CN103224994A - Foot and mouth disease virus typing diagnosis loop-mediated isothermal amplification kit and its use method - Google Patents
Foot and mouth disease virus typing diagnosis loop-mediated isothermal amplification kit and its use method Download PDFInfo
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Abstract
The invention discloses a loop-mediated isothermal amplification kit used for the foot and mouth disease virus typing diagnosis, and its use method. The kit is characterized in that the kit is formed by an O type, Asia I type, A type, C type, SAT1 type, SAT2 type and SAT3 type foot and mouth disease virus typing diagnosis loop-mediated isothermal amplimer liquid mixture, a loop-mediated isothermal amplification reaction premix liquid, and various types of foot and mouth disease positive quality control substances and negative quality control substances, and can be used for the typing diagnosis of O type, Asia I type or A type or C type or SAT1 type or SAT2 type or SAT3 type foot and mouth disease viruses, or two or three or four or five or six or seven serotype foot and mouth disease viruses thereof. The use method of the kit is characterized in that the real-time typing diagnosis of the foot and mouth disease viruses can be realized through an isothermal amplification fluorescence detection system or an isothermal amplification turbidity system. The method has a strong specificity and high sensitivity, and provides a simple and rapid way for the control of the foot and mouth disease viruses and the investigation analysis of the trends of various types.
Description
Technical field
The present invention relates to primer sets, test kit and real-time diagnosis method thereof that a cover is used for viral classification diagnosis, relate in particular to one and overlap loop-mediated isothermal amplification (LAMP) primer group, test kit and the using method thereof that is used to diagnose O type, AsiaI type, A type, C type, SAT1 type, SAT2 type, SAT3 type foot and mouth disease virus, belong to the foot and mouth disease virus detection range.
Background technology
Foot and mouth disease (Food and Mouth Disease, FMD) be by foot and mouth disease virus (Food and Mouth Disease Virus, FMDV) a kind of acute, hot, the height contagiousness that infects that cloven-hoofed animal causes and the animal epidemic of long-distance communications fast, can give livestock breeding industry, especially foreign trade brings about great losses.Foot and mouth disease virus has O type, AsiaI type, A type, C type, SAT1 type, SAT2 type, seven kinds of serotypes of SAT3 type, there is not cross-immune reaction between each serotype, the epidemic situation of O type, A type and AsiaI type once appearred in China, C type and African type also threaten bigger to China in the appearance of surrounding countries, therefore set up fast and accurately foot and mouth disease virus classification diagnosis method for the domestic epidemic situation of prevention and control and prevent that the invasion of external virus has the important strategic meaning.
At present, the laboratory diagnostic method of foot and mouth disease mainly comprises Pathogen Biology method, immunological method and molecular biology method three major types.Wherein, Pathogen Biology method and immunological method have certain dependency to actual sample, especially for immune animal, infect and the symptom animal arranged and infected but do not occur as yet that the disease symptom animal is difficult to distinguish and judge timely and effectively, and can fundamentally solve this difficult problem based on the molecular biology method of detection of nucleic acids, therefore, real-time fluorescence quantitative PCR nucleic acid molecule diagnostic method becomes the standard method of China's foot and mouth disease virus diagnosis.Yet traditional PCR reaction pair temperature has certain dependency, and the temperature cycle variation device has not only increased detection time as technical guarantee accurately, has also increased the cost that detects.
Ring mediated isothermal amplification (LAMP) method is a kind of new nucleic acid amplification technologies by Japanese doctor Notomi invention in 2000, have more easy, quick, susceptibility is high and advantage such as high specificity.Through ten years development, this method obtains constantly perfect, and especially aspect judgement as a result, the existing visual method of multiple naked eyes is used widely, can control the aerosol pollution of uncapping and causing from the source, be particularly suitable for field quick detection and field and detect by electrophoresis.Chinese invention patent 200810034124 and 201210221684 provides the loop-mediated isothermal amplification fast detection method of O type and C type foot and mouth disease virus respectively, and is highly sensitive, and detection time is short, is particularly suitable for the on-the-spot quick diagnosis of foot and mouth disease virus.Yet owing to the type differences of foot and mouth disease virus nucleotide sequence relatively disperses, and the design of loop-mediated isothermal amplification (LAMP) primer is relatively complicated, has not yet to see the report of relevant foot and mouth disease virus ring mediated isothermal amplification classification diagnosis research.
Summary of the invention
Main purpose of the present invention provides the loop-mediated isothermal amplification (LAMP) primer group that a cover is used for the foot and mouth disease virus classification diagnosis, by O type foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer group, AsiaI type foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer group, A type foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer group, C type foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer group, SAT1 type foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer group, SAT2 type foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer group, the loop-mediated isothermal amplification (LAMP) primer group of SAT3 type foot and mouth disease virus constitutes, and can be used for the loop-mediated isothermal amplification of O type foot and mouth disease virus or AsiaI type foot and mouth disease virus or A type foot and mouth disease virus or C type foot and mouth disease virus or SAT1 type foot and mouth disease virus or SAT2 type foot and mouth disease virus or SAT3 type foot and mouth disease virus or two kinds or three kinds or four kinds or five kinds or six kinds or seven kinds type foot and mouth disease viruses wherein.
