CN103276103A - Kit with RT-LAMP nucleic acid test strips for detecting porcine epidemic diarrhea virus and applications of kit - Google Patents
Kit with RT-LAMP nucleic acid test strips for detecting porcine epidemic diarrhea virus and applications of kit Download PDFInfo
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Abstract
The invention discloses a kit with RT-LAMP nucleic acid test strips for detecting a porcine epidemic diarrhea virus and applications of the kit. The kit comprises a primer group of a nucleic acid represented by SEQ ID No. 1-6 and test strips for detection of nucleic acid. The usage of the kit is as follows: first preparing a RT-LAMP reaction system comprising an AMV retrovirus, a 1* reaction buffer, a strand displacement DNA polymerase, a dNTP mixture, betaine, MgSO4, a FIP primer, a BIP primer, a LoopF primer, a LoopB primer, a F3 primer, a B3 primer and RNA of a sample to be measured; carrying out a reaction at a constant temperature, after testing the obtained products by using the nucleic-acid-detecting test strip, judging and reading directly: the positive result is that two red strips appear, and one strip is in the detection zone while the other strip is in the control zone. The kit has advantages of simple operation, low cost, easy observation of the reaction result, good specificity, easy popularization and application in large scope and being extremely suitable for export quarantine, food hygiene and on-site detection in animal husbandry.
Description
Technical field
The present invention relates to biological technical field, be specifically related to a kind of RT-LAMP nucleic acid test strip test kit and application that detects Porcine epidemic diarrhea virus.
Background technology
(porcine epidemic diarrhea is by Porcine epidemic diarrhea virus (porcine epidemic diarrhea virus, a kind of acute, the highly propagated intestinal tract disease of the pig that PEDV) causes PED) to porcine epizootic diarrhea.This disease is principal character with acute enteritis, vomiting, watery diarrhea and dehydration.The equal susceptible of each age pig is especially fed piglet, and sickness rate is 100%, and mortality ratio is 80～100%.
1971, Britain found porcine epizootic diarrhea first in fattening swinery and feeder pig.1978, obtain first identifying in Belgium and Britain.After this, except America, a plurality of country and the regional reports that all occurred about porcine epizootic diarrhea of raising pigs in Europe.Afterwards, the Asia is many raise pigs country also reported in succession should disease popular, especially in Japan, Korea S and China, the outburst of porcine epizootic diarrhea is more rampant.Harm at China's porcine epizootic diarrhea is more serious, in recent years, owing to the reasons such as appearance of the new variant of PEDV, has caused serious economy loss more for China's pig industry.Begin in immune swinery, to occur once again from 2006 from the beginning of, PEDV.Rise in December, 2010, and the trend of so far still not subduing the popular of PEDV appearred, in China province of mainly raising pigs in succession.
PEDV belongs to the many viraleses of Buddhist nun (Nidovirales) coronaviridaes (Coronaviridae) coronavirus genuses (Coronavirus), is the linear sub-thread positive chain RNA of non-segmented negative, present rarely seen serotype that has.The PEDV genome is near having 5 main open reading frame in the 3' end 5kb zone, and coding has typical coronavirus structural protein: N albumen, M albumen, sM albumen and S albumen.PEDV M gene is made up of 681nt, 226 the amino acid whose M albumen of encoding.M albumen is one and contains 3 transmembrane proteins of striding the film district, and it plays a significant role in virus particle assembling and virus are sprouted process.M albumen can induce the host to produce specific antibody (generally neutralization is not active for this antibody-like) and IFN-α, can also merge by mediated cell.The M gene is comparatively conservative, is to detect the important gene that PEDV infects and carries out epidemiology survey.
Can not diagnose PED according to clinical symptom and histopathology pathology, because cause other cause of disease of diarrhea of pigs, as TGEV, porcine rotavirus (porcine rotavirus, PoRV) etc. caused clinical symptom is extremely similar to PED with pathological change, can't make a definite diagnosis them, therefore in the laboratory PEDV be carried out differential diagnosis and be very important.Virus is separated the gold standard that often is counted as the diagnosis virus disease, yet PED virus is separated difficulty relatively.At present, there are many technology to can be used for the detection of PEDV, block ELISA as immunofluorescence technique, immunohistochemistry technology, direct Electronic Speculum, immuno-electron microscope, enzyme linked immunosorbent assay, blocking-up ELISA, Streptavidin-biotin technology, in situ hybridization method, competition.Yet these detection methods are not only time-consuming, and specificity and susceptibility are lower.
