CN105779645A - LAMP kit for detecting Ebola virus and dedicated primer thereof - Google Patents
LAMP kit for detecting Ebola virus and dedicated primer thereof Download PDFInfo
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Abstract
The invention discloses an LAMP kit for detecting Ebola virus, dedicated primer thereof, and an application of the LAMP kit in detecting Ebola virus. The LAMP primer for detecting Ebola virus is designed according to Ebola virus specific conserved sequence-N protein gene (GenBank: KM655246.1); is used to qualitatively detect Ebola virus in samples (pure virus, blood, manure, etc.); and is composed of six primers, which comprises three combinations: outer primers (F3 and B3), inner primers (FIP and BIP), and loop primers (LF and LB). The kit can rapidly, conveniently, efficiently, specifically, and sensitively detect Ebola virus under an isothermal condition; no complicated instrument is needed, a novel technology platform is provided for Ebola virus detection; and the kit can be used in animal husbandry production units, basic health service units, and various centers for disease control and prevention to screen and detect Ebola virus, has a wide market prospect, can generate great economic and social benefits, and is suitable for large scale popularization and application.
Description
Technical field
The invention belongs in biological technical field the molecular biology for detection of virus, particularly relate to the LAMP kit of a kind of Ebola virus and primer special thereof and its application in detecting Ebola virus.
Background technology
Ebola virus (Ebolavirus) belongs to filamentous virus section, and its genome is single strand RNA, encodes 7 kinds of albumen, rises in value in endochylema, with form release of sprouting.This virus is propagated in monkey group, passes to people by monkey, and propagates and popular between crowd.Ebola virus can cause the hemorrhagic fever of lethal, incubation period is 5-10 days, the similar influenza of their early stage,, there is the symptom such as Nausea and vomiting, abdominal pain diarrhea in state of an illness rapid progression later, and bleeding can occur subsequently, show as mucosal bleeding, hematemesis, melena etc., patient substantially becomes thin, collapses and insensitive, and often because of shock, multiple organ dysfunction death after 7-16 days after morbidity, case fatality rate is up to 50%-90%.
The second half year in 2014, Ebola virus is come into vogue from West Africa, and gradually to global spread.At present, not yet there are the safely and effectively vaccine for Ebola virus and medicine, therefore, it is thus achieved that a kind of prevention for Ebola virus and quick diagnosis technology are particularly significant.The methods such as virus purification, transmission electronic microscope technology, immunofluorescence, elisa (ELISA) have been used for the laboratory diagnosis that Ebola virus infects, but, these methods are time-consuming, and need a large amount of expensive equipment, therefore, the quick detection of Ebola virus it is not suitable for；RT-PCR is as the first-selected diagnostic techniques of detection Ebola virus, but RT-PCR detection technique needs high-precision thermal cycler to complete the alternating temperature process of complexity, it is impossible to for Site Detection.
Loop-mediated isothermal amplification (LAMP) is by T.Notomi (NotomiT, OkayamaH, MasubuchiH, etal.Loop-mediatedisothermalamplificationofDNA.NucleicAc idsRes2000；28 (12): 63) a kind of novel nucleic acids amplification technique invented, this technology relies on the primer of the special design of 4-6 bar and a kind of archaeal dna polymerase with strand displacement characteristic, under isothermal conditions can efficiently, quick, height expand target sequence specifically.The principle of LAMP detection method: primer expands after being combined with target sequence in a large number, is attended by the generation of by-product while a large amount of synthesis target dnas, i.e. the pyrophosphatase precipitation of white, it is possible to directly judge yin and yang attribute result by monitoring turbidity change.In recent years, abroad this technology is widely used in pathogen detection.MasakiImai et al. (ImaiM, NinomiyaA, MinekawaH, etal.RapiddiagnosisofH5N1avianinfluenzavirusinfectionbyn ewlydevelopedinfluenzaH5hemagglutiningene-specificloop-m ediatedisothermalamplificationmethod.JVirolMethods.2007；141 (2): 173-80.) LAMP is utilized to establish the Laboratory Diagnosed system of bird flu virus；NobuyukiHayashi et al. (HayashiN, AraiR, TadaS, TaguchiH, OgawaY.DetectionandidentificationofBrettanomyces/Dekkera sp.yeastswithaloop-mediatedisothermalamplificationmethod .FoodMicrobiol.2007；24 (7-8): 778-85) devise LAMP specific primer for the ITS sequence of four kinds of saccharomyces cerevisiaes, establish the saccharomycetic LAMP system of efficient detection.LAMP can also detect the virus that other is relevant to the mankind, such as Viral Hemorrhagic septicemia (VHS), cytomegalovirus (CMV), chronic burkitt's lymphoma virus (EBV), irido virus, mankind's herpus vivus 8 type, haematopoietic necrosis virus (HHNV), tomato spotted wilf virus, tomato yellow leaf curl virus etc..At present, the LAMP kit for detecting Ebola virus and primer special thereof it are showed no both at home and abroad.