Described various foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer group is the nucleotide sequence by seven kinds of serotype foot and mouth disease viruses of downloading among a large amount of comparison Genbank, designed at the VP1 fragment that specificity difference is bigger, every kind of primer sets includes a pair of outer primer and a pair of inner primer, comprise a ring primer (SEQ No.13) in the A type primer sets in addition, comprise a pair of ring primer (SEQ No.30, SEQ No.31) in the SAT3 type primer sets in addition.Each primer in the primer sets can be mixed with primer sets solution by a certain percentage, be used to prepare foot and mouth disease virus classification diagnosis test kit, wherein the concentration ratio of inner primer and outer primer determines that method is, concentration ratio by inner primer and outer primer is 2 respectively: 1-12: 1 preparation primer sets solution, constitute the loop-mediated isothermal amplification system, use the turbidity signal in ring mediated isothermal amplification turbidity detection system realization isothermal duplication and the real-time monitoring reaction pipe, obtain the isothermal duplication curve.Inner primer, the outer primer concentration ratio that can monitor the primer sets solution of amplified signal in the 30min are 6: 1-12: 1, time and reagent usage quantity according to acquisition turbidity signal are determined the best proportion of interior outer primer to concentration, preferred proportion is 8: 1, and the ratio of ring primer and inner primer is 1: 2.
Another object of the present invention provides a kind of foot and mouth disease virus classification diagnosis test kit, by O type foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer group solution, AsiaI type foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer group solution, A type foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer group solution, C type foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer group solution, SAT1 type foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer group solution, SAT2 type foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer group solution, SAT3 type foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer group solution and loop-mediated isothermal amplification premixed liquid constitute, it is characterized in that, can carry out the classification diagnosis of O type foot and mouth disease virus or AsiaI type foot and mouth disease virus or A type foot and mouth disease virus or C type foot and mouth disease virus or SAT1 type foot and mouth disease virus or SAT2 type foot and mouth disease virus or SAT3 type foot and mouth disease virus or two kinds or three kinds or four kinds or five kinds or six kinds or seven kinds type foot and mouth disease viruses wherein.This test kit is suitable for sample type tissues such as morbidity animal blister fluid, lymphoglandula; Virocyte nutrient solution, serum etc.
Described loop-mediated isothermal amplification premixed liquid, can be to make amplified production send a kind of commercial kit Isothermal Master mix of fluorescent signal or other test kits of equivalence, constitute by Bst polysaccharase, dNTP, trimethyl-glycine, MgSO4 and buffering solution.Also introduce one group of positive quality control product and negative quality control product in the test kit simultaneously, positive quality control product is the recombinant plasmid that is made up by various foot and mouth disease virus synthetic fragment, its concentration is 1ng/ μ L, and negative quality control product is the medium solution that does not contain the foot-and-mouth disease virus gene group.
The present invention also provides a kind of using method that is used for the loop-mediated isothermal amplification kit of foot and mouth disease virus classification diagnosis, it is characterized in that, do 7 reaction tubess as a whole, add O type foot and mouth disease virus respectively, AsiaI type foot and mouth disease virus, A type foot and mouth disease virus, C type foot and mouth disease virus, SAT1 type foot and mouth disease virus, SAT2 type foot and mouth disease virus, the loop-mediated isothermal amplification system of SAT3 type foot and mouth disease virus, use ring mediated isothermal amplification fluorescence detecting system is realized the fluorescent signal in synchronous isothermal duplication and the real-time monitoring reaction pipe, obtain isothermal duplication curve and solubility curve, also can use ring mediated isothermal amplification turbidity detection system to realize synchronous isothermal duplication and the real-time turbidity signal in the monitoring reaction pipe, obtain the isothermal duplication curve.
The cumulative volume of the loop-mediated isothermal amplification system of described various foot and mouth disease virus is 25 μ L, comprising various foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer group solution 5 μ L, loop-mediated isothermal amplification premixed liquid 15 μ L, each 5 μ L of sample to be tested or positive quality control product or negative quality control product; The amplification program of ring mediated isothermal amplification fluorescence detecting system is: 1. 65 ℃, and 60min, 2. 98 ℃-80 ℃, 0.05 ℃/s, 10min, the amplification program of ring mediated isothermal amplification turbidity detection system is: 65 ℃, 60min; The analysis of amplification and decision method are:
(1) the amplification curve platform fluorescent value that records of ring mediated isothermal amplification fluorescence detecting system is higher than 5 times of background noise signals and solubility curve is the positive findings that is judged to be of single sharp peak, amplification curve platform fluorescent value is lower than the negative findings that is judged to be of 5 times of background noise signals, and amplification curve platform fluorescent value is higher than 5 times of background noise signals but solubility curve nonspecific cross interference signal that is not being judged to be of single sharp peak.