Loop-mediated isothermal amplification (LAMP) is by the constant temperature nucleic acid amplification method of Japanese scholar Notomi in a kind of novelty of exploitation in 2000, this technology is utilized 6 specific regions of 4 kinds of different Auele Specific Primer identification target genes, and (Bst DNA polymerase) carries out the efficient rapid amplifying of target sequence under isothermal condition by a kind of strand displacement archaeal dna polymerase.In recent years, this technology is widely used in pathogen detection both at home and abroad.People (Development and evaluation of a novel loop-mediated isothermal amplification method for rapid detection of severe acute respiratory syndrome coronavirus.Clin Microbiol, 2004May such as Hong TC; 42 (5): 1956-61) in 2004 according to the LAMP principle design real-time quantitative RT-LAMP method, with rapid detection SARS-Cov, the sensitivity of RT-LAMP as a result is 100 times of RT-PCR; People (Rapid diagnosis of H5N1avian influenza virus infection by newly developed influenza H5hemagglutinin gene-specific loop-mediated isothermal amplification method.Virol Methods.2007May such as Masaki Imai; 141 (2): 173-80.) set up the RT-LAMP detection architecture of quick diagnosis H5N1 avian influenza virus in 2007.Because the amplified production of LAMP is white magnesium pyrophosphate precipitation, with the naked eye is difficult to observe, Japan has developed the turbidimeter of specially the LAMP product being monitored in real time.But the use of turbidimeter has not only increased expense, and is inconvenient to carry; There is the scholar to utilize to add in the reacted system method of SYBR Green I dyestuff to detect amplified production, but the observation processing of must uncapping again like this, the high efficiency of LAMP amplification and hypersensitivity make and contain a large amount of purpose fragments in the product, uncap and add dyestuff contaminate environment extremely easily.Afterwards, there is research that the LAMP method is further improved, after fluorexon and Manganous chloride tetrahydrate are added in the basis of original LAMP reaction reagent, can derive another more easy visual LAMP, one step can finish LAMP amplification and the result judges, the LAMP product need not to uncap and analyzes the result that can detect by an unaided eye.False positive rate is too high, the differentiation of weak positive findings is not accurate enough and the shortcomings such as concentration proportioning system instability of fluorexon and mn ion are restricted the application of this method but this method is because existing.
Summary of the invention
Primary and foremost purpose of the present invention provides one and has highly sensitive, high specific, visual, RT-LAMP nucleic acid test strip test kit that working method simply detects Porcine epidemic diarrhea virus.
Another object of the present invention is to provide the application of the RT-LAMP nucleic acid test strip test kit of realizing above-mentioned detection Porcine epidemic diarrhea virus.
Purpose of the present invention is achieved through the following technical solutions: a kind of RT-LAMP nucleic acid test strip test kit that detects Porcine epidemic diarrhea virus comprises following primer sets and detection of nucleic acids test strip:
The nucleotide sequence of described primer sets is as follows:
F3：5’-GGCGCAGGACACATTCTTG-3’；
B3：5’-CTTGGCGACTGTGACGAA-3’；
FIP：5’-Biotin-CCAAGCACTGGAATGCAGACCTCTGAAACAGACGCGCTTCTC-3’；
BIP：5’-FITC-AGCACCAACTGGTGTAACGCTCCTGTACGCCAGTAGCAAC-3’；
LoopF：5’-CGGCCCATCACAGAAGTAGT-3’；
LoopB：5’-AACACTCCTTAGTGGTACATTGC-3’；
Described detection of nucleic acids test strip is the universal nucleic acid test strip;
The RT-LAMP nucleic acid test strip test kit of described detection Porcine epidemic diarrhea virus preferably comprises above-mentioned primer sets and full closed target nucleic amplifier fast testing device;
Described full closed target nucleic amplifier fast testing device is Yousida Biological Technology Co., Ltd., Hangzhou's product; This proofing unit obtains in the palm plastics proofing unit for the universal nucleic acid test strip is inserted;
The RT-LAMP nucleic acid test strip test kit of described detection Porcine epidemic diarrhea virus also comprises dNTP mixture solution, MgSO
4Solution, reaction buffer, strand displacement archaeal dna polymerase (Bst DNA polymerase), trimethyl-glycine (Betaine) solution and AMV reversed transcriptive enzyme;
It is that the strand displacement archaeal dna polymerase of 8U/L, the dNTP mixture solution that concentration is 2.5mmol/L, alkali solution of beet, the concentration that concentration is 10mol/L are the MgSO of 100mmol/L that described test kit more preferably comprises AMV reversed transcriptive enzyme, 10 times of reaction buffers (10 * ThermoPol Reaction Buffer), concentration that concentration is 5U/ μ L
4Solution, concentration are that primers F IP, the concentration of 10 μ mol/L is that the primer BIP of 10 μ mol/L, primers F 3, concentration that concentration is 10 μ mol/L are that primer B3, the concentration of 10 μ mol/L is that primer LoopF and the concentration of 10 μ mol/L is the primer LoopB of 10 μ mol/L;
The application of the RT-LAMP nucleic acid test strip test kit of described detection Porcine epidemic diarrhea virus comprises following steps:
(1) preparation RT-LAMP reaction system is pressed final concentration and is calculated, and the AMV reversed transcriptive enzyme is 10
5U/L, 10 times of reaction buffers (10 * ThermoPol Reaction Buffer) are that 1 times (1 *), strand displacement archaeal dna polymerase are that 0.32U/L, dNTP mixture are that 0.4～0.6mmol/L, trimethyl-glycine are 0～2.5mol/L, MgSO
4Be that 3～6mmol/L, FIP primer are that 1.2 μ mol/L, BIP primer are that 1.2 μ mol/L, F3 primer are that 0.2 μ mol/L, B3 primer are that 0.2 μ mol/L, LoopF primer are that 0.8 μ mol/L, LoopB primer are 0.8 μ mol/L, testing sample RNA is 12ng/ μ L; Isothermal reaction;
(2) reaction: with the RT-LAMP reaction system isothermal reaction that step (1) obtains, the product that obtains detects with the detection of nucleic acids test strip, the 10min observations;
(3) interpretation as a result: directly naked eyes interpretation,
1. negative (-): only a red stripes occurs at Quality Control district (C), the redfree band occurs in detection zone (T), proves that the sample that detects does not have Porcine epidemic diarrhea virus to infect;
2. positive (+): two red stripes occur, one is positioned at detection zone (T), and another is positioned at Quality Control district (C), proves that the sample that detects is that Porcine epidemic diarrhea virus infects;
3. invalid: all redfree band appearance in Quality Control district (C) and the detection zone (T) show that the nucleic acid test strip lost efficacy.
