CN109055617A - Lassa fever virus detection kit and its primer special - Google Patents
Lassa fever virus detection kit and its primer special Download PDFInfo
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- CN109055617A CN109055617A CN201811035787.XA CN201811035787A CN109055617A CN 109055617 A CN109055617 A CN 109055617A CN 201811035787 A CN201811035787 A CN 201811035787A CN 109055617 A CN109055617 A CN 109055617A
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- 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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- 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
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
Abstract
The invention discloses Lassa fever virus detection kit and its primer specials.The primer is used to carry out RT-LAMP detection to Lassa fever virus or carries out LAMP detection to Lassa fever virus genome cDNA, the primer is designed according to the conservative target sequence of Lassa fever virus specificity, and the conservative target sequence of Lassa fever virus specificity is as shown in SEQ ID NO:1.Using primer or detection kit of the invention, can under isothermal conditions quickly, conveniently, efficiently, high specific, detect Lassa fever virus in high sensitivity, do not need complex instrument, can be applied to carry out RT-LAMP detection to Lassa fever virus geneome RNA.
Description
Technical field
The invention belongs to molecular biology for detection viral in field of biotechnology, more particularly to Lassa fever virus
Reverse transcription-ring mediate isothermal nucleic acid amplification (RT-LAMP) detection primer special, the detection kit comprising the primer and
The application of the primer and detection kit in detection Lassa fever virus.
Background technique
Lassa fever virus (Lassa virus) belongs to Arenaviridae (Arenaviridae), arenavirus genus
(Arenavirus), to have the two segment sub-thread minus-stranded rna virus of coating, because treatment difficulty and infectiousness are listed in bio-safety
Level Four (Biosafety level 4, BSL-4) virus, in addition, Lassa fever virus is also listed in A class biological weapons agent.It draws husky
Heat is a kind of common disease of 1-4 weeks people and animals of course of disease, and the main area that occurs is western for Africa, belongs to Nigeria to Sierra Leone
The endemic infection disease of one band is popular in Benin, Guinea, Ghana, Liberia, Mali, Sierra Leone and Nigeria.
But because the rodent type of transmitted virus is western throughout Africa, actually occur area perhaps traveled to it is other
The country of African western area.Total case fatality rate is 1%, but reach as high as in inpatient 15% (WHO, 2017.http: //
Who.int/mediacentre/factsheets/fs179/zh/), case fatality rate is more than 50% and in certain outburst cases.It draws
The case load that husky fever virus infects estimates in West Africa in 100,000 to 300,000 people every year, about 5,000 people is dead.It is drawn in plug
The some areas in Leon and Nigeria, about 10%-16% to hospital seek the patient of medical treatment with Lassa fever disease, and which show drawings
Serious impact caused by the common people of the husky heat to this region.When about 80% common people infect Lassa fever virus, symptom may not
Obviously, the symptoms such as fever, vomiting, diarrhea, pharyngitis are shown as, remaining 20% infection Lassa fever virus when, there is serious multisystem
There is multiple organs dysfunction, failure, such as liver, kidney and spleen so as to cause death in disease, several organs of virus attack body.
As international tourism crowd increasingly increases, and the viral transmission speed is fast, it is wide to involve range, explores sensitiveer, quick, special
Different, convenient detection method can effectively resist epidemic situation except the gateway of a country, and can quickly take measures to control epidemic situation, have weight
Big meaning.In addition, a kind of sensitive, quick, special, convenient detection method is constructed in the laboratory research virus, it can be quickly
Ground determines cause of disease, shortens search time, is of great significance to the biological characteristics and genome signature of studying the virus.
The laboratory testing of Lassa fever virus is generally virus purification, immunization based on cell culture and measures to be exempted from as enzyme-linked
Epidemic disease adsorption test (ELISA), indirect immunofluorescence assay (IFA) and nucleic acid molecules detection reverse transcription PCR (RT-PCR).Cell training
Supporting is influenced very little by virus variation, and can be described in detail.But it needs the related facility of BSL-4, and
Culture period is 7-10 days, and the period is long.Although being reliably that it does not have other detection sides by the detection that ELISA captures antigen
Method is sensitive.RT-PCR and real-time quantitative fluorescence PCR (Real-time PCR) technology are the sensitivity sides of quick detection Lassa fever virus
Method, however due to needing expensive heat circulating equipment to be difficult in small-size laboratory and on-site test using these methods.
Summary of the invention
To solve the problems, such as to detect for Lassa fever virus in the prior art, the first purpose of this invention is to mention
For a kind of primer, for carrying out RT-LAMP detection to Lassa fever virus or carrying out LAMP inspection to Lassa fever virus genome cDNA
It surveys, is designed according to the conservative target sequence of Lassa fever virus specificity, the conservative target sequence such as SEQ of Lassa fever virus specificity
Shown in ID NO:1.