(2) turbidity value of the amplification curve that records of ring mediated isothermal amplification turbidity detection system surpasses 0.1 the positive findings that is judged to be, and the turbidity value of amplification curve is lower than 0.1 the negative findings that is judged to be.
(3) the whole positive results of each reaction tubes of positive quality control product, the whole negative results of each reaction tubes of negative quality control product are considered as efficiency test, otherwise invalidate the test carries out again.
(4) the whole negative results of each reaction tubes of sample to be tested then do not detect foot and mouth disease virus; The positive result of any pipe of each reaction tubes of sample to be tested then is that this type foot and mouth disease virus infects; Any two pipe or the positive results of multitube of each reaction tubes of sample to be tested then are foot and mouth disease virus polyinfection.Reading when wherein the amplification curve of O type foot and mouth disease virus, C type foot and mouth disease virus, SAT1 type foot and mouth disease virus, SAT2 type foot and mouth disease virus, SAT3 type foot and mouth disease virus is with 20min is a benchmark, and the reading of the amplification curve of AsiaI type foot and mouth disease virus and A type foot and mouth disease virus during with 60min is benchmark.
A kind of using method that is used for the loop-mediated isothermal amplification kit of foot and mouth disease virus classification diagnosis, also comprise the check of sensitivity, its method is, positive quality control product (1ng/ μ L) with every kind of serotype foot and mouth disease virus of DEPC water dilution, concentration gradient is 1ng/ μ L, 100pg/ μ L, 10pg/ μ L, 1pg/ μ L, 100fg/ μ L, 10fg/ μ L, 1fg/ μ L, carry out sensitivity experiment, respectively by the fluorescent signal in ring mediated isothermal amplification fluorescence detecting system and ring mediated isothermal amplification turbidity detection system realization isothermal duplication and the real-time monitoring reaction pipe, the relatively sensitivity of two kinds of detection methods.
Description of drawings
The influence of inside and outside primer mole ratio in Fig. 1 O type foot and mouth disease virus primer sets solution.
Fig. 2 real-time fluorescence detection method O type foot and mouth disease virus primer specificity experimental result.
(a) real-time fluorescence amplification curve, (b) real-time fluorescence solubility curve
The real-time turbidity detection method of Fig. 3 O type foot and mouth disease virus primer specificity experimental result.
Fig. 4 real-time fluorescence detection method O type foot and mouth disease virus primer susceptibility experimental result.
The real-time turbidity detection method of Fig. 5 O type foot and mouth disease virus primer susceptibility experimental result.
Fig. 6 O type foot and mouth disease suckling mouse strain real-time fluorescence detection method confirmatory experiment result that goes down to posterity.
(a) real-time fluorescence amplification curve, (b) real-time fluorescence solubility curve
Fig. 7 real-time fluorescence detection method AsiaI type foot and mouth disease virus primer specificity experimental result.
The real-time turbidity detection method of Fig. 8 AsiaI type foot and mouth disease virus primer specificity experimental result.
Fig. 9 real-time fluorescence detection method AsiaI type foot and mouth disease virus primer susceptibility experimental result.
The real-time turbidity detection method of Figure 10 AsiaI type foot and mouth disease virus primer susceptibility experimental result.
Figure 11 AsiaI type foot and mouth disease suckling mouse strain real-time fluorescence detection method confirmatory experiment result that goes down to posterity.
Figure 12 real-time fluorescence detection method A type foot and mouth disease virus primer specificity experimental result.
The real-time turbidity detection method of Figure 13 A type foot and mouth disease virus primer specificity experimental result.
Figure 14 real-time fluorescence detection method A type foot and mouth disease virus primer susceptibility experimental result.
The real-time turbidity detection method of Figure 15 A type foot and mouth disease virus primer susceptibility experimental result.
Figure 16 A type foot and mouth disease suckling mouse strain real-time fluorescence detection method confirmatory experiment result that goes down to posterity.
Figure 17 real-time fluorescence detection method C type foot and mouth disease virus primer specificity experimental result.
The real-time turbidity detection method of Figure 18 C type foot and mouth disease virus primer specificity experimental result.
Figure 19 real-time fluorescence detection method C type foot and mouth disease virus primer susceptibility experimental result.
The real-time turbidity detection method of Figure 20 C type foot and mouth disease virus primer susceptibility experimental result.
Figure 21 real-time fluorescence detection method SAT1 type foot and mouth disease virus primer specificity experimental result.
The real-time turbidity detection method of Figure 22 SAT1 type foot and mouth disease virus primer specificity experimental result.