The final concentration of the dNTP mixture described in the step (1) is preferably 0.4mmol/L;
The final concentration of the trimethyl-glycine described in the step (1) is preferably 1mol/L;
MgSO described in the step (1)
4Final concentration be preferably 3mmol/L;
The time of the isothermal reaction described in the step (2) is preferably 10～40min;
The condition optimization of the isothermal reaction described in the step (2) is 62 ℃ of reaction 40min.
Compared with prior art, the present invention has the following advantages and beneficial effect:
(1) RT-LAMP nucleic acid test strip detection method is with low cost, utilizes Bst DNA polymerase to realize isothermal duplication at 62 ℃, does not need complexity and expensive PCR instrument, and therefore, test kit use cost provided by the invention is low.
(2) test kit provided by the present invention is swift in response, and utilizes the AMV ThermoScript II to realize single stage method RT-LAMP, need not to increase by the transcriptive process,reversed of 42 ℃ of 1h, can finish reaction in 40min, can finish in 10min the soonest.
(3) reaction result that obtains of test kit provided by the invention is intuitively accurate, need not to carry out complex operations.Though producing a large amount of magnesium pyrophosphate precipitations in the DNA cloning process, LAMP make reaction solution present muddiness, after reaction finishes, need not agarose gel electrophoresis can direct visual inspection reaction tubes in muddiness whether judge the positive, but weak positive findings still brings big difficulty to differentiation, if carry out the specific biological mark at primer 5 ' end, and then with full closed target nucleic amplifier fast testing device product is detected, both can be accurately to the result carry out intuitively, interpretation fast, can prevent from again polluting.
(4) test kit specificity provided by the invention is good, to the reaction that all is negative such as transmissible gastro-enteritis virus, Porcine epidemic diarrhea virus; Highly sensitive, the minimum RNA template that can detect 10pg, consistent with the detectability of RT-LAMP agarose gel electrophoresis, the visual RT-LAMP method of fluorexon, detect 10 times of limits for height than RT-PCR method.Even several virus particle also can be detected fast and accurately.
(5) RT-LAMP nucleic acid test strip test kit of the present invention can detect Porcine epidemic diarrhea virus fast, delicately, need not expensive instrument, only needs a thermostat water bath can finish reaction.This test kit is simple to operate, with low cost, and reaction result is easy to observe, and specificity is good, and the scene that is highly suitable for export quarantine, food sanitation and livestock-raising field is detected, and is easy to apply on a large scale.
Description of drawings
Fig. 1 is RT-LAMP reaction system optimization figure as a result, wherein:
A is different dNTP concentration and electrophoresis brightness relationship figure, and swimming lane M is DNA Marker DL2000, and swimming lane 1～6 corresponding dNTP concentration successively is respectively the reaction product that 0.1mM, 0.2mM, 0.3mM, 0.4mM, 0.5mM and 0.6mM obtain;
B is different B etaine concentration and electrophoresis brightness relationship figure, and swimming lane M is DNA Marker DL2000, and swimming lane 1～6 corresponding Betaine concentration successively is respectively the reaction product that 0M, 0.5M, 1.0M, 1.5M, 2.0M and 2.5M obtain;
C is different Mg SO
4Concentration and electrophoresis brightness relationship figure, swimming lane M is DNA Marker DL2000, swimming lane 1～8 is corresponding MgSO successively
4Concentration is respectively the reaction product that 0mM, 0.5mM, 1mM, 2mM, 3mM, 4mM, 5mM and 6mM obtain;
D is inside and outside primer concentration different ratios and different concns outer primer and electrophoresis brightness relationship figure, swimming lane M is DNA Marker DL2000, swimming lane 1～11 corresponding inner primer (BIP+FIP) and outer primer (B3+F3) successively is 2:1 by concentration ratio, 4:1,6:1,8:1,10:1,12:1,14:1,6:1,6:1, the reaction product that 6:1 and 6:1 obtain, the outer primer concentration of swimming lane 1～7 is 0.2 μ mol/L, the outer primer concentration of swimming lane 8 is 0.1 μ mol/L, the outer primer concentration of swimming lane 9 is 0.2 μ mol/L, the outer primer concentration of swimming lane 10 is 0.3 μ mol/L, and the outer primer concentration of swimming lane 11 is 0.4 μ mol/L;
E is ring primer and outer primer different concns ratio and electrophoresis brightness relationship figure, swimming lane M is DNA Marker DL2000, swimming lane 1～6 corresponding ring primer (LoopF+LoopB) and outer primer (B3+F3) successively is the reaction product that 1:1,2:1,3:1,4:1,5:1 and 6:1 obtain by concentration ratio, and outer primer concentration is 0.2 μ mol/L.