Preferably, it is above-mentioned for Lassa fever virus carry out RT-LAMP detection or to Lassa fever virus genome cDNA into
Row LAMP detection primer be primer combine, the nucleotide sequence of five primers respectively such as SEQ ID NO:2, SEQ ID NO:3,
Shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.
It is provided by the present invention to be used to carry out Lassa fever virus RT-LAMP detection or to Lassa fever virus genome cDNA
The primer for carrying out LAMP detection is able to achieve the Lassa fever virus of batch detection and screening when studying to(for) Lassa fever virus, improves
The specificity and sensitivity of detection.
A second object of the present invention is to provide a kind of detection kits, for carrying out RT-LAMP inspection to Lassa fever virus
It surveys or LAMP detection is carried out to Lassa fever virus genome cDNA, it includes the primers of aforementioned present invention.
Preferably, detection kit of the invention includes the reagent for being used for 25 μ l detection architectures below: 20.0mM Tris-
HCl (PH 8.8), 10.0mM KCl, 2.0mM MgSO4, 10.0mM (NH4)2SO4, 0.1%Triton X-100,0.8mM beet
Alkali, 6mM MgSO4, every kind of dNTP each 1.4mM, 8U Bst archaeal dna polymerase, addition 7.5U WarmStart when template is RNA
RTx reverse transcriptase, primer additional amount are as follows: each 0.1 μ l of primer shown in 100 μM of SEQ ID NO:2 and SEQ ID NO:3,100 μM
Each 0.8 μ l of primer shown in SEQ ID NO:4 and SEQ ID NO:5,0.3 μ l of primer shown in 100 μM of SEQ ID NO:6.
Third object of the present invention is to provide above-mentioned for carrying out RT-LAMP detection to Lassa fever virus or to Lassa fever
Full-length cDNA carries out the primer of LAMP detection in the RT-LAMP detection reagent or Lassa fever virus for preparing Lassa fever virus
Application in the LAMP detection reagent of genome cDNA.
In above-mentioned application, detection reagent is in the RT-LAMP detection of Lassa fever virus, detection method to may include following
Step:
(1) using the geneome RNA of sample to be tested as template, under the guidance of above-mentioned primer or use above-mentioned detection kit
Carry out RT-LAMP amplification；
(2) result judgement is carried out after reaction: in the case where adding calcein indicator in reaction solution, according to anti-
Answer the color change judging result of liquid, green indicates in sample to be tested there are Lassa fever virus, in orange expression sample to be tested not
There are Lassa fever virus, (principle: calcein is Metal ion indicator, the Mn in calcein and reagent2+It quenches in conjunction with being in
It goes out state, when the pyrophosphoric acid and Mn that amplified reaction releases2+In conjunction with release calcein, cancellation state is released, and is issued yellow
Green fluorescence)；Or in the case where not adding calcein indicator, the turbid of reaction front and back reaction solution directly is detected with transmissometer
Degree variation carrys out judging result, and turbidity, which rises (> 0.1), to be indicated in sample to be tested there are Lassa fever virus, turbidity is unchanged indicate to
There is no Lassa fever virus, (principle: can generate magnesium pyrophosphate during LAMP reaction, magnesium pyrophosphate is a kind of white in sample
Color precipitating, transmissometer can judge that LAMP is reacted according to the variation of turbidity).
In above-mentioned application, 25 μ l RT-LAMP reaction systems are used in step 1), may include:
2 μ l, 20.0mM Tris-HCl, 10.0mM KCl, the 2.0mM MgSO of geneome RNA of sample to be tested4, 10.0mM
(NH4)2SO4, 0.1%Triton X-100,0.8mM glycine betaine, 8mM MgSO4, every kind of dNTP each 1.4mM, 8U Bst DNA are poly-
Synthase, 7.5U WarmStart RTx reverse transcriptase, primer additional amount are as follows: 100 μM of SEQ ID NO:2 and SEQ ID NO:3 institutes
Show each 0.1 μ l of primer, primer each 0.8 μ l, 100 μM of SEQ ID NO:6 shown in 100 μM of SEQ ID NO:4 and SEQ ID NO:5
Shown 0.3 μ l of primer.