Figure 23 real-time fluorescence detection method SAT1 type foot and mouth disease virus primer susceptibility experimental result.
The real-time turbidity detection method of Figure 24 SAT1 type foot and mouth disease virus primer susceptibility experimental result.
Figure 25 real-time fluorescence detection method SAT2 type foot and mouth disease virus primer specificity experimental result.
The real-time turbidity detection method of Figure 26 SAT2 type foot and mouth disease virus primer specificity experimental result.
Figure 27 real-time fluorescence detection method SAT2 type foot and mouth disease virus primer susceptibility experimental result.
The real-time turbidity detection method of Figure 28 SAT2 type foot and mouth disease virus primer susceptibility experimental result.
Figure 29 real-time fluorescence detection method SAT3 type foot and mouth disease virus primer specificity experimental result.
The real-time turbidity detection method of Figure 30 SAT3 type foot and mouth disease virus primer specificity experimental result.
Figure 31 real-time fluorescence detection method SAT3 type foot and mouth disease virus primer susceptibility experimental result.
The real-time turbidity detection method of Figure 32 SAT3 type foot and mouth disease virus primer susceptibility experimental result.
Reference numeral
Among Fig. 12: 1,4: 1,6: 1,8: 1,10: 1,12: 1 are represented inside and outside primer mole ratio in the primer sets solution respectively
O among Fig. 2-Figure 32, AS, A, C, S1, S2, S3 represent O type foot and mouth disease virus, AsiaI type foot and mouth disease virus, A type foot and mouth disease virus, C type foot and mouth disease virus, SAT1 type foot and mouth disease virus, SAT2 type foot and mouth disease virus, SAT3 type foot and mouth disease virus respectively, and water is represented negative quality control product.
Wherein, in the primer specificity experimental result picture *-* all representative is with the foot and mouth disease virus template of the foot and mouth disease virus primer sets solution amplification latter representative serotype of the former representative serotype, in the primer susceptibility experimental result picture *-* all represent the increase template of different concns of corresponding serotype foot and mouth disease virus of foot and mouth disease virus primer sets solution with the former representative serotype.
Specific implementation method
Be example and in conjunction with the accompanying drawings with the specific implementation method of O type foot and mouth disease virus below, the present invention done describing in further detail, advantage of the present invention and characteristics will be more clear along with description.But these embodiment only are exemplary; scope of the present invention is not constituted any restriction; what those skilled in the art should understand that is; down can make amendment or replace without departing from the spirit and scope of the present invention, but these modifications and replacing all fall within the scope of protection of the present invention the details of technical solution of the present invention and form.
Embodiment
1. test materials and method
1.1 test materials
Seven kinds of serotype foot and mouth disease virus VP3-VP1-2A synthetic fragments, be with reference to reported sequence (sequence number of the O type foot and mouth disease virus of institute's reference is HM008917.1) synthetic on the Genbank, fragment is connected on the pMD18-T Simple carrier, and be converted into competent cell DH5 α, store with the form of the bacterium liquid of 30% glycerine.
The suckling mouse of O type foot and mouth disease virus the strain that goes down to posterity is provided by the Lanzhou veterinary institute.
1.2 design of primers
Utilization DNAMAN software is compared to the VP3-VP1-2A fragment of seven kinds of serotype foot and mouth disease viruses, select the bigger VP1 fragment of various otherness as target sequence, use the loop-mediated isothermal amplification (LAMP) primer group of online software PrimerExplorer V4 (http://primerexplorer.jp/e/) design O type foot and mouth disease virus, comprise a pair of outer primer (SEQ No.1, SEQ No.2) and a pair of inner primer (SEQ No.3, SEQ No.4), its primer sequence is:
1.3 the extraction of recombinant plasmid and checking
Add the glycerol stock liquid that inserts 100uL in the LB substratum of penbritin in advance at 3mL, 37 ℃, 250rpm shaking culture 8-10h extracts plasmid with reference to the explanation of the little extraction reagent kit of plasmid, carry out that enzyme is cut, PCR identifies, puts into-20 ℃ of preservations after all the other bacterium liquid add 30% glycerine.Measure plasmid concentration with ultraviolet spectrophotometry.
1.4 loop-mediated isothermal amplification
The cumulative volume of loop-mediated isothermal amplification system is 25 μ L, comprises primer mixed solution 5 μ L, reaction premixed liquid 15 μ L, response sample 5 μ L, in the test of negative control with DEPC water surrogate response sample.After adding sample, can select in following three kinds of methods any one to carry out loop-mediated isothermal amplification.
(1) reaction tubes is placed the ring mediated isothermal amplification fluorescence detecting system, amplification program is set is: 1. 65 ℃, 20min, 2. 98 ℃-80 ℃, 0.05 ℃/s, 10min, fluorescent signal in the real-time monitoring reaction pipe of system is judged test-results by the isothermal duplication curve and the solubility curve that obtain.
