making linearisation to exclude downstream RNA product with Not I digested plasmid, then with MAXIscript T7Kit
It is transcribed in vitro, generates purpose RNA, then carry out DNase processing with TurboDNase, remove the Plasmid DNA that do not transcribed
Template finally uses ethanol precipitation, purifies RNA product, as positive control RNA template, is stored in -70 DEG C of ultra low temperature freezers and waits for
With.Same method obtains the RNA segment of Ebola virus VP35.
2. the acquisition of blood sample
The whole blood sample 1.0ml of port detection patient is taken, centrifugal treating obtains serum.
3. the extraction of geneome RNA
Dengue fever RNA, Huang are extracted using blood sample geneome RNA extracts kit (centrifugal column type, Qiagen company)
Hot RNA, west nile virus RNA.100uL serum sample is taken, extracts RNA according to the extraction step in kit specification.
The measurement of 4.RNA concentration
The concentration of RNA segment is measured by Nanodrop 2000c, and the copy of RNA is calculated according to the size of RNA segment
Number.
5. the amplification of target gene
The design of 5.1 primers
Using the peculiar gene VP35 gene of Marburg virus as target gene, each three based on loop-mediated isothermal amplification technology design
To primer: inner primer FIP/BIP, outer primer F3/B3, ring primer LF/LB, to sequence of the VP35 gene in a variety of Marburg virus
Column are compared, and choosing the highest one section of design primer of homology can be improved the specificity of Marburg virus detection.Three pairs special
Property primer are as follows:
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
It is expanded on LAMP amplification instrument, amplification system are as follows: 12.5 μ l of 2X reaction buffer, specific primer mixed liquor
2.5 μ l, 1.0 μ l, dd H of enzyme solution23.0 μ l of O, 1.0 μ l, RNA extracting solution of fluorescence visual observation agent, 5 μ l.
The reaction condition of isothermal reaction: 63 DEG C of reaction 60min.
6. result interpretation
A is expanded on LAMP amplification instrument, is judged by expanding histogram
B is expanded in thermostatical instrument, is judged by amplification system color change, and negative control is brown, and the positive is
Green.
7. detection architecture is specific
According to the measurement of RNA concentration, 10E+6copies/ul and two Marburg virus V35 nucleic acid of 1copies/ul are obtained
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 is detected according to 5.2 steps, observe its specificity.
8. system sensitivity detects
Each dilution is measured according to 5.2 method, and calculates the Monitoring lower-cut of detection method.
9. detection time: according to the curve of detection, determining detection time
Two, result
1. the detectability of Marburg virus
It is tested with the Marburg RNA sample synthesized in vitro, the result is shown in Figure 1.It is found through experiments that, from 1copy/ul
To 109The sample of copies/ul may detect that, and testing result by machine fluorescence intensity and visually observes anti-
Answer the result of variations of liquid color consistent.Absolutely prove that this method is easy to operate, as long as there is a water-bath to can be carried out reacting,
And observe result.It, may be accurate not as good as machine compared with machine acquires fluorescence intensity.
2. sample specific test:
If Fig. 2 is shown, two Marburg virus V35 nucleic acid fragment 10E+6copies/ul and 1copies/ul height two
Concentration can detecte, and other viral RNAs and the artificial synthesized V35 nucleic acid fragment of Ebola are not detected.
3, sensitivity experiments
100copies/ul, 10copies/ul, 1copies/ul, 0.1copies/ul, 0.01copies/ are detected respectively
The sample of ul, 0.001copies/ul and 0.0001copies/ul difference diluted concentration gradient carries out method sensitivity experiments,
If Fig. 3 is shown, the sample of 1copies/ul concentration, sample size 5ul, Monitoring lower-cut 5copies can detecte.
4, detection time measurement
It can detecte when the sample of 10,100copies/ul is in 28min, and 1copy/ul sample can in 42min
To detect, it is specifically shown in Fig. 4.In addition other data show that 10E+6copies/ul sample can even be examined in 16min
It measures, there is fabulous timeliness.
Claims (1)
1. the primer based on RT-LAMP rapid sensitive detection Marburg virus, which is characterized in that with Marburg virus VP35 gene
Segment is target gene, based on loop-mediated isothermal amplification technology three pairs of specific primers of design are as follows:
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)。
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| 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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| 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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| 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 |
Non-Patent Citations (3)
| Title |
|---|
| Development and evaluation of a simple assay for Marburg virus detection using a reverse transcription-loop-mediated isothernal amplification method;Yohei Kurosaki et al.;《Clinical Microbiology》;20100731;第48卷(第7期);摘要部分、第2332页左栏第4段-右栏第2段和表1 * |
| Marburg hemorrhagic fever associated with multiple genetic lineages of virus;Daniel G. Bausch et al.;《The New England journal of medicine》;20060831(第355期);第912页左栏第2段-右栏第2段 * |
| 马尔堡病毒的实时荧光RT-PCR检测方法研究;洪烨;《中国国境卫生检疫杂志》;20111231;第34卷(第6期);第435-438页 * |
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