RT-LAMP amplification condition in above-mentioned application, in step 1) are as follows: set 63 DEG C of constant temperature 45-55min, preferably
52min。
In above-mentioned application, the additive amount of calcein indicator can be that (end reaction system is 26 μ to 1 μ l in step 2)
l)。
To sum up, the present invention provides one kind for carrying out RT-LAMP detection to Lassa fever virus or to Lassa fever virus base
Because group primer, detection kit and primer of cDNA progress LAMP detection and detection kit are preparing Lassa fever virus
RT-LAMP detection reagent or Lassa fever virus genome cDNA LAMP detection reagent in application.The present invention has following
Advantage:
1) high specific: the identification of the specific regions of target sequence in five primer pair Lassa fever virus or its genome cDNA
Ensure that the high degree of specificity of amplification, can from difference only one nucleotide gene sample in find out corresponding target sequence into
Row amplification；
2) highly sensitive: as regular-PCR, detection is limited up to 8.27pg/ μ l for sensitivity；
3) result judgement is easy: result (the presence or absence of calcein colour developing or observation white precipitate) can be observed by the naked eye,
Transmissometer judging result can also directly be used；
4) it is easy to operate: as long as sample to be tested (target nucleic acid) and detection reagent are put into togerther 52 in 63 DEG C of thermostat water baths
It may determine that result after minute；
5) it quick, efficient amplification: can be completed in entire amplified reaction one hour.
In conclusion using primer or detection kit of the invention, can under isothermal conditions quickly, conveniently, efficiently,
High specific detects Lassa fever virus in high sensitivity, does not need complex instrument, provides newly for the detection of Lassa fever virus
Technology platform, can be used for each research unit and laboratory at different levels detect Lassa fever virus under study for action and studies the life of the virus
Object characteristic and genome signature, it can also be used to Animal husbandry production unit, primary care health unit, quarantine port at different levels and each
Disease prevention and control center's screening and detection Lassa fever virus, have a vast market foreground with biggish economical, societal benefits,
Suitable for promoting and applying on a large scale.
The present invention is described in further details combined with specific embodiments below.
Detailed description of the invention
Fig. 1 shows 5 sets of primer LAMP response curve figures；
Fig. 2 shows the figures of detection optimum reaction condition measurement experiment result；
Fig. 3 shows the figure that primer specificity tests real-time transmissometer result；
Fig. 4 shows the figure of primer specificity experiment calcein coloration result；
Fig. 5 shows the figure of the real-time transmissometer result of primer sensitivity experiment；
Fig. 6 shows the figure of primer sensitivity experiment calcein result；
Fig. 7 shows the figure of PCR sensitivity experiment result.
Specific embodiment
Ring mediate isothermal amplification (LAMP) be Notomi et al. (Notomi T, Okayama H,
MasubuchiH, et al. .Loop-mediated isothermal amplification of DNA.Nucleic Acids
Res 2000；28 (12): the novel nucleic acids amplification method 63.) being initially reported, can under isothermal conditions quickly and efficiently, it is high
Spend specifically DNA amplification.This simple and quick technology utilizes a kind of Bst archaeal dna polymerase with high strand-displacement activity,
By the synthesis of the strand displacement DNA of automatic cycle, the amplification in vitro of nucleic acid is realized.Isothermy of the reaction at 60 DEG C to 65 DEG C
Lower progress, therefore do not need expensive heat circulating equipment.Since thermal change does not have the loss of time, the amplification efficiency of LAMP method is non-
Chang Gao.
The reaction is high degree of specificity to target sequence, because a group-specific primers identify at least six differences on it
Site.It can be further improved LAMP reaction rate by the way that Loop primer is added.The modification reduces the reaction time and has
Improve the potentiality of sensitivity.In addition, LAMP method generates turbidity increase in positive sample, by based on the turbid of reaction mixture
The real-time monitoring of degree and agarose gel electrophoresis is detected.Therefore, compared with other nucleic acid amplification methods, LAMP measuring method
There is advantage in terms of specificity and rapidity.This method has been successfully applied to the inspection of human pathogen's DNA virus and RNA virus
It surveys.
LAMP reaction has high degree of specificity to target sequence, and the determination of different virus amplimer is the pass of this method application
Key.Not yet see that LAMP method can apply in the detection to Lassa fever virus before making the present invention.
The present invention explores through great efforts, provides in this application a kind of for carrying out RT-LAMP inspection to Lassa fever virus
Survey or carry out to Lassa fever virus genome cDNA primer, detection kit and the primer and detection reagent of LAMP detection
Box answering in the LAMP detection reagent of the RT-LAMP detection reagent or Lassa fever virus genome cDNA that prepare Lassa fever virus
With.
The present invention is described in detail in a manner of specific embodiment below.Embodiment carries out reality under the premise of the technical scheme of the present invention
It applies, the detailed implementation method and specific operation process are given, but the disclosure is not limited to the following embodiments.
Method therefor is conventional method unless otherwise instructed in following embodiments.