(2) reaction tubes is placed ring mediated isothermal amplification turbidity detection system, amplification program is set is: 65 ℃, 30min, the turbidity signal in the real-time monitoring reaction pipe of system is judged test-results by the isothermal duplication curve that obtains.
(3) reaction tubes is placed thermostat water bath or other constant temperature systems, amplification program is set is: 1. 65 ℃, 20min, 2. 80 ℃, 10min judges test-results by the band of gel electrophoresis.
1.5 the optimization of inside and outside primer mole ratio in the primer mixed solution
It is 2 in inside and outside primer mole number ratio respectively: 1-12: 1 preparation primer mixed solution, add the loop-mediated isothermal amplification system, turbidity signal in use ring mediated isothermal amplification turbidity detection system realization isothermal duplication and the real-time monitoring reaction pipe is according to the best proportion of time that obtains the turbidity signal and the definite interior outer primer mole number of reagent usage quantity.
1.6 the specificity of primer mixed solution test
Recombinant plasmid with O type, AsiaI type, A type, C type, SAT1 type, SAT2 type, seven kinds of serotype foot and mouth disease viruses of SAT3 type is a template respectively, carry out the detection of real-time fluorescence system, real-time turbidity system and water bath with thermostatic control-gel electrophoresis system respectively with O type foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer mixed solution, replace plasmid template as negative control with DEPC water simultaneously, the specificity of check primer mixed solution.
1.7 sensitivity test
The O type foot and mouth disease virus plasmid of measuring concentration is carried out 10 times of gradient dilutions (1ng/ μ L, 100pg/ μ L, 10pg/ μ L, 1pg/ μ L, 100fg/ μ L, 10fg/ μ L, 1fg/ μ L) successively, above-mentioned each concentration plasmid is detected respectively by real-time fluorescence detection system and real-time turbidity detection system.
The strain 1.8 the suckling mouse that detects O type foot and mouth disease virus with the ring mediated isothermal amplification fluorescence detecting system is gone down to posterity
From the suckling mouse of O type foot and mouth disease virus is gone down to posterity strain, extract RNA nucleic acid with reference to the little extraction reagent kit of RNA, and carry out reverse transcription generation cDNA, as template with reference to single stage method reverse transcription test kit.Use the fluorescent signal in ring mediated isothermal amplification fluorescence detecting system realization isothermal duplication and the real-time monitoring reaction pipe, judge test-results by the isothermal duplication curve and the solubility curve that obtain.
2. test-results
2.1 the influence of inside and outside primer mole ratio in the primer mixed solution
In pressing respectively, outer primer mole number ratio is 2: 1-12: 1 preparation primer mixed solution, add the loop-mediated isothermal amplification system, use the turbidity signal in ring mediated isothermal amplification turbidity detection system realization isothermal duplication and the real-time monitoring reaction pipe, result such as Fig. 1 (2: 1,4: 1,6: 1,8: 1,10: 1, the mole number ratio of outer primer in being respectively in the primer mixed solution at 12: 1), in, the ratio of outer primer mole number is 6: 1-12: all can monitor amplified signal in 30min at 1 o'clock, comprehensive time and the consideration of reagent usage quantity that obtains the turbidity signal, in, the preferred proportion of outer primer mole number is 8: 1.
2.2 real-time fluorescence detection method primer specificity experimental result
Respectively with the O type, the AsiaI type, the A type, the C type, the SAT1 type, the SAT2 type, the recombinant plasmid of seven kinds of serotype foot and mouth disease viruses of SAT3 type is a template, carry out the detection of real-time fluorescence system with O type foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer mixed solution, replace plasmid template as negative control with DEPC water simultaneously, test-results that real-time fluorescence detects such as (a) of Fig. 2 and (b) (O-O, O-AS, O-A, O-C, O-SAT1, O-SAT2, O-SAT3 is respectively the O type, Asi aI type, the A type, the C type, the SAT1 type, the SAT2 type, the template of SAT3 type, the contrast of O-water belongs with yin), have only the recombinant plasmid template of O type foot and mouth disease virus amplified signal to occur, and solubility curve is single sharp peak, illustrates that the specificity of O type foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer mixed solution is good.
2.3 real-time turbidity detection method primer specificity experimental result
Respectively with the O type, the AsiaI type, the A type, the C type, the SAT1 type, the SAT2 type, the recombinant plasmid of seven kinds of serotype foot and mouth disease viruses of SAT3 type is a template, carry out the detection of real-time turbidity system with O type foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer mixed solution, replace plasmid template as negative control with DEPC water simultaneously, test-results such as Fig. 3 (O-O of turbidity detection in real time, O-AS, O-A, O-C, O-SAT1, O-SAT2, O-SAT3 is respectively the O type, the AsiaI type, the A type, the C type, the SAT1 type, the SAT2 type, the template of SAT3 type, the contrast of O-water belongs with yin), have only the recombinant plasmid template of O type foot and mouth disease virus amplified signal to occur, illustrate that the specificity of O type foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer mixed solution is good.