Fig. 2 is RT-LAMP reaction condition optimization figure as a result, wherein:
A is the electrophoresis brightness relationship figure that reacts under the differing temps, and swimming lane M is DNA Marker DL2000, and swimming lane 1～8 corresponding temperature of reaction successively is the product of 59 ℃, 60 ℃, 61 ℃, 62 ℃, 63 ℃, 64 ℃, 65 ℃ and 66 ℃;
B is DNA Marker DL2000 for the electrophoresis brightness relationship figure under the differential responses time, swimming lane M, and the corresponding reaction times is the product of 10min, 20min, 30min, 40min, 50min, 60min, 90min to swimming lane 1～7 successively.
Fig. 3 is the agarose gel electrophoresis figure as a result in the RT-LAMP nucleic acid test strip test kit specificity provided by the invention experiment, wherein:
M is DNA Marker DL2000; The 1st, be the RT-LAMP reaction product of template with the PEDV genome; The 2nd, be the RT-LAMP reaction product of template with the TGEV genome; The 3rd, be the RT-LAMP reaction product of template with the PRRSV genome; The 4th, be the RT-LAMP reaction product of template with the PCV-2 genome; The 5th, be the RT-LAMP reaction product of template with the PRV genome; The 6th, be the LAMP reaction product of template with the bacillus coli gene group; The 7th, negative contrast.
Fig. 4 is the sensitivity detected result figure of RT-LAMP and RT-PCR, wherein:
A is the figure as a result that the fluorexon method detects RT-LAMP product provided by the invention under the ultraviolet condition;
B is that the product that utilizes RT-LAMP nucleic acid test strip test kit provided by the invention to obtain carries out the figure as a result that agarose gel electrophoresis obtains;
C is that the product that utilizes the RT-PCR method to obtain carries out the figure that agarose gel electrophoresis obtains;
Be among the figure: 1 template is that PEDV CV777 strain 100ng RNA carries out 10 times of dilutions; 2 template is that PEDV CV777 strain 100ng RNA carries out 10
2Doubly dilution; 3 template is that PEDV CV777 strain 100ng RNA carries out 10
3Doubly dilution; 4 template is that PEDV CV777 strain 100ng RNA carries out 10
4Doubly dilution; 5 template is that PEDV CV777 strain 100ng RNA carries out 10
5Doubly dilution; 6 template is that PEDV CV777 strain 100ng RNA carries out 10
6Doubly dilution; 7 negative contrasts.
Embodiment
The present invention is described in further detail below in conjunction with embodiment and accompanying drawing, but embodiments of the present invention are not limited thereto.
Primer used in following examples is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd; The big fragment of BstDNA polysaccharase is available from New England company; Trimethyl-glycine (Betaine) and MgSO
4Available from Sigma company; Trizol, AMV ThermoScript II, RNA enzyme inhibitors, random primer, Ex-Taq archaeal dna polymerase, dNTP(2.5mM), agarose is available from Takara company; Full closed target nucleic amplifier fast testing device is available from Yousida Biological Technology Co., Ltd., Hangzhou's (article No.: 20120420-32).
One, design of primers
According to LAMP design of primers principle, at PEDV M gene conservative regional sequence, according to LAMP primer design principle, use online primer-design software PrimerExporer4.0 and carry out the LAMP design of primers.The PrimerSelect instrument of using the Larsergene7.0 biosoftware simultaneously carries out preliminary screening to primer, guarantee each primer between the dimer that produces less.Obtain three cover primers of theoretical value optimum by preliminary screening, as shown in table 1 (at this moment, LoopF and LoopB do not carry out Biotin and FITC mark), primer is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd, primer after synthetic is diluted to 10pmol/ μ L solution with the sterilization tri-distilled water, and-20 ℃ keep in Dark Place.This three covers primer is carried out respectively after temperature of reaction optimizes, under peak optimization reaction temperature separately, carry out the optimization in reaction times, and the reaction times optimum result of three cover primers are compared, reaction system is as shown in table 2, and primer uses and respectively to overlap primer corresponding in the primer.The peak optimization reaction temperature of the first cover primer is 61 ℃, and reaction 40min product amount can reach the highest.The peak optimization reaction temperature of the second cover primer is 61 ℃, and the reaction times is that 60min just has product to form.The peak optimization reaction temperature of the 3rd cover primer is 62 ℃, reacts for 60min just has product to form.By screening, obtain the first the shortest cover primer (as shown in table 1) of reaction times, comprise 1 pair of outside primer (F3 and B3), 1 pair of inner primer (FIP and BIP) and 1 pair of ring primer (LoopF and LoopB).FIP is by the complementary sequence of F1c(F1) and the F2 sequence form; BIP is by the complementary sequence of B1c and B2(B2c) form.Respectively 5 ' end of the FIP primer in the first cover primer and LoopF primer is carried out the Biotin(vitamin H by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd) mark, respectively 5 ' of BIP primer and LoopB primer held again and carried out the FITC(fluorescein isothiocyanate) mark.FIP and BIP primer mark group and LoopF and LoopB primer mark group are carried out the blank sample test of RT-LAMP respectively, and product detects with the nucleic acid test strip.Found that the blank sample nucleic acid test strip tests positive of LoopF and LoopB primer mark group, and FIP and BIP primer target blank sample nucleic acid test strip detect and are negative, so select for use 5 ' end of FIP and BIP primer to carry out the Biotin(vitamin H respectively) and the FITC(fluorescein isothiocyanate) be labeled as optimum mark mode (as shown in table 1).