1. design of primers of embodiment
1. target sequence is chosen
From the U.S., gene data library searching obtains Lassa fever virus sequence (No. GenBank: AY628207.1), passes through
BLAST carries out homology analysis, obtains one section of specific conservative's target sequence (such as SEQ ID NO:1 of Lassa fever virus NP gene
It is shown).
2. design of primers
Target-gene sequence is guarded according to this, with software Primer design V5 designed for carrying out to Lassa fever virus
RT-LAMP detection or the primer that LAMP detection is carried out to Lassa fever virus genome cDNA, design 5 sets of primer LSR-1 extremely
LSR-5 (referring to table 1) (primer is synthesized by holding up section's biology Wuhan combining unit) is right by experiment (experimental method is referring to embodiment 2)
Than (referring to Fig. 1), best primer combination (i.e. LSR-1) is finally had chosen, 5 sets of primer sequences for comparing are as follows:
Table 1: primer LSR-1 to LSR-5
By comparing experiment (referring to embodiment 3), with the spirit for the result that LSR-1 this set primer detection Lassa fever virus obtains
Sensitivity is 8.27pg/ μ l, than current sensitivity 1ng (i.e. 0.5ng/ μ l, referring to Fukuma A et al., Rapid reported in the literature
detection of Lassa virus by reverse transcription-loop-mediated isothermal
Amplification.Microbiol Immunol.2011,55 (1): 44-50.) high about 60 times or so.
The foundation of 2. detection method of embodiment
1. the foundation of optimum reaction condition
Five primers (LSR-1) for carrying out RT-LAMP detection to Lassa fever virus obtained with embodiment 1 are husky to drawing
((plasmid origin is in holding up for Lassa fever virus NP gene full genome synthetic product insertion pUC57 plasmid for fever virus genome cDNA segment
Section's biology Wuhan combining unit) obtain pUC57-LSR-NP plasmid) LAMP detection is carried out, to obtain optimum reaction condition, specific side
Method is as follows:
Under identical reaction system, respectively to Lassa fever virus NP gene masculine plasmid in the application at 58-65 DEG C
LAMP detection is carried out, to determine optimum reaction condition, comprising the following steps:
1) using the Lassa fever virus NP gene PUC57 plasmid synthesized in the application as template, at five of the acquisition of embodiment 1
Under the guidance of primer carry out LAMP amplification, wherein 25 μ l LAMP reaction systems include: pUC57-LSR-NP plasmid (final concentration >
1ng/ μ l) 2 μ l, 2.5 μ l 10 × ThermoPol Reaction Buffer (New England Biolabs Inc.,
Massachusetts, USA, 1 × ThermoPol Reaction Buffer, including 20.0mM Tris-HCl (PH 8.8),
10.0mM KCl, 2.0mM MgSO4, 10.0mM (NH4)2SO4, 0.1%Triton X-100), 0.8mM glycine betaine (Sigma-
Aldrich Inc., St Louis, USA), 6mM MgSO4(New England Biolabs Inc.,Massachusetts,
USA), every kind of dNTP each 1.4mM (TaKaRa Bio Inc., Clontech Laboratories, Inc., Dalian, China), 8U
Bst archaeal dna polymerase (New England Biolabs Inc., Massachusetts, USA), primer additional amount are as follows: 100 μM
Each 0.1 μ l (final concentration 4pmol) of primer shown in SEQ ID NO:2 and SEQ ID NO:3,100 μM of SEQ ID NO:4 and SEQ
Each 0.8 μ l (final concentration 32pmol) of primer shown in ID NO:5,0.3 μ l (final concentration of primer shown in 100 μM of SEQ ID NO:6
12pmol)。
2) carry out result judgement after reaction: (end reaction system is 26 μ l) that 1 μ l is added in reaction solution is contained
The calcein color indicator of 0.5mM calcein and 10mM manganese chloride is green according to the color change judging result of reaction solution
Color table shows in institute's sample that there are Lassa fever virus genome cDNA segment (positive), there is no draw in orange expression institute sample
Husky fever virus genome cDNA segment (feminine gender)；Or do not add calcein indicator, directly with transmissometer (LA-320C,Rong Yan Biotechnology Co., Ltd) detection reaction front and back reaction solution turbidity variation come judging result, turbidity
Rising (> 0.1) indicates that, there are Lassa fever virus genome cDNA segment (positive) in institute's sample, the unchanged expression of turbidity is surveyed
Lassa fever virus genome cDNA segment (feminine gender) is not present in sample.
As depicted in figs. 1 and 2, LAMP optimal detection condition can are as follows: 63 DEG C, 45-55min (because of negative control in 54min and
Peak is also started after it, so detection time selects 52min.)
