CN103937912A - LAMP (Loop-Mediated Isothermal Amplification) primer composition for detecting infectious pancreas necrosis virus and application of LAMP primer composition - Google Patents
LAMP (Loop-Mediated Isothermal Amplification) primer composition for detecting infectious pancreas necrosis virus and application of LAMP primer composition Download PDFInfo
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Abstract
The invention discloses an LAMP (Loop-Mediated Isothermal Amplification) primer composition for detecting an infectious pancreas necrosis virus and an application of the LAMP primer composition. The LAMP primer composition is composed of a forward inner primer (FIP) with a sequence as shown in SEQ ID No.1, a backward inner primer with a sequence as shown in SEQ ID No.2, a forward outer primer F3 with a sequence as shown in SEQ ID No.3 and a backward outer primer B3 with a sequence as shown in SEQ ID No.4. The LAMP primer composition disclosed by the invention is used for rapidly detecting the infectious pancreas necrosis virus in a sample, favorable in specificity and sensitivity for the infectious pancreas necrosis virus, simple in required detecting instruments, capable of rapidly, efficiently and accurately detecting the infectious pancreas necrosis virus and suitable for being popularized to port check and examination organizations and basic laboratories.
Description
Technical field
The invention belongs to genetically engineered field, relate to LAMP primer sets compound and application thereof for detection of infectious pancreas necrosis virus.
Background technology
Infectious pancreas necrosis virus (Infectious pancreatic necrosis virus, IPNV) is one very important fish disease pathogenic agent economically, can cause and serious infection in salmon fishes, causes high mortality ratio.Initial only in North America and some countries in Europe popular, along with the increase of hydrocoles import trade, infectious pancreatic necrosis has imported China popular in some areas into, has caused serious financial loss in recent years.At present, China mainly contains viral separation and authentication method, reverse transcription-polymerase chain reaction method, enzyme-linked immunologic adsorption test method etc. to the detection method of this disease pathogen IPNV, though these detection methods have reached diagnosis accurately, but length consuming time, cost are high, plant and instrument requires high, detecting step complexity, can not meet fast, accurately detect this viral demand.Therefore, need to set up a kind of method of quick, effective, the accurate and applicable field condition operation for IPNV, for the Salmons cultivation of China provides scientific and effective epidemic disease epidemic situation prevention thinking.
LAMP (Loop-Mediated Isothermal Amplification) method is the constant temperature nucleic acid amplification method by a kind of novelty of the inventions such as Notomi in 2000.The method adopts the Bst archaeal dna polymerase of identifying specifically four primers (two outer primer, two inner primers) in six regions on target sequence and having strand displacement activity, carries out the exponential amplification of nucleic acid 65 DEG C of left and right, and its amplification efficiency can reach 10
9-10
10individual copy number magnitude.Whole reaction only needs 1 hour, and in amplified reaction, by product magnesium pyrophosphate precipitation is combined with fluorexon, produces yellow-green fluorescence, need to just can not distinguish experimental result by other instruments.But the method is not also applied to the detection of IPNV at present.
Summary of the invention
The object of the present invention is to provide a kind of for fast, accurately detecting the LAMP primer sets compound of infectious pancreas necrosis virus (IPNV).
Another object of the present invention is to the application of this LAMP primer sets compound.
Object of the present invention can realize by following technical solution:
For detection of a LAMP primer sets compound for infectious pancreas necrosis virus, this primer sets compound is that forward inner primer FIP, the sequence shown in SEQ ID No.1 is that reverse inner primer BIP, the sequence shown in SEQ ID No.2 is that forward outer primer F3, the sequence shown in SEQ ID No.3 is that the reverse outer primer B3 shown in SEQ ID No.4 forms by sequence.
The present invention also provides above-mentioned LAMP primer sets compound in the application detecting in infectious pancreas necrosis virus, comprises the following steps:
(1) extract the RNA in sample;
(2) taking the middle RNA extracting of step (1) as template, carry out RT-LAMP amplified reaction;
(3) RT-LAMP amplified production step (3) being obtained detects.
In above-mentioned steps (2), RT-LAMP amplification reaction condition is: at 60-65 DEG C, react 30-90 minute, stopped reaction; Highly preferred reaction conditions is: at 62 DEG C, react stopped reaction 60 minutes.