Summary of the invention
Present invention provide for Ebola virus is carried out the primer of LAMP detection, to realize the batch detection of Ebola virus, improve specificity and the sensitivity of detection.
LAMP primer for detecting Ebola virus provided by the present invention, design according to Ebola virus specific and conserved sequence-N protein gene (GenBank:KM655246.1), in order to the Ebola virus in the samples such as the pure virus of qualitative detection, blood, feces, described LAMP primer is made up of six primers, including the combination of outer primer F3 and B3, inner primer FIP and BIP and ring primer LF and LB；Described Ebola virus specific conservative target sequence-N protein gene is such as shown in SEQ ID NO:1.
Specifically, shown in the nucleotide sequence such as SEQ ID NO:2 (F3) of described six primers for Ebola virus being carried out LAMP detection, SEQIDNO:3 (B3), SEQIDNO:4 (FIP), SEQIDNO:5 (BIP), SEQIDNO:6 (LF) and SEQIDNO:7 (LB).
Described LAMP primer, for the compositions of F3 and B3, FIP and BIP and LF and LB 5:40:20 in molar ratio.
Second purpose of the present invention is to provide a kind of test kit for Ebola virus carries out LAMP detection.
Test kit provided by the present invention, including the above-mentioned primer for Ebola virus carries out LAMP detection.
Specifically, described test kit includes following reagent: 20mMTris HCl (pH8.8), 10mMKCl, 10mM (NH4)2SO4, 0.1%Tween20,0.8M glycine betaine (betaine), 8mMMgSO4, 1.4mMdNTPeach, 8UBstDNA Large fragment polymerase and 8UAMV reverse transcriptase, primer addition is: 5pmol primers F 3 and B3,40pmol primers F IP and BIP, 20pmol primer LF and LB.
For convenience of detection, may also include positive control and negative control in described test kit, described positive control is the cloned plasmids carrying Ebola virus N protein gene, and described negative control is the LAMP amplification system without DNA, such as distilled water.
Above-mentioned LAMP primer or test kit application in Ebola virus LAMP detects fall within protection scope of the present invention.
3rd purpose of the present invention is to provide the LAMP detection method of a kind of Ebola virus.
Detection method provided by the present invention, it may include following steps:
1) with testing sample geneome RNA for template, carrying out LAMP amplification under the guiding of above-mentioned primer, 25 μ lLAMP reaction systems include: testing sample geneome RNA 2 μ l, 20mMTris HCl (pH8.8), 10mMKCl, 10mM (NH4)2SO4, 0.1%Tween20,0.8M glycine betaine (betaine), 8mMMgSO4, 1.4mMdNTPeach, 8UBstDNA Large fragment polymerase and 8UAMV reverse transcriptase, primer addition is: 5pmol primers F 3 and B3,40pmol primers F IP and BIP, 20pmol primer LF and LB；LAMP amplification condition is: put 60-65 DEG C of constant temperature 45-55min；
2) reaction carries out result judgement after terminating: add calcein indicator in reactant liquor, changes judged result according to the color of reactant liquor, there is Ebola virus, be absent from Ebola virus in orange expression testing sample in green expression testing sample；Or directly carrying out judged result with the turbidity change of reactant liquor before and after transmissometer detection reaction without calcein indicator, turbidity rises and represents in testing sample there is Ebola virus, is absent from Ebola virus in the unchanged expression testing sample of turbidity.
In above-mentioned detection method, described step 1) in LAMP reaction system in be additionally provided with positive control and negative control, described positive control is the cloned plasmids 2 μ l carrying Ebola virus N protein gene, and described negative control is the LAMP amplification system 2 μ l without DNA, such as distilled water；Described LAMP amplification condition is preferably: put 65 DEG C of constant temperature 50min.
Described step 2) in the addition of calcein indicator can be 1 μ l (end reaction system is 26 μ l), containing 0.5mM calcein and 10mM manganese chloride.
The invention provides the LAMP detection method of a kind of Ebola virus and primer special thereof and test kit.