Table 1
Below the first cover primer design is described in detail that (following sequence is PEDV M Gene Partial sequence, and Genbank number: FJ196194.1): the position of F3 sequence is as follows, and B3 is the complementary sequence of B3c; FIP is by the complementary sequence of F1c(F1) and the F2 sequence form; BIP is by the complementary sequence of B1c and B2(B2c) form; LoopF is the complementary sequence of LoopFc, and the position of LoopB sequence is as follows.
GCTGTGGATAATGTACTTTGTCAATAGCATTCGGTTGT
TTCTTGGTGGTCTTTCAATC
TTGTAGAGGGCTATAAG
GTTGCTA 
TAAGTCAATTACCTAAC
GCCACTACAACAATTGTCTACGGACGTGTTGGTCGTTCAGTCAATGCTTCATCTGGCA。
Two, RT-LAMP reaction (the first cover primer carries out following test in the use table 1)
1, the extracting of viral RNA:
Method (the Hofmann that transmissible gastroenteritis of swine, porcine epizootic diarrhea bigeminal live vaccine (available from biotechnology development company of Harbin dimension section) are provided by document, M.and R.Wyler, Propagation of the virus of porcine epidemic diarrhea in cell culture.J Clin Microbiol, 1988.26 (11): p.2235-9.) after African green monkey kidney cell Vero cell (available from Shanghai Inst. of Life Science, CAS cell resource center) carries out the virus separation, obtain Porcine epidemic diarrhea virus CV777 strain.Get Porcine epidemic diarrhea virus CV777 strain, use Trizol Reagent extracting RNA, carry out extracting according to following steps:
(1) 250 μ L liquid samples is added in the 1.5mL centrifuge tube, add the RNAiso Reagent(TaKaRa of 750 μ L ice precooling again);
(2) with behind the violent mixing of sample, leave standstill 5min in room temperature;
(3) add 250 μ L chloroforms, thermal agitation 10s, make the abundant mixing of liquid be milky white shape (no noted phase separation phenomena) after, room temperature leaves standstill 5min again;
(4) under 4 ℃ of conditions, with the centrifugal 15min of 12000r/min;
(5) with in upper water phase transition to the new centrifuge tube, add isopyknic Virahol, the mixing that turns upside down leaves standstill 10～15min then under 4 ℃ of conditions;
(6) under 4 ℃ of conditions, with the centrifugal 15min of 12000r/min, the careful supernatant that also removes as far as possible;
(7) with ethanolic soln washing RNA precipitation and the tube wall of 1mL volume percent 75%, under 4 ℃ of conditions, with the centrifugal 8min of 12000r/min, carefully discard ethanol then;
(8) RNA precipitation is carried out with 10 μ L RNase-free water RNA is dissolved after drying (can not complete drying) handles, add 0.5 μ LRNA enzyme inhibitors (TaKaRa company) (40U), store for future use in-80 ℃ of refrigerators.
2, the foundation of RT-LAMP detection architecture:
With reference to (Notomi such as Notomi, T., Okayama, H., Masubuchi, H., et al.Loop-mediated isothermal amplification of DNA[J] .Nucleic Acids Res, 2000, the method that 28:E63) provides makes up 25 μ L RT-LAMP reaction systems, successively to dNTP, Betaine, MgSO
4, inner primer, outer primer, ring primer concentration ratio be optimized, the result who obtains carries out sepharose (mass volume ratio 2%) electrophoresis detection.