In above-mentioned steps (3), whether LAMP amplified production contains infectious pancreas necrosis virus in can adopting and judging sample with the following method: 1. amplified production carries out agarose gel electrophoresis, under ultraviolet lamp, observe, if there is scalariform band away from point sample hole, prove that the virus detecting is infectious pancreas necrosis virus, contains this virus in sample; 2. after adding double-stranded DNA (dsDNA) dyestuff PicoGreen developer in amplified production, observe fluorescence color variation, if reacted reaction solution shows green fluorescence, prove that the virus detecting is infectious pancreas necrosis virus, contains this virus in sample; 3. from LAMP amplified reaction zero hour by Loopamp turbidimeter detection reaction liquid, finish to amplified reaction, observe LAMP turbidimeter and detect figure, if turbidity increases gradually, the color of module changes redness into gradually by green, prove that the virus detecting is infectious pancreas necrosis virus, contains this virus in sample.
Be with respect to prior art beneficial effect of the present invention: LAMP primer sets compound of the present invention, infectious pancreas necrosis virus (IPNV) in can rapid detection sample, this LAMP primer sets compound only carries out specific amplification to IPNV, with other virus no cross reaction, the more conventional PCR method of the specificity to IPNV and sensitivity is high.Utilize LAMP combination of primers quality testing of the present invention to survey IPNV, detection time is short, the specificity to IPNV and highly sensitive, and required detecting instrument is simple, can fast, efficiently, accurately detect infectious pancreas necrosis virus, be applicable to Check and Examination of Port quarantine mechanism and laboratories and promote.Method of the present invention, for China fish can export the country such as European Union, the U.S. and area lays the foundation smoothly, is also to find in time fish disease epidemic situation hidden danger, and improving epidemic situation early warning and prevention and control ability provides theoretical foundation.
Brief description of the drawings
Fig. 1 is the agarose electrophoresis figure that utilizes the LAMP amplified reaction result that LAMP composition of the present invention carries out under differing temps, wherein swimming lane M is DL2000Marker, LAMP amplification when swimming lane 1-6 is followed successively by 60 DEG C of temperature of reaction, 61 DEG C, 62 DEG C, 63 DEG C, 64 DEG C and 65 DEG C.
Fig. 2 is the agarose electrophoresis figure that utilizes the LAMP amplified reaction result that LAMP primer sets compound of the present invention carries out under the differential responses time, wherein swimming lane M is DL2000Marker, LAMP amplification when swimming lane 1-5 respectively is isothermal reaction 30min, 45min, 60min, 75min and 90min.
Fig. 3 is the detected result of utilizing the LAMP amplified reaction result that LAMP primer sets compound of the present invention carries out, wherein Fig. 3 A is by the result of agarose gel electrophoresis interpretation: in electrophorogram, swimming lane M is DL2000Marker, and swimming lane 1 and 2 is respectively IPNV sample and negative control; Fig. 3 B changes the result of interpretation by detecting turbidity: from LAMP amplified reaction zero hour by Loopamp turbidimeter detection reaction liquid, finish to amplified reaction, in Fig. 3 B, X-coordinate 1 and 2 is respectively IPNV sample and negative control.
Fig. 4 utilizes LAMP combination of primers quality testing of the present invention to survey the agarose gel electrophoresis figure of the specificity analyses of IPNV, wherein swimming lane M is DL2000Marker, swimming lane 1-5 respectively is the amplified production of the RT-LAMP of IPNV, IHNV, SVCV, IPNV and ISA, the negative contrast of swimming lane 6.
Fig. 5 utilizes LAMP combination of primers quality testing of the present invention to survey the agarose gel electrophoresis figure of the sensitivity analysis of IPNV, wherein swimming lane M is DL2000Marker, amplified production when swimming lane 1-8 respectively is IPNV rna content 1.0 μ g, 0.1 μ g, 0.01 μ g, 1.0ng, 0.1ng, 0.01ng, 0.01pg, 0.001pg in reaction system.
Embodiment
Following non-limiting example can make the present invention of those of ordinary skill in the art's comprehend, but does not limit the present invention in any way.In following embodiment, if no special instructions, the experimental technique using is ordinary method, and agents useful for same etc. all can be bought from biological or chemical reagent company.
The design of embodiment 1 LAMP primer sets of the present invention compound and synthetic
According to the nucleotide sequence (Genbank NO:AF342728.1) of the A fragment polyprotein of encoding in Genbank IPNV genome, use Primer Explore V3 and Primer Premier5.0 software for 6 regions (F3c of 3 ' end wherein, F2c, the B1 of F1c and 5 ' end, B2, B3) design LAMP primer, different primers group is carried out LAMP amplification experiment and detected and analyze, it is high that finishing screen is selected rate of amplification, one group of LAMP primer that specificity is good, be respectively forward inner primer FIP, oppositely inner primer BIP, forward outer primer F3, oppositely outer primer B3, its sequence is as table 1.Primer is synthetic by Dalian precious biotechnology company limited.