2. the foundation of Lassa fever virus RT-LAMP detection method
Based on optimum reaction condition determined above, Lassa fever virus RT-LAMP detection method be can comprise the following steps that
(1) geneome RNA of Lassa fever virus NP gene pseudovirus RNA or sample to be tested is obtained；
(2) using the RNA of acquisition as template, RT- is carried out under determining optimum reaction condition under the guidance of LSR-1 primer
LAMP amplification；
(3) result judgement is carried out after reaction: in the case where adding calcein indicator in reaction solution, according to anti-
Answer the color change judging result of liquid, green indicates in institute's sample there are Lassa fever virus, in orange expression institute sample not
There are Lassa fever virus；Or in the case where not adding calcein indicator, the reaction of reaction front and back directly is detected with transmissometer
The turbidity variation of liquid carrys out judging result, and turbidity, which rises, indicates that there are Lassa fever virus, the unchanged expression institutes of turbidity in institute's sample
Lassa fever virus is not present in sample.
Here, 25 μ l RT-LAMP reaction systems may include: Lassa fever virus NP gene pseudovirus RNA (or sample to be tested
Geneome RNA) 2 μ l, 20.0mM Tris-HCl, 10.0mM KCl, 2.0mM MgSO4,10.0mM (NH4) 2SO4,0.1%
Triton X-100,0.8mM glycine betaine, every kind of each 1.4mM of 8mM MgSO4, dNTP, 8U Bst archaeal dna polymerase, 7.5U
WarmStart RTx reverse transcriptase, primer additional amount are as follows: primer shown in 100 μM of SEQ ID NO:2 and SEQ ID NO:3 is each
0.1 μ l, each 0.8 μ l of primer shown in 100 μM of SEQ ID NO:4 and SEQ ID NO:5, primer shown in 100 μM of SEQ ID NO:6
0.3μl。
RT-LAMP amplification condition can are as follows: sets 63 DEG C of constant temperature 45-55min.
The additive amount of calcein indicator can be that (end reaction system is 26 μ l to 1 μ l, and the reaction time is in result judgement
52min)。
Wherein, the acquisition modes of Lassa fever virus NP gene pseudovirus RNA: by the overall length NP gene of Lassa fever virus
(No. GenBank: AY628207.1) is inserted into plasmid (Cao Xuefeng etc., containing 5 that packaging virus is constructed in Lentiviral
The preparation military medicine of the pseudovirus positive reference product of the strong hemorrhagic fever viruse of kind, 2016,40 (9): 713-737.).With packing
Virus particle transfects 293T cell respectively, and culture collects supernatant after observing within 48 hours and 72 hours fluorescence, obtains Lassa fever
The pseudovirus of virus.RNA is extracted from the pseudovirus.
The acquisition modes (must carry out in the laboratory BSL-4) of the geneome RNA of sample to be tested: clinical blood sample, brain are taken
The body fluid samples such as spinal fluid sample, lymph extract its geneome RNA with existing commercialization RNA extracts kit, be placed in -80 DEG C it is standby
With.
3. specificity of embodiment, sensitivity technique
One, specific detection
Respectively with Lassa fever virus NP gene plasmid pUC57-LSR-NP, Hepatitis C Virus HCV full-length genome plasmid
(Diseases Preventing and Controlling Institute), hepatitis b virus hbv full-length genome plasmid (Chinese People's Liberation Army's disease
Sick prevention and control institute), people's acquired immunity defact virus HIV full-length genome plasmid (Chinese People's Liberation Army's disease prevention control
Institute processed), the sars coronavirus end envelope protein S1C lack (the Chinese people's liberation of 19 AA plasmid pCAGGS-SARS-S1 Δs -19
Prevention and control of diseases institute of army), Middle East respiratory syndrome virus M ERS spike protein plasmid (Chinese People's Liberation Army's disease prevention control
Institute processed), the Coronavirus OC43 end envelope protein S1C lack (the Chinese people's liberation of 17 AA plasmid pCAGGS-OC43-S1 Δs -17
Prevention and control of diseases institute of army), Ebola virus Ebola envelope glycoprotein plasmid (Chinese People's Liberation Army's prevention and control of diseases
Institute), influenza A genome cDNA (Wuhan Virology Institute,Chinan academy of Sciences's influenza virus subject group), meningitis Neisser
B groups of 29022 genomic DNAs of B CMCC (Diseases Preventing and Controlling Institute) of bacterium, Lassa fever virus N gene plasmid
PUC57-NiV-MN (Diseases Preventing and Controlling Institute), yellow fever virus positive plasmid pGSI-YF-NS (Chinese
People's liberation army prevention and control of diseases institute) it is template, using no enzyme water as negative control, the optimum condition of the detection acquisition of embodiment 2
The specificity of detection method.