The invention have the advantages that
1) high specific: the identification of 8 specific regions of 6 primer pair Ebola virus target sequences ensure that the LAMP high degree of specificity expanded, namely LAMP can find out corresponding target sequence and expands from the gene specimen of difference only one nucleotide；
2) high sensitivity: remolding sensitivity regular-PCR is high 10 times；
3) result identifies simplicity: can pass through visual results (calcein colour developing), it is also possible to directly use transmissometer judged result；
4) it is simple to operate: as long as detection sample (target nucleic acid) and detectable are put in 60-65 DEG C of thermostat water bath after 45-55min it may determine that result together；
5) quickly, efficient amplification: whole LAMP amplified reaction can complete in 1 hour, and productivity can reach 0.5mg/mL.
In sum, the present invention can under isothermal conditions quickly, convenient, efficiently, high special, Ebola virus detected with sensitivity, do not need complex instrument, detection for Ebola virus provides new technology platform, can be used for Animal husbandry production unit, primary care health unit and the examination of each disease prevention and control center and detection Ebola virus, there are wide market prospect and bigger economical, societal benefits, be suitable to popularization and application on a large scale.
Below in conjunction with specific embodiment, the present invention is described in further details.
Accompanying drawing explanation
Fig. 1 is the physical map of the cloned plasmids carrying Ebola virus N protein gene；
Fig. 2 is the LAMP response curve of five set primers；
Fig. 3 is the specific transmissometer testing result of Ebola virus LAMP detection method；
Fig. 4 is the calcein indicator testing result that Ebola virus LAMP detection method is specific；
Fig. 5 is the transmissometer testing result of Ebola virus LAMP detection method sensitivity；
Fig. 6 is the calcein indicator testing result of Ebola virus LAMP detection method sensitivity；
Fig. 7 is the testing result of Ebola virus regular-PCR detection method sensitivity.
Detailed description of the invention
In following embodiment, method therefor is conventional method if no special instructions, concrete steps can be referring to: " MolecularCloning:ALaboratoryManual " (Sambrook, J., Russell, DavidW., MolecularCloning:ALaboratoryManual, 3rdedition, 2001, NY, ColdSpringHarbor).
Described in embodiment to the acquirement approach of various biomaterials be only to provide a kind of approach testing acquisition to reach concrete disclosed purpose, should not become the restriction to biological material source of the present invention.It is true that the source of used biomaterial is widely, any biomaterial that can obtain with moral ethics that keeps on the right side of the law can be replaced according to the prompting in embodiment and use.
The primer synthesizes by Beijing Sheng Gong biotech firm, and standard substance plasmid is by the synthesis of Bo Maide biological (Beijing) company limited.
Embodiment is carried out under premised on technical solution of the present invention, gives detailed embodiment and concrete operating process, and embodiment will assist in understands the present invention, but protection scope of the present invention is not limited to following embodiment.
Embodiment 1, design for carrying out the primer of LAMP detection to Ebola virus
Ebola virus sequence is obtained from U.S.'s gene data library searching, homology analysis is carried out by BLAST software, obtain the specific and conserved sequence-N protein gene (GenBank:KM655246.1 of Ebola virus, SEQ ID NO:1) design, according to this conservative target-gene sequence, with software PrimerdesignV4 design for Ebola virus being carried out the primer of LAMP detection, design 5 set primers altogether, through Experimental comparison, finally have chosen combination of primers EBL2, sequence is as shown in table 1.
Table 1 for carrying out the primer sequence of LAMP detection to Ebola virus
Embodiment 2, set up the LAMP detection method of Ebola virus
One, the cloned plasmids (positive control) carrying Ebola virus N protein gene is built
The conservative target-gene sequence of the specific conservative's N protein gene according to Ebola virus, external synthesis Ebola virus N protein gene (633bp, SEQ ID NO:8), this genetic fragment is connected with expression vector pBM19-B (purchased from Bo Maide company), connection site is LacZ, obtaining carrying the recombinant vector of Ebola virus N protein gene, called after pBM19-T, shown in its physical map such as Fig. 1 (NP is N protein gene).Plasmid is extracted with the plasmid extraction kit of Takara company.