Table 1.IPNV RT-LAMP detects primer sets
The optimization of embodiment 2RT-LAMP reaction conditions
1. the preparation of sample RNA template
Infectious pancreatic necrosis virus (IPNV) strain isolated using in the present invention, by document (Hu Xiaoli, Li Wei, start intelligent monarch, Wu Bin. separation and the qualification of rainbow trout infectious pancreatic necrosis virus, Chinese Animal Quarantine, 2012,29 (3): 27-30) described method separates and obtains, and is stored in Dong Jian laboratory, Liaoning Entry-Exit Inspection and Quarantine Bureau technique center.Adopt Takara MiniBEST Viral RNA/DNA Extraction Kit Ver.4.0 test kit, from this virocyte culture, extract and obtain sample RNA, concrete operation step reference reagent box specification sheets.
2.RT-LAMP reaction system
RT-LAMP amplified reaction adopts Yeast Nucleic Acid amplification kit (RT-LAMP), Shenzhen Bo Ruixiang Bioisystech Co., Ltd, concrete operation step reference reagent box specification sheets.
RT-LAMP adopts 25 μ L reaction systems, and the component dosage adding is as follows:
When preparation reaction system, with the nuclease free H of same volume
2o substitutes RNA template as negative control.
The amplified reaction of 3.RT-LAMP and result are judged
Carry out after RT-LAMP amplified reaction according to above-mentioned steps 2 ready reaction systems, amplified production is judged by following 3 kinds of methods.
Method 1: by agarose gel electrophoresis sentence read result: get amplified production 8 μ L and carry out gel electrophoresis, under UV projection, observe, there is hangover near point sample hole, occur that away from point sample hole the sample of scalariform band is judged to the positive, do not occur that the sample of gradient band is judged to feminine gender.
Method 2: change sentence read result (fluorescence developing method) by observing fluorescence color: add double-stranded DNA (dsDNA) dyestuff PicoGreen in reaction tubes, reacted reaction solution shows the positive that is judged to of green fluorescence, and reacted reaction solution shows the feminine gender that is judged to of light orange fluorescence.
Method 3: change sentence read result by detecting turbidity: from LAMP amplified reaction zero hour by Loopamp turbidimeter (meeting of Japanese Rong Yan chemistry strain formula, LA-320C; Parameter: light source is ED (wavelength 650mm), and analyzer is photorectifier, temperature regulating range is region (55-70 DEG C), light shield (65-90 DEG C), and measuring interval is 6 seconds, and detection reaction liquid to amplified reaction finishes.Detect in figure at LAMP turbidimeter, along with the increase of reaction product turbidity, module color transition is red, shows that IPNV gene is amplified, and is judged to be the positive; And reaction solution turbidity unchanged (being bordering on 0) while increasing, it is green that the color of module is still, and shows not exist the amplification of IPNV gene, is judged to be feminine gender.
In aforesaid method, described " positive " represents, contains infectious pancreas necrosis virus in sample; Described " positive " represents not contain infectious pancreas necrosis virus in sample.
The optimization of 4.RT-LAMP amplified reaction optimal reaction temperature
Prepare 6 identical reaction systems (same template, the configuration of identical component) according to step 2, reaction system is respectively at the temperature of 60 DEG C, 61 DEG C, 62 DEG C, 63 DEG C, 64 DEG C and 65 DEG C, and isothermal reaction 60min, to determine optimal reaction temperature;
From the reaction tubes having reacted, get 8 μ L amplified productions and carry out gel electrophoresis, electrophoresis result is as Fig. 1, reaction tubes amplified production when wherein swimming lane 1-6 is followed successively by 60 DEG C of temperature of reaction, 61 DEG C, 62 DEG C, 63 DEG C, 64 DEG C and 65 DEG C.Swimming lane M is DL2000Marker.Under UV projection, observe, the amplified production of reaction tubes 1-6, all occurring scalariform band away from point sample hole, is positive, and when wherein temperature of reaction is 62 DEG C, scalariform feature band is the brightest, illustrates that when temperature of reaction is 62 DEG C, expanding effect is best.