By dNTP:0.1mM, 0.2mM, 0.3mM, 0.4mM, 0.5mM, the 0.6mM that different final concentrations are set, the consumption of other compositions (result is shown in Figure 1A) as shown in table 2; Betaine:0M, 0.5M, 1.0M, 1.5M, 2.0M, the 2.5M of different final concentrations, the consumption of other compositions (result is as shown in Figure 1B) as shown in table 2; The MgSO of different final concentrations
4: 0mM, 0.5mM, 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, the consumption of other compositions (result is shown in Fig. 1 C) as shown in table 2; (inner primer is BIP+FIP to the inside and outside primer concentration of different final concentrations, outer primer is B3+F3) ratio and outer primer concentration: 2:1,4:1,6:1,8:1,10:1,12:1,14:1,6:1,6:1,6:1,6:1, this moment, concrete primer concentration ratio was: 0.4 μ M:0.2 μ M, 0.8 μ M:0.2 μ M, 1.2 μ M:0.2 μ M, 1.6 μ M:0.2 μ M, 2.0 μ M:0.2 μ M, 2.4 μ M:0.2 μ M, 2.8 μ M:0.2 μ M, 0.6 μ M:0.1 μ M, 1.2 μ M:0.2 μ M, 1.8 μ M:0.3 μ M, 2.4 μ M:0.4 μ M, the consumption of other compositions (result is shown in Fig. 1 D) as shown in table 2.The ring primer (LoopF+LoopB) of different final concentrations and outer primer (B3+F3) concentration ratio: 1:1,2:1,3:1,4:1,5:1 and 6:1, this moment, concrete primer concentration ratio was: 0.2 μ M:0.2 μ M, 0.4 μ M:0.2 μ M, 0.6 μ M:0.2 μ M, 0.8 μ M:0.2 μ M, 1.0 μ M:0.2 μ M, 1.2 μ M:0.2 μ M, the consumption of other compositions (result is shown in Fig. 1 E) as shown in table 2.Testing conditions is 62 ℃ of constant temperature 40min.According to the experimental result of gained, final definite detection architecture of optimizing (25 μ L) is as shown in table 2.
The detection architecture that table 2 is optimized
| Composition | Consumption | Final concentration |
| Template ribonucleic acid 100ng/ μ L | 3.0μL | 12ng/μL |
| AMV(5U/μL) | 0.5μL | 0.1U/ |
| 10×ThermoPol?Reaction?Buffer | 2.5 |
1×/L |
| dNTP2.5mmol/L | 4.0μL | 0.4mmol/L |
| Betaine10mol/L | 2.5μL | 1mol/L |
| FIP10μmol/L | 3.0μL | 1.2μmol/L |
| BIP10μmol/L | 3.0μL | 1.2μmol/L |
| LoopF10μmol/L | 2.0μL | 0.8μmol/L |
| LoopB10μmol/L | 2.0μL | 0.8μmol/L |
| F310μmol/L | 0.5μL | 0.2μmol/L |
| B310μmol/L | 0.5μL | 0.2μmol/L |
| MgSO 4100mmol/L | 0.5μL | 2mmol/L |
| Bst?DNA?polymerase8U/L | 1.0μL | 0.32U/L |
3, the optimization of RT-LAMP testing conditions
In order to obtain optimized temperature of reaction, the RT-LAMP reaction is placed 59 ℃, 60 ℃, 61 ℃, 62 ℃, 63 ℃, 64 ℃, 65 ℃, 66 ℃ respectively, the reaction times is 60min, reaction system is as shown in table 2.From repeatedly determining optimal reaction temperature the revision test, detect by mass volume ratio 2% agarose gel electrophoresis, the result illustrates that optimal reaction temperature is 62 ℃ shown in Fig. 2 A.
With optimal reaction temperature (62 ℃), to increase progressively by 10min, 20min, 30min, 40min, 50min, 60min, 90min in the reaction times, reaction system is as shown in table 2, from repeatedly determining optimum reacting time (result is shown in Fig. 2 B) the revision test, the presentation of results optimum reacting time of Fig. 2 B is 40min.Testing conditions after the optimization is 62 ℃ of constant temperature 40min.
4, detection architecture specificity, sensitivity analysis
Use Porcine epidemic diarrhea virus CV777 strain (transmissible gastroenteritis of swine, the porcine epizootic diarrhea bigeminal live vaccine adapts to Vero passage purifying), the magnificent strain of transmissible gastro-enteritis virus (TGEV) (by document provide method " Zhong Wang etc.; the isolation identification of transmissible gastro-enteritis virus NC strain and biological characteristics test. Chinese animal doctor's magazine; 2012 (11): the 13-17 page or leaf. " with transmissible gastroenteritis of swine, the porcine epizootic diarrhea bigeminal live vaccine adapts to porcine kidney cell line PK-15 cell (brilliant bio tech ltd is ground in Shanghai) and carries out virus separation acquisition), high-pathogenicity porcine reproductive and respiration syndrome living vaccine JXA1-R strain (PRRSV, high-pathogenicity porcine reproductive and respiration syndrome living vaccine, Guangdong Dahuanong Animal Healthcare Product Ltd), porcine circovirus 2 type (PCV-2, the porcine circovirus 2 type oil emulsion inactivated vaccine, the sharp bio tech ltd in sea, Shanghai), Pseudorabies virus (PRV, the pseudoabies virus live vaccine, the sharp bio tech ltd in sea, Shanghai), intestinal bacteria are (in order to guarantee the consistence of reaction conditions, this step will add the AMV enzyme, this bacterial strain document " Pan Wen etc.; foundation and the application of ox Bacillus brucellae and Mycobacterium bovis dual-PCR method. Agricultural University Of South China's journal; 2010 (4): 100-103 and 107 pages " open) be the specificity of template detection system, simultaneously with the alternative nucleic acid-templated negative control that arranges of water.Reaction system is as shown in table 2, and reaction conditions is the optimal conditions that step 3 is determined, uses full closed target nucleic amplifier fast testing device and concentration as the agarose gel electrophoresis of mass volume ratio 2% product to be detected.Use full closed target nucleic amplifier fast testing device to detect (seeing the result behind the 10min), the result who obtains is: be two red stripes of RT-LAMP reaction product appearance of template with PEDV CV777 pnca gene group, article one, be positioned at detection zone (T), another is positioned at Quality Control district (C); Be respectively template with TGEV genome, PRRSV genome, PCV-2 genome, PRV genome, bacillus coli gene group and water and obtain the RT-LAMP reaction product, only a red stripes occurs at Quality Control district (C), the redfree band occurs in detection zone (T).Use the detected result (Fig. 3) of agarose gel electrophoresis (taking 40min) as follows: only as the RT-LAMP reaction product of template the goal gene band to be arranged with PEDV CV777 pnca gene group.The result shows that the specificity of detection architecture is good, can detect Porcine epidemic diarrhea virus specifically, and detects with the detection of nucleic acids test strip, more convenient operation, saves time.