Testing result as shown in Figure 3 and Figure 4 (1: Lassa fever virus NP gene plasmid pUC57-LSR-NP；2: hepatitis C
Viral HCV full-length genome plasmid；3: hepatitis b virus hbv full-length genome plasmid；4: people's acquired immunity defact virus
HIV full-length genome plasmid；The 5:SARS coronavirus end envelope protein S1C lacks 19 AA plasmid pCAGGS-SARS-S1 Δs-
19；6: Middle East respiratory syndrome virus M ERS spike protein plasmid；7: the Coronavirus OC43 end envelope protein S1C lacks 17 AA
Plasmid pCAGGS-OC43-S1 Δ -17；8: Ebola virus Ebola envelope glycoprotein plasmid；9:A type influenza virus gene group
cDNA；10: B groups of 29022 genomic DNAs of B CMCC of Neisseria meningitidis；11: Lassa fever virus N gene plasmid pUC57-NiV-
MN；12: yellow fever virus positive plasmid pGSI-YF-NS；13: negative control, in Fig. 4: "+" is the positive, and "-" is feminine gender), from Fig. 3
In as can be seen that only 1 have occurred LAMP reaction, remaining does not occur, in Fig. 4, calcein indicator dyeing make 1 display
For green, remaining be it is orange, it is consistent with the result of transmissometer detection method, it is higher to show that LAMP detection method of the invention has
Specificity, can specifically detect Lassa fever virus genome cDNA segment.
Two, sensitivity technique
Compare the sensitivity of LAMP detection method of the present invention and regular-PCR method detection Lassa fever virus genome cDNA,
Method are as follows: Lassa fever virus NP gene plasmid pUC57-LSR-NP is extracted, then with 10 times of gradients (1 times, 10 times, 102Again, 103
Again, 104Again, 105Again, 106Times) be diluted to obtain sample 1-7, and sample 8 is obtained by negative control of no enzyme water, then with warp
The Plasmid DNA of gradient dilution is template, respectively with LAMP detection method and regular-PCR method (primer sequence LSA- of the invention
F:5 '-GGCAAGTGGACCTCAATGAT-3 ' and LSA-R:5 '-TGGGTTTCTTTCACCAGGAG-3 ') carry out sensitivity inspection
It surveys.
Testing result is as illustrated in figs. 5-7 (after extraction Lassa fever virus NP gene plasmid pUC57-LSR-NP quantitatively, with 10 times
Gradient is diluted, make total DNA in 1-7 template concentration be respectively 82.7ng/ μ l, 8.27ng/ μ l, 0.827ng/ μ l,
0.0827ng/ μ l, 8.27pg/ μ l, 0.827pg/ μ l, 0.0827pg/ μ l, template 8 are using no enzyme water as the negative control of template.
Fig. 5: real-time transmissometer testing result.Fig. 6: calcein indicator color reaction testing result, in Fig. 6: "+" is the positive, "-"
For feminine gender.Sensitivity .M, the DNA marker D2000. of Fig. 7: PCR method), LAMP detection method of the invention can be detected
8.27pg/ μ l, regular-PCR method can also detect 8.27pg/ μ l, and calcein indicator colouring method and transmissometer inspection
The result of survey method is consistent, shows that LAMP detection method of the invention is consistent with the sensitivity of regular-PCR detection method.
4. detection kit of embodiment
Based on Examples 1 and 2, detection kit provided by the present invention, comprising for carrying out RT- to Lassa fever virus
LAMP detection or five primers (sequence such as SEQ ID NO:2-6 institute that LAMP detection is carried out to Lassa fever virus genome cDNA
Show).
Specifically, which includes the reagent for being used for 25 μ l reaction systems below: 20.0mM Tris-HCl
(PH 8.8), 10.0mM KCl, 2.0mM MgSO4, 10.0mM (NH4)2SO4, 0.1%Triton X-100,0.8mM glycine betaine,
6mM MgSO4, every kind of dNTP each 1.4mM, 8U Bst archaeal dna polymerase, 7.5U WarmStart RTx reverse transcriptase (New
England Biolabs Inc., Massachusetts, USA, template are added when being RNA, i.e. when RT-LAMP adds), primer
Additional amount are as follows: each 0.1 μ l (final concentration 4pmol) of primer shown in 100 μM of SEQ ID NO:2 and SEQ ID NO:3,100 μM of SEQ
Each 0.8 μ l (final concentration 32pmol) of primer shown in ID NO:4 and SEQ ID NO:5, primer shown in 100 μM of SEQ ID NO:6
0.3 μ l (final concentration 12pmol).