In addition, in the reaction tubes that contains amplified production 6 μ L, add 1 μ L double-stranded DNA (dsDNA) dyestuff PicoGreen developer, the color of observing response liquid, the reaction solution that can observe 6 reaction tubess all shows the green fluorescence of varying strength, and wherein temperature of reaction is that the green fluorescence that produces in the reaction tubes of 62 DEG C is the darkest.
Comprehensive for the above-mentioned result of determination to the amplified production at differential responses temperature, can determine that 62 DEG C of temperature of reaction are to utilize the suitableeest RT-LAMP temperature of reaction of LAMP primer sets compound amplification IPNV gene of the present invention.
The optimization of 5.RT-LAMP amplified reaction optimum reacting time
Prepare 5 identical reaction systems (same template, identical component configure) according to step 2, reaction system is at the temperature of 62 DEG C, and isothermal reaction 30min, 45min, 60min, 75min and 90min respectively, to determine optimum reacting time.
From the reaction tubes having reacted, get 8 μ L amplified productions and carry out gel electrophoresis, as shown in Figure 2, wherein swimming lane M is DL2000Marker to electrophoresis result, and swimming lane 1-5 respectively is the LAMP reaction amplified production after isothermal reaction 30min, 45min, 60min, 75min and 90min.Visible in Fig. 2, in 45min-90min reaction time range, all can see scalariform feature band, but the brightest band producing during for reaction times 60min can determine that optimum reacting time is 60min.
Therefore the optimum reaction condition that in the present invention, adopts selected LAMP combination of primers quality testing to survey the RT-LAMP amplified reaction of infectious pancreas necrosis virus is: at 62 DEG C of isothermal reaction 60min.
Embodiment 3 different methods are judged the RT-LAMP amplified production that utilizes LAMP primer sets compound to carry out
Prepare to add example reaction system (being called for short: IPNV sample) and the negative control reaction system of infectious pancreas necrosis virus strain isolated RNA template according to the method for step 2 in embodiment 2, after 62 DEG C of isothermal reaction 60min, stopped reaction, adopts the LAMP reaction result decision method described in step 3 in embodiment 2 to judge in sample whether contain IPNV.
Method 1: from the reaction tubes having reacted, get 8 μ L amplified productions and carry out gel electrophoresis, electrophoresis result is as Fig. 3 A, and wherein swimming lane M is DL2000Marker, and swimming lane 1 and 2 is respectively IPNV sample and negative control.The demonstration of Fig. 3 result, there is scalariform specific band in the swimming lane of IPNV sample, and there is not scalariform specific band in negative control, in known IPNV sample, IPNV gene is amplified, and contains IPNV in sample.
Method 2: add 1 μ L double-stranded DNA (dsDNA) dyestuff PicoGreen developer in the reaction tubes that contains amplified production 6 μ L, the color of observing response tube reaction liquid, the reaction solution that can observe IPNV sample shows very strong green fluorescence, IPNV is amplified, in sample, contain IPNV, and the reaction solution of negative control shows light orange fluorescence, without IPNV amplification, in sample, there is no IPNV.
Method 3: from LAMP amplified reaction zero hour by Loopamp turbidimeter (meeting of Japanese Rong Yan chemistry strain formula, LA-320C; Parameter: light source is ED (wavelength 650mm), analyzer is photorectifier, temperature regulating range is region (55-70 DEG C), light shield (65-90 DEG C), and measuring interval is 6 seconds) detection reaction liquid, finishes to amplified reaction.LAMP turbidimeter detects figure as shown in Figure 3 B.The X-coordinate 1 and 2 of Fig. 3 B is respectively IPNV sample and negative control, can find out that IPNV sample turbidity reaches 0.5, and the color of module is red, illustrates that IPNV is amplified; And the turbidity of negative control is 0, it is green that the color of module is still, and illustrates that reaction solution increases.
From the result of method 1-3, these 3 kinds of methods all can be for utilizing the judgement of IPNV gene amplification result of LAMP primer sets compound of the present invention, and the difference between the amplification of the positive that contains IPNV and negative control (without IPNV) is fairly obvious.Can adopt one or more the combination of method in above-mentioned 3 kinds of methods for utilizing the judgement of IPNV gene LAMP amplification of LAMP primer sets compound of the present invention amplification.
Embodiment 4 utilizes LAMP combination of primers quality testing of the present invention to survey the specificity analyses of IPNV
The RNA for preparing IPNV (infectious pancreas necrosis virus), IHNV (infectious hematopoietic necrosis's poison), SVCV (carp spring virus), VHSV (viral haemorrhagic septicaemia virus) and ISA (infectious salmon anaemia syndrome virus) according to the method described in embodiment 2 step 1-2 is as template, carry out respectively RT-LAMP amplified reaction, amplified reaction temperature is 62 DEG C, and the amplified reaction time is 60min.