From 250 μ L Porcine epidemic diarrhea virus CV777 strains, extract virus total RNA, measure its rna content (100ng/ μ L) at ultraviolet spectrophotometer, RNA is carried out 10 times of gradient dilutions, and it is as shown in table 2 that the sample after the dilution is carried out the RT-LAMP(reaction system respectively, except template changes; Reaction conditions is the optimal conditions that step 3 is determined), the visual RT-LAMP(of fluorexon method adds the Calcein(fluorexon again in the reaction system of table 2) (250 μ mol/L Calcein) and MnCl
2(25mmol/L MnCl
2), final concentration separately is respectively 25 μ mol/L and 0.5mmol/L, and template changes; Reaction conditions is the optimal conditions that step 3 is determined).The employed primer of RT-PCR is
P1：5′-CCTAGACTTCAACCTTACGA-3′；
P2：5′-CAGGAAAAAGAGTACGAAAA-3′；
Get the RNA solution 10 μ L of 10 times of gradient dilutions, place the centrifuge tube of handling through DEPC, other composition that adds reverse transcription more successively carries out reverse transcription.Concrete reverse transcription system is as shown in table 3:
Table 3
| 5×Buffer | 4μL |
| dNTPs(10mmol/L) | 2μL |
| RNase enzyme inhibitors (40U/ μ L) | 1μL |
| Random primer (50pmol/ μ L) | 2μL |
| AMV(5U/μL) | 1μL |
| RNA solution (100ng/ μ L) | 10μL |
| Amount to | 20μL |
Use LX-100 palm type whizzer instantaneous centrifugal behind the mixing, liquid is concentrated on manage at the end, put in 42 ℃ of water-baths, reacted 1 hour.
Reverse transcription is template with this cDNA after finishing, and P1 and P2 are that primer carries out PCR, and concrete system is as shown in table 4:
Table 4
| 10×PCR?Buffer | 2.5μL |
| dNTPs(2.5mmol/L) | 2μL |
| P1(10μmol/L) | 0.75μL |
| P2(10μmol/L) | 0.75μL |
| Ex-Taq(5U/μL) | 0.15μL |
| CDNA template (100ng/ μ L) | 3μL |
| ddH 2O mends extremely | 25μL |
The PCR program is as follows: 94 ℃ of pre-sex change 2min; 94 ℃ of 1min, 50 ℃ of 1min, 72 ℃ of 1min are a circulation, move 30 circulations, last 72 ℃ are extended 10min, 4 ℃ of preservations.
The relatively sensitivity of RT-LAMP nucleic acid test strip detection kit, the visual RT-LAMP of fluorexon method, RT-LAMP agarose gel electrophoresis, four kinds of detection methods of RT-PCR agarose gel electrophoresis.The result as shown in Figure 4, RT-LAMP nucleic acid test strip detection kit and RT-LAMP agarose gel electrophoresis, the visual RT-LAMP of fluorexon method, RT-PCR agarose gel electrophoresis method for detecting are consistent to the detectability of PEDV geneome RNA, the minimum PEDV RNA that can detect 10pg.As seen, RT-LAMP nucleic acid test strip test kit provided by the invention is highly sensitive, operates simplyr, and the time spent is shorter, and the result is more directly perceived, is easy to detect, and need not electrophoresis.
4, the result of detection architecture identifies
1. negative (-): only a red stripes occurs at Quality Control district (C), the redfree band occurs in detection zone (T).The sample that proof detects does not have Porcine epidemic diarrhea virus to infect;
2. positive (+): two red stripes occur.Article one, be positioned at detection zone (T), another is positioned at Quality Control district (C).The sample that proof detects is that Porcine epidemic diarrhea virus infects.
3. invalid: all redfree band appearance in Quality Control district (C) and the detection zone (T) show that the nucleic acid test strip lost efficacy.