Two, the best combination of primers for Ebola virus LAMP detection method is determined
The cloned plasmids (positive control) carrying Ebola virus N protein gene step one built by 5 set combination of primers of embodiment 1 design carries out LAMP detection, and to obtain best combination of primers, concrete grammar is as follows:
1) with carry Ebola virus N protein gene cloned plasmids for template, LAMP amplification is carried out under the guiding of 5 set primers in embodiment 1, wherein, 25 μ lLAMP reaction systems include: carry the cloned plasmids 2 μ l of Ebola virus N protein gene, 20mMTris HCl (pH8.8), 10mMKCl, 10mM (NH4)2SO4, 0.1%Tween20,0.8M glycine betaine (betaine), 8mMMgSO4, 1.4mMdNTPeach, 8UBstDNA Large fragment polymerase and 8UAMV reverse transcriptase, primer addition is: 5pmol primers F 3 and B3,40pmol primers F IP and BIP, 20pmol primer LF and LB；Reaction condition is: put 50min in 65 DEG C of thermostat water baths；
2) reaction carries out result and judges after terminating: add the 1 μ l (end reaction system as 26 μ l) the calcein indicator containing 0.5mM calcein and 10mM manganese chloride in reactant liquor, color change judged result (principle: calcein is Metal ion indicator, Mg in energy Indicator Reaction liquid according to reactant liquor2+Change), green represent in testing sample there is Ebola virus (positive), orange expression testing sample be absent from Ebola virus (feminine gender)；Or directly carry out judged result with the turbidity change of reactant liquor before and after transmissometer detection reaction without calcein indicator and (process of principle: LAMP reaction can produce magnesium pyrophosphate, magnesium pyrophosphate is a kind of white precipitate, according to the change of turbidity, transmissometer can judge that LAMP reacts), turbidity rises and represents in testing sample there is Ebola virus (positive), the unchanged expression testing sample of turbidity is absent from Ebola virus (feminine gender), (abscissa is the product turbidity value at 650nm to LAMP response curve such as Fig. 2 of 5 set primers, vertical coordinate is the response time) shown in, as can be seen from the figure the reaction effect of combination of primers EBL2 is best.
Therefore, the best combination of primers of the present invention is: primers F 3 (SEQIDNO:2) and B3 (SEQIDNO:3): primers F IP (SEQIDNO:4) and BIP (SEQIDNO:5): primer LF (SEQIDNO:6) and LB (SEQIDNO:7) are 5:40:20 in molar ratio.
The 25 μ lLAMP reaction systems of best detection Ebola virus include: testing sample geneome RNA 2 μ l, 20mMTris HCl (pH8.8), 10mMKCl, 10mM (NH4)2SO4, 0.1%Tween20,0.8M glycine betaine (betaine), 8mMMgSO41.4mMdNTPeach, 8UBstDNA Large fragment polymerase and 8UAMV reverse transcriptase, primer addition is: 5pmol primers F 3 and B3 (shown in SEQIDNO:2 and SEQIDNO:3 primer), 40pmol primers F IP and BIP (shown in SEQIDNO:4 and SEQIDNO:5 primer), 20pmol primer LF and LB (shown in SEQIDNO:6 and SEQIDNO:7 primer).
Three, the best amplification condition for Ebola virus LAMP detection method is determined
With combination of primers EBL2, Ebola virus being carried out LAMP detection, to obtain best amplification condition, concrete grammar is as follows:
1) carrying out LAMP amplification under the guiding of combination of primers EBL2,25 μ lLAMP reaction systems include: carry the cloned plasmids 2 μ l, 20mMTris HCl (pH8.8) of Ebola virus N protein gene, 10mMKCl, 10mM (NH4)2SO4, 0.1%Tween20,0.8M glycine betaine (betaine), 8mMMgSO41.4mMdNTPeach, 8UBstDNA Large fragment polymerase and 8UAMV reverse transcriptase, primer addition is: 5pmol primers F 3 and B3 (shown in SEQIDNO:2 and SEQIDNO:3 primer), 40pmol primers F IP and BIP (shown in SEQIDNO:4 and SEQIDNO:5 primer), 20pmol primer LF and LB (shown in SEQIDNO:6 and SEQIDNO:7 primer)；LAMP amplification condition is: put 60-65 DEG C of constant temperature 45-55min；
2) reaction carries out result judgement after terminating: identical with step one.
Result is under the LAMP amplification condition of 60-65 DEG C of constant temperature 45-55min, and combination of primers EBL2 all achieves good reaction effect, and particularly under the LAMP amplification condition of 65 DEG C of constant temperature 50min, reaction effect is best.
The LAMP amplification condition of best detection Ebola virus is: put 60-65 DEG C of constant temperature 45-55min, it is preferred to put 65 DEG C of constant temperature 50min.