From the reaction tubes having reacted, get respectively 8 μ L amplified productions and carry out gel electrophoresis, electrophoresis result is as Fig. 4, and wherein swimming lane M is DL2000Marker, swimming lane 1-5 respectively is the amplified production of the RT-LAMP of IPNV, IHNV, SVCV, IPNV and ISA, the negative contrast of swimming lane 6.The result of Fig. 4 is visible, only have the swimming lane 1 of amplification IPNV to occur scalariform specific band, gene is amplified, and all there is not trapezoid-shaped strips in all the other swimming lanes, gene is not amplified, show that LAMP primer sets compound of the present invention has only carried out specific amplification to IPNV, with other virus no cross reaction.
Embodiment 5 utilizes LAMP combination of primers quality testing of the present invention to survey the sensitivity analysis of IPNV
Prepare IPNV RNA according to the method described in embodiment 2 steps 1, and use nuclease free H
2o dilutes, and obtains the IPNV RNA of different concns, the 25 μ l LAMP reaction systems that preparation contains different concns IPNV RNA respectively.In reaction system, IPNV RNA total content is respectively 1.0 μ g (10
0doubly dilution), 0.1 μ g (10
-1doubly dilution), 0.01 μ g (10
-2doubly dilution), 1.0ng (10
-3doubly dilution), 0.1ng (10
-4doubly dilution), 0.01ng (10
-5doubly dilution), 0.01pg (10
-8doubly dilution), 0.001pg (10
-9doubly dilution).Described reaction system, carries out respectively RT-LAMP amplified reaction, and amplified reaction temperature is 62 DEG C, and the amplified reaction time is 60min.
From the reaction tubes having reacted, to get respectively 8 μ L amplified productions and carry out gel electrophoresis, electrophoresis result is as Fig. 5, and wherein swimming lane M is DL2000Marker, and swimming lane 1-8 respectively is IPNV RNA total content and is respectively 1.0 μ g (10
0doubly dilution), 0.1 μ g (10
-1doubly dilution), 0.01 μ g (10
-2doubly dilution), 1.0ng (10
-3doubly dilution), 0.1ng (10
-4doubly dilution), 0.01ng (10
-5doubly dilution), 0.01pg (10
-8doubly dilution), 0.001pg (10
-9doubly dilution) time amplified production.The result of Fig. 5 is visible, and scalariform specific band all appears in swimming lane 1-7, but along with the reduction of template ribonucleic acid content, the brightness of signature band also weakens, and trapezoid-shaped strips does not appear in swimming lane 8.The minimum RNA amount that occurs IPNV gene masculine result (occurring scalariform specific band) is 0.01pg RNA.
Claims (5)
1. the LAMP primer sets compound for detection of infectious pancreas necrosis virus, it is characterized in that, this LAMP primer sets compound is that forward inner primer FIP, the sequence shown in SEQ ID No.1 is that reverse inner primer BIP, the sequence shown in SEQ ID No.2 is that forward outer primer F3, the sequence shown in SEQ ID No.3 is that the reverse outer primer B3 shown in SEQ ID No.4 forms by sequence.
2. LAMP primer sets compound claimed in claim 1 is in the application detecting in infectious pancreas necrosis virus.
3. application according to claim 2, is characterized in that, comprises the following steps:
(1) extract the RNA in sample;
(2) taking the middle RNA extracting of step (1) as template, carry out RT-LAMP amplified reaction;
(3) LAMP amplified production step (2) being obtained detects.
4. application according to claim 3, is characterized in that, in step (2), RT-LAMP amplification reaction condition is: at 60-65 DEG C, react 30-90 minute, stopped reaction.
5. application according to claim 4, is characterized in that, described RT-LAMP amplification reaction condition is: at 62 DEG C, react stopped reaction 60 minutes.
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| CN105176979A (en) * | 2015-08-31 | 2015-12-23 | 孙翠言 | Method for detecting infectious pancreatic necrosis virus |
| CN109234458A (en) * | 2018-11-09 | 2019-01-18 | 中国水产科学研究院黑龙江水产研究所 | Detect the reagent set of infectious pancreas necrosis virus |
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| RU2732726C1 (en) * | 2019-09-03 | 2020-09-22 | Александр Юрьевич Михайлов | Diagnostic technique for acute infected pancreatonecrosis |
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| CN103937912B (en) | 2016-07-20 |
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