2.4 detected through gel electrophoresis method primer specificity experimental result
Respectively with the O type, the AsiaI type, the A type, the C type, the SAT1 type, the SAT2 type, the recombinant plasmid of seven kinds of serotype foot and mouth disease viruses of SAT3 type is a template, carry out the detection of water bath with thermostatic control-gel electrophoresis system with O type foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer mixed solution, replace plasmid template as negative control with DEPC water simultaneously, (M is Ladder Marker for the test-results of water bath with thermostatic control-detected through gel electrophoresis such as Fig. 4 in real time, 1-7 is respectively the O type, the AsiaI type, the A type, the C type, the SAT1 type, seven kinds of foot and mouth disease virus templates of SAT2 type and SAT3 type, 8 negative contrasts), have only the recombinant plasmid template of O type foot and mouth disease virus amplified signal to occur, illustrate that the specificity of O type foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer mixed solution is good.
2.5 real-time fluorescence detection method primer susceptibility experimental result
The O type foot and mouth disease virus plasmid of measuring concentration is carried out 10 times of gradient dilutions (1ng/ μ L, 100pg/ μ L, 10pg/ μ L, 1pg/ μ L, 100fg/ μ L, 10fg/ μ L, 1fg/ μ L) successively, by the real-time fluorescence detection system above-mentioned each concentration plasmid is detected respectively, result such as Fig. 5 (O-1, O-2, O-3, O-4, O-5, O-6, O-7 are respectively the gradient dilution multiple of template), finally when 10-7 (1fg/ μ L) dilution gradient, still faint amplified signal can be arranged, can reach the susceptibility of 1fg/ μ L.
2.6 real-time turbidity detection method primer susceptibility experimental result
The O type foot and mouth disease virus plasmid of measuring concentration is carried out 10 times of gradient dilutions (1ng/ μ L, 100pg/ μ L, 10pg/ μ L, 1pg/ μ L, 100fg/ μ L, 10fg/ μ L, 1fg/ μ L) successively, by the real-time fluorescence detection system above-mentioned each concentration plasmid is detected respectively, result such as Fig. 6 (O-1, O-2, O-3, O-4 are respectively the gradient dilution multiple of template), finally when 10-4 (100fg/ μ L) dilution gradient, still faint amplified signal can be arranged, can reach the susceptibility of 100fg/ μ L.
The strain real-time fluorescence detection method confirmatory experiment result 2.7O type foot and mouth disease suckling mouse is gone down to posterity
Extract the go down to posterity RNA of strain of suckling mouse, and reverse transcription generates cDNA, as template, use the fluorescent signal in ring mediated isothermal amplification fluorescence detecting system realization isothermal duplication and the real-time monitoring reaction pipe, (a) of result such as Fig. 7 and (b), amplified signal in 20min, occurs, and solubility curve is single sharp peak.
Claims (5)
1. a cover is used for the loop-mediated isothermal amplification (LAMP) primer group of foot and mouth disease virus classification diagnosis, by O type foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer group, AsiaI type foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer group, A type foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer group, C type foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer group, SAT1 type foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer group, SAT2 type foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer group, the loop-mediated isothermal amplification (LAMP) primer group of SAT3 type foot and mouth disease virus constitutes, it is characterized in that the loop-mediated isothermal amplification when can be used for O type foot and mouth disease virus or AsiaI type foot and mouth disease virus or A type foot and mouth disease virus or C type foot and mouth disease virus or SAT1 type foot and mouth disease virus or SAT2 type foot and mouth disease virus or SAT3 type foot and mouth disease virus or wherein two kinds or three kinds or four kinds or five kinds or six kinds or seven kinds of type foot and mouth disease viruses diagnosis.