Embodiment 3, the specificity of LAMP detection method of Ebola virus, susceptiveness detect
One, the specific detection of the LAMP detection method of Ebola virus
Respectively with the respiratory tract disease virus gene extract of the cloned plasmids (positive control) carrying Ebola virus N protein gene and other non-Ebola virus of 25 strain for template, with distilled water for negative control, the specificity of the best Ebola virus LAMP detection method that detection embodiment 2 obtains.
(abscissa is the product turbidity value at 650nm to the specific transmissometer testing result such as Fig. 3 of Ebola virus LAMP detection method, vertical coordinate is the response time) shown in, No. 1 curve is the cloned plasmids (positive control) carrying Ebola virus N protein gene, 2-26 curve is Respirovirus 1-25 strain, No. 27 is distilled water (negative control), as can be seen from the figure, the cloned plasmids (positive control) only carrying Ebola virus N protein gene there occurs that LAMP reacts, and all the other all do not occur；(1 is the cloned plasmids (positive control) carrying Ebola virus N protein gene to the specific calcein indicator testing result such as Fig. 4 of Ebola virus LAMP detection method；2-26 is the 1-25 strain of other Respirovirus, 27 is negative control (distilled water)) shown in, calcein indicator testing result is consistent with transmissometer testing result, show that the LAMP detection method of Ebola virus has higher specificity, it is possible to Ebola virus detected specifically.
Two, the sensitivity technique of the LAMP detection method of Ebola virus
By carry the cloned plasmids (positive control) of Ebola virus N protein gene with 10 times of gradients (1 times, 10 times, 102Again, 103Again, 104Again, 105Again, 106Again, 107Times) be diluted, make 1-7 template is carried the concentration respectively 23ng/ μ l of the cloned plasmids (positive control) of Ebola virus N protein gene, 2.3ng/ μ l, 0.23ng/ μ l, 23pg/ μ l, 2.3pg/ μ l, 0.23pg/ μ l, 0.023pg/ μ l, again with the plasmid through gradient dilution for template, with distilled water for negative control, respectively the sensitivity of LAMP detection method and regular-PCR detection method (primer sequence is F3 and B3) is detected.
null(template concentrations of 1-7 is respectively as follows: 23ng/ μ l to the specific transmissometer testing result such as Fig. 5 of Ebola virus LAMP detection method、2.3ng/μl、0.23ng/μl、23pg/μl、2.3pg/μl、0.23pg/μl、0.023pg/μl，8 is negative control) shown in，(template concentrations of 1-7 is respectively as follows: 23ng/ μ l to the specific calcein indicator testing result such as Fig. 6 of Ebola virus LAMP detection method、2.3ng/μl、0.23ng/μl、23pg/μl、2.3pg/μl、0.23pg/μl、0.023pg/μl，8 is negative control) shown in，(template concentrations of swimming lane 1-7 is respectively as follows: 23ng/ μ l to the testing result of regular-PCR such as Fig. 7、2.3ng/μl、0.23ng/μl、23pg/μl、2.3pg/μl、0.23pg/μl、0.023pg/μl，Swimming lane 8 is negative control，Swimming lane M is DNAmarkerD2000) shown in，Target product clip size is 216bp，Can be seen that，The LAMP detection method of Ebola virus can detect that 2.3pg/ μ l carries the cloned plasmids (10 of Ebola virus N protein gene4Times diluted concentration), and regular-PCR method is only capable of the cloned plasmids (10 that 23pg/ μ l carries Ebola virus N protein gene being detected3Times diluted concentration), and calcein indicator detection method is consistent with the result of transmissometer detection method, it was shown that the LAMP detection method of Ebola virus ratio highly sensitive 10 times of regular-PCR detection method.
The test kit that embodiment 4, LAMP for Ebola virus detect
By 20mMTris HCl (pH8.8), 10mMKCl, 10mM (NH4)2SO4, 0.1%Tween20,0.8M glycine betaine (betaine), 8mMMgSO4, 1.4mMdNTPeach, 8UBstDNA Large fragment polymerase and 8UAMV reverse transcriptase, primer addition is: 5pmol primers F 3 and B3 (shown in SEQIDNO:2 and SEQIDNO:3 primer), 40pmol primers F IP and BIP (shown in SEQIDNO:4 and SEQIDNO:5 primer), 20pmol primer LF and LB (shown in SEQIDNO:6 and SEQIDNO:7 primer), the cloned plasmids 2 μ l carrying Ebola virus N protein gene as positive control, and the distilled water 2 μ l as negative control packs jointly, obtain the LAMP detection kit of Ebola virus for 25 μ l reaction systems.