Above-described embodiment is preferred implementation of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (7)
1. a RT-LAMP nucleic acid test strip test kit that detects Porcine epidemic diarrhea virus is characterized in that: comprise following primer sets and detection of nucleic acids test strip;
The nucleotide sequence of described primer sets is as follows:
F3：5’-GGCGCAGGACACATTCTTG-3’；
B3：5’-CTTGGCGACTGTGACGAA-3’；
FIP：5’-Biotin-CCAAGCACTGGAATGCAGACCTCTGAAACAGACGCGCTTCTC-3’；
BIP：5’-FITC-AGCACCAACTGGTGTAACGCTCCTGTACGCCAGTAGCAAC-3’；
LoopF：5’-CGGCCCATCACAGAAGTAGT-3’；
LoopB：5’-AACACTCCTTAGTGGTACATTGC-3’。
2. the RT-LAMP nucleic acid test strip test kit of detection according to claim 1 Porcine epidemic diarrhea virus, it is characterized in that: described detection of nucleic acids test strip is the universal nucleic acid test strip.
3. the RT-LAMP nucleic acid test strip test kit of detection Porcine epidemic diarrhea virus according to claim 1 is characterized in that: comprise the described primer sets of full closed target nucleic amplifier fast testing device and claim 1; Full closed target nucleic amplifier fast testing device obtains for the universal nucleic acid test strip is inserted in the palm plastics proofing unit.
4. the RT-LAMP nucleic acid test strip test kit of detection Porcine epidemic diarrhea virus according to claim 1 is characterized in that: also comprise dNTP mixture solution, MgSO
4Solution, reaction buffer, strand displacement archaeal dna polymerase, alkali solution of beet and AMV reversed transcriptive enzyme.
5. the RT-LAMP nucleic acid test strip test kit of detection according to claim 4 Porcine epidemic diarrhea virus is characterized in that: the strand displacement archaeal dna polymerase that is 8U/L that comprises concentration and be the AMV reversed transcriptive enzyme of 5U/ μ L, 10 times of reaction buffer, concentration, the dNTP mixture solution that concentration is 2.5mmol/L, alkali solution of beet, the concentration that concentration is 10mol/L are the MgSO of 100mmol/L
4Solution, concentration are that primers F IP, the concentration of 10 μ mol/L is that the primer BIP of 10 μ mol/L, primers F 3, concentration that concentration is 10 μ mol/L are that primer B3, the concentration of 10 μ mol/L is that primer LoopF and the concentration of 10 μ mol/L is the primer LoopB of 10 μ mol/L.
6. the application of the RT-LAMP nucleic acid test strip test kit of each described detection Porcine epidemic diarrhea virus of claim 1～5 is characterized in that comprising following steps:
(1) preparation RT-LAMP reaction system is pressed final concentration and is calculated, and the AMV reversed transcriptive enzyme is 10
5U/L, 10 times of reaction buffers are that 1 times, strand displacement archaeal dna polymerase are that 0.32U/L, dNTP mixture are that 0.4～0.6mmol/L, trimethyl-glycine are 0～2.5mol/L, MgSO
4Be that 3～6mmol/L, FIP primer are that 1.2 μ mol/L, BIP primer are that 1.2 μ mol/L, F3 primer are that 0.2 μ mol/L, B3 primer are that 0.2 μ mol/L, LoopF primer are that 0.8 μ mol/L, LoopB primer are 0.8 μ mol/L, testing sample RNA is 12ng/ μ L; Isothermal reaction;
(2) reaction: with the RT-LAMP reaction system isothermal reaction that step (1) obtains, the product that obtains detects with the detection of nucleic acids test strip, the 10min observations;
(3) interpretation as a result: directly naked eyes interpretation,
1. negative: a red stripes only occurs in the Quality Control district, the redfree band occurs in detection zone, proves that the sample that detects does not have Porcine epidemic diarrhea virus to infect;
2. positive: two red stripes occur, one is positioned at detection zone, and another is positioned at the Quality Control district, proves that the sample that detects is that Porcine epidemic diarrhea virus infects;
3. invalid: all redfree band appearance in Quality Control district and the detection zone show that the nucleic acid test strip lost efficacy.
7. the application of the RT-LAMP nucleic acid test strip test kit of detection Porcine epidemic diarrhea virus according to claim 6 is characterized in that:
The final concentration of the dNTP mixture described in the step (1) is 0.4mmol/L;
The final concentration of the trimethyl-glycine described in the step (1) is 1mol/L;
MgSO described in the step (1)
4Final concentration be 3mmol/L;
The time of the isothermal reaction described in the step (2) is 10～40min;
The condition of the isothermal reaction described in the step (2) is 62 ℃ of reaction 40min.
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| CN105349707A (en) * | 2015-12-16 | 2016-02-24 | 广西壮族自治区兽医研究所 | RT-LAMP (reverse transcriptase loop-mediated isothermal amplification) kit for porcine epidemic diarrhea viruses and applications thereof |
| CN108315499A (en) * | 2018-05-15 | 2018-07-24 | 绵阳师范学院 | A kind of RT-LAMP Primer compositions and its application based on PEDV M genes |
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| CN109609695A (en) * | 2018-12-29 | 2019-04-12 | 华南农业大学 | Detect the RPA-LED visualizing agent box of Porcine epidemic diarrhea virus |
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