5. sample detection example of embodiment
It is carried out in the laboratory BSL-4.The body fluid such as blood, cerebrospinal fluid, the lymph of desirable patient or suspected patient, mention respectively
Its geneome RNA is taken, concentration is measured and is diluted to after suitable concentration using it as template, while with Lassa fever virus NP gene packaging
Pseudovirus is positive control, carries out RT-LAMP detection by negative control of Lentiviral.
Sequence table
<110>Wuhan Virology Institute,Chinan academy of Sciences
Diseases Preventing and Controlling Institute
<120>Lassa fever virus detection kit and its primer special
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 480
<212> DNA
<213>SEQ ID NO:1 (artificial sequence)
<400> 1
agaacatcaa ttactgggag agcttgggaa aacactcttg ttgatttgga atctgacaac 60
aaaccaccga aactaaatag cgctggttcc aataaaagct tgcaggctgc aggtctaaat 120
gctgggttga cctattctca actgatgaca ctaaaagatt gtatgttaca gttggaccca 180
agtagcaaaa catggataga tattgagggc aggcctgaag acccagtgga gattgctttg 240
tttcaaccaa gctctggttg ttacatacac ttttttagag aacctactga ccttaagcaa 300
ttcaaacagg atgctaagta ctcacatggg atagatgtta cagatctttt ttccacacaa 360
cccgggctga caagtgctgt catagaggca cttccccgca atatggtgtt gacctgccag 420
gggtcagatg acatcaaaaa gctgttggag tctcaaggga gaagagacat caaattgatt 480
<210> 2
<211> 19
<212> DNA
<213>SEQ ID NO:2 (artificial sequence)
<400> 2
tgagcaactc caaggaaat 19
<210> 3
<211> 18
<212> DNA
<213>SEQ ID NO:3 (artificial sequence)
<400> 3
ttcagatccc tcagtcgc 18
<210> 4
<211> 49
<212> DNA
<213>SEQ ID NO:4 (artificial sequence)
<400> 4
gcatctttga caacttgcaa ctttattttc tttggactca gtccttacg 49
<210> 5
<211> 45
<212> DNA
<213>SEQ ID NO:5 (artificial sequence)
<400> 5
tcctgcatgg ccttgacttt tttttttagg tctgcatcat ctctc 45
<210> 6
<211> 18
<212> DNA
<213>SEQ ID NO:6 (artificial sequence)
<400> 6
aggaaatcaa atcattcc 18
Claims (10)
1. a kind of primer, for carrying out RT-LAMP detection to Lassa fever virus or being carried out to Lassa fever virus genome cDNA
LAMP detection, which is characterized in that it is designed according to the conservative target sequence of Lassa fever virus specificity, and the Lassa fever virus is special
The conservative target sequence of property is as shown in SEQ ID NO:1.
2. primer according to claim 1, it is characterised in that: the nucleotide sequence of five primers is respectively such as SEQ ID
NO:2, SEQ ID NO:3, SEQ ID NO:4, shown in SEQ ID NO:5 and SEQ ID NO:6.
3. a kind of detection kit, for carrying out RT-LAMP detection to Lassa fever virus or to Lassa fever virus genome cDNA
LAMP detection is carried out, it includes primers according to claim 1 or 2.
4. detection kit according to claim 3, which is characterized in that the detection kit includes to be used for 25 μ l below
Detection architecture reagent: 20.0mM Tris-HCl, 10.0mM KCl, 2.0mM MgSO4, 10.0mM (NH4)2SO4, 0.1%
Triton X-100,0.8mM glycine betaine, 6mM MgSO4, every kind of dNTP each 1.4mM, 8U Bst archaeal dna polymerase, template RNA
Shi Tianjia 7.5U WarmStart RTx reverse transcriptase, primer additional amount are as follows: 100 μM of SEQ ID NO:2 and SEQ ID NO:3
Shown each 0.1 μ l of primer, primer each 0.8 μ l, 100 μM of SEQ ID shown in 100 μM of SEQ ID NO:4 and SEQ ID NO:5
The μ of primer 0.3 shown in NO:6 l.
5. detection kit described in primer of any of claims 1 or 2 or claim 3 or 4 is preparing Lassa fever virus
Application in RT-LAMP detection reagent or the LAMP detection reagent of Lassa fever virus genome cDNA.
6. application according to claim 5, the reagent is used in the RT-LAMP detection of Lassa fever virus, detection method
The following steps are included:
1) it using the geneome RNA of sample to be tested as template, is wanted under the guidance of primer as claimed in claim 1 or 2 or using right
Detection kit described in asking 3 or 4 carries out RT-LAMP amplification；
2) result judgement is carried out after reaction: in the case where adding calcein indicator in reaction solution, according to reaction solution
Color change judging result, green indicates that there are Lassa fever virus in the sample to be tested, orange to indicate the sample to be tested
In be not present Lassa fever virus；Or in the case where not adding calcein indicator, reaction front and back directly is detected with transmissometer
The turbidity variation of reaction solution carrys out judging result, and turbidity, which rises, indicates that there are Lassa fever virus in the sample to be tested, and turbidity is without change
Changing indicates that Lassa fever virus is not present in the sample to be tested.