Described O type foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer group is characterized in that, is made up of a pair of outer primer (SEQ No.1, SEQ No.2) and a pair of inner primer (SEQ No.3, SEQ No.4), and its primer sequence is:
Described AsiaI type foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer group is characterized in that, is made up of a pair of outer primer (SEQ No.5, SEQ No.6) and a pair of inner primer (SEQ No.7, SEQ No.8), and its primer sequence is:
Described A type mouth hoof is established the virus loop-mediated isothermal amplification primer sets, it is characterized in that, be made up of a pair of outer primer (SEQ No.9, SEQ No.10), a pair of inner primer (SEQ No.11, SEQ No.12) and a ring primer (SEQ No.13), its primer sequence is:
Described C type foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer group is characterized in that, is made up of a pair of outer primer (SEQNo.14, SEQNo.15) and a pair of inner primer (SEQ No.16, SEQ No.17), and its primer sequence is:
Described SAT1 type foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer group is characterized in that, is made up of a pair of outer primer (SEQNo.18, SEQNo.19) and a pair of inner primer (SEQ No.20, SEQ No.21), and its primer sequence is:
Described SAT2 type foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer group is characterized in that, is made up of a pair of outer primer (SEQNo.22, SEQNo.23) and a pair of inner primer (SEQ No.24, SEQ No.25), and its primer sequence is:
Described SAT3 type foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer group, it is characterized in that, be made up of a pair of outer primer (SEQNo.26, SEQNo.27), a pair of inner primer (SEQNo.28, SEQNo.29) and a pair of ring primer (SEQNo.30, SEQNo.31), its primer sequence is:
2. O type foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer group as claimed in claim 1, AsiaI type foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer group, A type foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer group, C type foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer group, SAT1 type foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer group, SAT2 type foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer group, SAT3 type foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer group, it is characterized in that, the method that is used for the foot and mouth disease virus diagnosis comprises: 1) direct fluorescence visual means, it is characterized in that, in amplified reaction forward direction system, add fluorexon as fluorescent indicator, can be directly by the naked-eye observation result, the greeny positive result of reaction solution in the reaction tubes, not greeny negative result; 2) UV-irradiation fluorescence visual means is characterized in that, in the amplified reaction afterreaction pipe reaction solution under UV-irradiation, the positive result of the fluorescence that takes on a red color, the negative result who does not take on a red color; 3) gel electrophoresis method is characterized in that, the positive result of feature trapezoid-shaped strips person, the negative result of no feature trapezoid-shaped strips person are arranged during reaction afterreaction liquid electrophoresis; 4) the real-time fluorescence detection method is characterized in that, the fluorescent signal during reaction in the real-time detection reaction pipe obtains isothermal duplication curve and solubility curve, and then judges detected result; 5) real-time turbidity detection method, the turbidity signal during reaction in the real-time monitoring reaction pipe obtains the isothermal duplication curve, and then judges detected result; 6) micro-total analysis method, it is characterized in that, sample dissociation, nucleic acid extraction purifying, isothermal duplication and detection all are integrated on the micro-total analysis system to be finished automatically, decision method comprises aforesaid direct fluorescence day vision method, UV-irradiation fluorescence visual means, gel electrophoresis method, real-time fluorescence detection method, turbidity detection method in real time as a result, this micro-total analysis system is driven by micro diaphragm pump (valve), by the unlatching and the closed driving control that realizes microfluid sample of time variable control micro diaphragm pump (valve).
3. a various foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer group as claimed in claim 1 is used for the test kit of foot and mouth disease virus classification diagnosis, mainly by O type foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer group solution, AsiaI type foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer group solution, A type foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer group solution, C type foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer group solution, SAT1 type foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer group solution, SAT2 type foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer group solution, SAT3 type foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer group solution, the loop-mediated isothermal amplification premixed liquid, the negative quality control product of various foot and mouth disease virus positive quality control product and various foot and mouth disease virus constitutes, it is characterized in that, adopt real-time fluorescence detection method and real-time turbidity detection method, realize the foot and mouth disease virus classification diagnosis, can carry out the classification diagnosis of O type foot and mouth disease virus or AsiaI type foot and mouth disease virus or A type foot and mouth disease virus or C type foot and mouth disease virus or SAT1 type foot and mouth disease virus or SAT2 type foot and mouth disease virus or SAT3 type foot and mouth disease virus or two kinds or three kinds or four kinds or five kinds or six kinds or seven kinds type foot and mouth disease viruses wherein.
Described O type, AsiaI type, C type, SAT1 type, SAT2 type foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer group solution is characterized in that, be by wherein inner primer and outer primer in 6: 1-12: 1 ratio is mixed with, and preferred proportion is 8: 1; Described A type foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer group solution, it is characterized in that, be to dispose according to a certain percentage by a pair of inner primer, a pair of outer primer and a ring primer to form, wherein inner primer and outer primer are in 6: 1-12: 1 ratio is mixed with, preferred proportion is 8: 1, and the ratio of ring primer and inner primer is 1: 2; Described SAT3 type foot and mouth disease virus loop-mediated isothermal amplification (LAMP) primer group, it is characterized in that, be to dispose according to a certain percentage by inner primer, outer primer and a pair of ring primer to form, wherein inner primer and outer primer are in 6: 1-12: 1 ratio is mixed with, preferred proportion is 8: 1, and the ratio of ring primer and inner primer is 1: 2.Described loop-mediated isothermal amplification premixed liquid is characterized in that ,-by Bst polysaccharase, dNTP, trimethyl-glycine, MgSO
4, buffered soln and colouring reagents constitute.Described various foot and mouth disease virus positive quality control product is characterized in that, can be the recombinant plasmid that is made up by various foot and mouth disease virus synthetic fragment, and its concentration is 1ng/ μ L.The negative quality control product of described various foot and mouth disease virus is characterized in that, does not contain foot-and-mouth disease virus gene group fragment.