Claims (10)
1. for detecting the LAMP primer of Ebola virus, design according to Ebola virus specific and conserved sequence-N protein gene (GenBank:KM655246.1), in order to the Ebola virus in the samples such as the pure virus of qualitative detection, blood, feces, described LAMP primer is made up of six primers, including the combination of outer primer F3 and B3, inner primer FIP and BIP and ring primer LF and LB；Described Ebola virus specific conservative target sequence-N protein gene is such as shown in SEQ ID NO:1.
2. LAMP primer according to claim 1, it is characterised in that: shown in the nucleotide sequence such as SEQ ID NO:2 (F3) of described six primers for Ebola virus being carried out LAMP detection, SEQIDNO:3 (B3), SEQIDNO:4 (FIP), SEQIDNO:5 (BIP), SEQIDNO:6 (LF) and SEQIDNO:7 (LB).
3. LAMP primer according to claim 2, it is characterised in that: for the compositions of F3 and B3, FIP and BIP and LF and LB 5:40:20 in molar ratio.
4. for Ebola virus being carried out a test kit for LAMP detection, including described in claim 1 or 2 or 3 for Ebola virus being carried out the primer of LAMP detection.
5. test kit according to claim 4, it is characterised in that: described test kit includes the following reagent for 25 μ l reaction systems: 20mMTris HCl (pH8.8), 10mMKCl, 10mM (NH4)2SO4, 0.1%Tween20,0.8M glycine betaine (betaine), 8mMMgSO4, 1.4mMdNTPeach, 8UBstDNA Large fragment polymerase and 8UAMV reverse transcriptase, primer addition is: 5pmol primers F 3 and B3,40pmol primers F IP and BIP, 20pmol primer LF and LB.
6. test kit according to claim 5, it is characterized in that: described test kit also includes positive control and negative control, described positive control is the cloned plasmids carrying Ebola virus N protein gene, and described negative control is the LAMP amplification system without DNA, such as distilled water.
7. the LAMP primer described in claim 1 or 2 or 3 or the application in the LAMP of Ebola virus detects of the test kit described in claim 4 or 5 or 6.
8. application according to claim 7, it is characterised in that: the LAMP for Ebola virus detects, and comprises the following steps:
1) with testing sample geneome RNA 2 μ l for template, LAMP amplification is carried out under the guiding of primer described in claim 1 or 2 or 3,25 μ lLAMP reaction systems include: testing sample geneome RNA 2 μ l20mMTris HCl (pH8.8), 10mMKCl, 10mM (NH4)2SO4, 0.1%Tween20,0.8M glycine betaine (betaine), 8mMMgSO4, 1.4mMdNTPeach, 8UBstDNA Large fragment polymerase and 8UAMV reverse transcriptase, primer addition is: 5pmol primers F 3 and B3,40pmol primers F IP and BIP, 20pmol primer LF and LB；LAMP amplification condition is: put 60-65 DEG C of constant temperature 45-55min；
2) reaction carries out result judgement after terminating: add calcein indicator in reactant liquor, changes judged result according to the color of reactant liquor, there is Ebola virus, be absent from Ebola virus in orange expression testing sample in green expression testing sample；Or directly carrying out judged result with the turbidity change of reactant liquor before and after transmissometer detection reaction without calcein indicator, turbidity rises and represents in testing sample there is Ebola virus, is absent from Ebola virus in the unchanged expression testing sample of turbidity.
9. application according to claim 8, it is characterized in that: described step 1) in LAMP reaction system in be additionally provided with positive control and negative control, described positive control is the cloned plasmids 2 μ l carrying Ebola virus N protein gene, described negative control is the LAMP amplification system 2 μ l without DNA, such as distilled water；Described LAMP amplification condition is preferably: put 65 DEG C of constant temperature 50min.
10. application according to claim 8, it is characterised in that: described step 2) in the addition of calcein indicator can be 1 μ l (end reaction system is 26 μ l), containing 0.5mM calcein and 10mM manganese chloride.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109022621A (en) * | 2018-09-03 | 2018-12-18 | 中国科学院武汉病毒研究所 | Nipah virus detection kit and its primer special |
| CN109022621B (en) * | 2018-09-03 | 2020-12-11 | 中国科学院武汉病毒研究所 | Nipah virus detection kit and special primer thereof |
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