7. application according to claim 6, it is characterised in that: use 25 μ l RT-LAMP reactants in the step 1)
System, it includes: 2 μ l, 20.0mM Tris-HCl, 10.0mM KCl, the 2.0mM MgSO of geneome RNA of sample to be tested4,
10.0mM(NH4)2SO4, 0.1%Triton X-100,0.8mM glycine betaine, 8mM MgSO4, every kind of dNTP each 1.4mM, 8U Bst
Archaeal dna polymerase, 7.5U WarmStart RTx reverse transcriptase, primer additional amount are as follows: 100 μM of SEQ ID NO:2 and SEQ ID
Primer shown in NO:3 each 0.1 μ l, primer each 0.8 μ l, 100 μM of SEQ shown in 100 μM of SEQ ID NO:4 and SEQ ID NO:5
The μ l of primer 0.3 shown in ID NO:6.
8. application according to claim 6, it is characterised in that: the RT-LAMP amplification condition in the step 1) are as follows: set 63
DEG C constant temperature 45-55min.
9. application according to claim 8, it is characterised in that: the RT-LAMP amplification condition in the step 1) are as follows: set 63
DEG C constant temperature 52min.
10. application according to any one of claims 6 to 9, it is characterised in that: calcein indicates in the step 2)
The additive amount of agent is 1 μ l.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811035787.XA CN109055617A (en) | 2018-09-06 | 2018-09-06 | Lassa fever virus detection kit and its primer special |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811035787.XA CN109055617A (en) | 2018-09-06 | 2018-09-06 | Lassa fever virus detection kit and its primer special |
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| Publication Number | Publication Date |
|---|---|
| CN109055617A true CN109055617A (en) | 2018-12-21 |
Family
ID=64760687
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|---|---|---|---|
| CN201811035787.XA Pending CN109055617A (en) | 2018-09-06 | 2018-09-06 | Lassa fever virus detection kit and its primer special |
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| WO2016011280A1 (en) * | 2014-07-16 | 2016-01-21 | Tangen Biosciences, Inc. | Isothermal methods for amplifying nucleic acid samples |
| CN105483293A (en) * | 2016-01-29 | 2016-04-13 | 中国人民解放军疾病预防控制所 | Fulminating-infectious-disease pathogen detecting primer pair and kit |
| EP3098326A1 (en) * | 2015-05-27 | 2016-11-30 | Institut Pasteur | Isothermal amplification assay for rapid and accurate detection of hemorrhagic fever viruses in clinical samples |
| CN107937612A (en) * | 2017-12-21 | 2018-04-20 | 北京卓诚惠生生物科技股份有限公司 | A kind of primer, probe and kit for detecting Marburg virus |
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2018
- 2018-09-06 CN CN201811035787.XA patent/CN109055617A/en active Pending
Patent Citations (4)
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|---|---|---|---|---|
| WO2016011280A1 (en) * | 2014-07-16 | 2016-01-21 | Tangen Biosciences, Inc. | Isothermal methods for amplifying nucleic acid samples |
| EP3098326A1 (en) * | 2015-05-27 | 2016-11-30 | Institut Pasteur | Isothermal amplification assay for rapid and accurate detection of hemorrhagic fever viruses in clinical samples |
| CN105483293A (en) * | 2016-01-29 | 2016-04-13 | 中国人民解放军疾病预防控制所 | Fulminating-infectious-disease pathogen detecting primer pair and kit |
| CN107937612A (en) * | 2017-12-21 | 2018-04-20 | 北京卓诚惠生生物科技股份有限公司 | A kind of primer, probe and kit for detecting Marburg virus |
Non-Patent Citations (4)
| Title |
|---|
| FUKUMA A ET.AL: "Rapid detection of Lassa virus by reverse transcription-loop-mediated isothermal amplification", 《MICROBIOLOGY AND IMMUNOLOGY》 * |
| KUROSAKI Y ET.AL: "Development and Evaluation of Reverse Transcription-Loop-Mediated Isothermal Amplification (RT-LAMP) Assay Coupled with a Portable Device for Rapid Diagnosis of Ebola Virus Disease in Guinea", 《PLOS NEGLECTED TROPICAL DISEASES》 * |
| 吴昊等: "LAMP技术在病毒检测中的应用", 《医药前沿》 * |
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