4. foot and mouth disease virus classification diagnosis test kit as claimed in claim 3, it is characterized in that, do 7 reaction tubess as a whole, the loop-mediated isothermal amplification (LAMP) primer group solution 5 μ L that add O type foot and mouth disease virus, AsiaI type foot and mouth disease virus, A type foot and mouth disease virus, C type foot and mouth disease virus, SAT1 type foot and mouth disease virus, SAT2 type foot and mouth disease virus, SAT3 type foot and mouth disease virus respectively, various loop-mediated isothermal amplification premixed liquid 15 μ L, each 5 μ L of sample to be tested or positive quality control product or negative quality control product.Described real-time fluorescence detection method, it is characterized in that, use ring mediated isothermal amplification fluorescence detecting system is realized the fluorescent signal in synchronous isothermal duplication and the real-time detection reaction pipe, obtain isothermal duplication curve and solubility curve, the amplification program of fluorescence detecting system is: 1. 65 ℃, and 60min, 2. 98 ℃-80 ℃, 0.05 ℃/s, 10min.Described real-time turbidity detection method, it is characterized in that use ring mediated isothermal amplification turbidity detection system realizes the turbidity signal in synchronous isothermal duplication and the real-time detection reaction pipe, obtains the isothermal duplication curve, the amplification program of turbidity detection system is: 65 ℃, and 60min.
5. the using method of a foot and mouth disease virus classification diagnosis test kit as claimed in claim 2 is characterized in that, the analysis of amplification and decision method are:
When (1) platform appears in the amplification curve that records of ring mediated isothermal amplification fluorescence detecting system, its fluorescent value is higher than 5 times of background noise signals and solubility curve is the positive findings that is judged to be of single sharp peak, and its fluorescent value is higher than 5 times of background noise signals but solubility curve nonspecific cross interference signal that is not being judged to be of single sharp peak; Its fluorescent value is lower than the negative findings that is judged to be of 5 times of background noise signals.
(2) turbidity value of the amplification curve that records of ring mediated isothermal amplification turbidity detection system surpasses 0.1 the positive findings that is judged to be, and the turbidity value of amplification curve is lower than 0.1 the negative findings that is judged to be.
(3) whole positive results of each reaction tubes of positive quality control product and the whole negative results of each reaction tubes of negative quality control product be considered as efficiency test, otherwise invalidate the test carries out again.
(4) whole negative as a result the time when each reaction tubes of sample to be tested, then do not detect foot and mouth disease virus; The positive result of any pipe of each reaction tubes of sample to be tested then is that this type foot and mouth disease virus infects; Any two pipe or the positive results of multitube of each reaction tubes of sample to be tested then are the polyinfection of corresponding type foot and mouth disease virus.Reading when wherein the amplification curve of O type foot and mouth disease virus, C type foot and mouth disease virus, SAT1 type foot and mouth disease virus, SAT2 type foot and mouth disease virus, SAT3 type foot and mouth disease virus is with 20min is a benchmark, and the reading of the amplification curve of AsiaI type foot and mouth disease virus and A type foot and mouth disease virus during with 60min is benchmark.
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| CN105671201A (en) * | 2016-03-03 | 2016-06-15 | 广西壮族自治区兽医研究所 | Primer combination for identifying foot-and-mouth disease virus and vesicular stomatitis virus and application thereof |
| CN106636476A (en) * | 2017-03-03 | 2017-05-10 | 张薇 | Loop-mediated isothermal amplification primer group used for detecting foot-and-mouth diseases, and kit |
| WO2019151764A1 (en) * | 2018-01-31 | 2019-08-08 | 경북대학교 산학협력단 | Primer set for diagnosis of seven serotypes of foot-and-mouth disease virus and use thereof |
| CN113564276A (en) * | 2021-05-19 | 2021-10-29 | 中国农业科学院兰州兽医研究所 | Establishment of foot-and-mouth disease virus visual RT-LAMP detection system and method |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105671201A (en) * | 2016-03-03 | 2016-06-15 | 广西壮族自治区兽医研究所 | Primer combination for identifying foot-and-mouth disease virus and vesicular stomatitis virus and application thereof |
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| CN106636476A (en) * | 2017-03-03 | 2017-05-10 | 张薇 | Loop-mediated isothermal amplification primer group used for detecting foot-and-mouth diseases, and kit |
| WO2019151764A1 (en) * | 2018-01-31 | 2019-08-08 | 경북대학교 산학협력단 | Primer set for diagnosis of seven serotypes of foot-and-mouth disease virus and use thereof |
| CN113564276A (en) * | 2021-05-19 | 2021-10-29 | 中国农业科学院兰州兽医研究所 | Establishment of foot-and-mouth disease virus visual RT-LAMP detection system and method |
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