CN105177179A - RT-LAMP (reverse transcription loop-mediated isothermal amplification) primer set and kit for detecting porcine epidemic diarrhea virus and application thereof - Google Patents
RT-LAMP (reverse transcription loop-mediated isothermal amplification) primer set and kit for detecting porcine epidemic diarrhea virus and application thereof Download PDFInfo
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Abstract
The invention discloses an RT-LAMP (reverse transcription loop-mediated isothermal amplification) primer set and kit for detecting porcine epidemic diarrhea virus and application thereof. The primer set is composed of a pair of outer primers, a pair of inner primers and a pair of loop primers. Sequences of the paired outer primers are shown in SEQ ID No.1 and SEQ ID No.2. Sequences of the paired inner primers are shown in SEQ ID No.3 and SEQ ID No.4. Sequences of the paired loop primers are shown in SEQ ID No.5 and SEQ ID No.6. The primer set is effective in detecting the porcine epidemic diarrhea virus. The kit comprises the afore-mentioned primers and is applicable to detecting the porcine epidemic diarrhea virus. The invention further provides a method of detecting the porcine epidemic diarrhea virus with the kit; detection results of the method may be visually detected according to changes in the color of reaction liquid, quickly and accurately. The primer set, the kit and the method are highly specific, highly sensitive and quick and accurate in detecting the porcine epidemic diarrhea virus and are particularly applicable to quick field detection.
Description
Technical field
The present invention relates to the detection technique field of Porcine epidemic diarrhea virus.
Background technology
Porcine epizootic diarrhea (porcineepidemicdiarrheavirus, PED) be by Porcine epidemic diarrhea virus (porcineepidemicdiarrheavirus, the one of the pig PEDV) caused is with diarrhoea, and vomiting and dehydration are the high degree in contact sexually transmitted disease of main clinical characteristics.1971 starting in Britain, and early 1980s, China successively this disease occurred.Many main pig-raising countries have the report of this disease in the world at present.
PEDV detection method mainly comprises: virus purification, RT-PCR, real-time fluorescence quantitative PCR.These methods have played vital role in Viral diagnosis, but the shortcomings such as the plant and instrument that ubiquity complex operation, sense cycle are long, needs are expensive, be not suitable for the rapid detection at basic unit or scene.
Reverse transcription loop-mediated isothermal amplification technique (ReverseTranscriptionLoop-MediatedIsothermalAmplification, RT-LAMP) is specifically designed to rapid amplifying RNA.Its ultimate principle is employing 4 or 6 special primers and the archaeal dna polymerase with strand displacement function, and increase to nucleic acid under isothermal conditions, round of visits is very short, can complete reaction in one hour.After having reacted, only need carry out observations by the magnesium pyrophosphate precipitation of its by product white, or add fluorescence dye in advance, just can judged result by visual inspection.
RT-LAMP method has easy, quick, responsive, special advantage, is particluarly suitable for basic unit or rapid detection is carried out at scene.In RT-LAMP technology, primer determines detected result sensitivity and specific key factor.Need badly at present set up a kind of can the RT-LAMP detection means of sensitive, special, efficient detection Porcine epidemic diarrhea virus.
Summary of the invention
For the problems referred to above, the Porcine epidemic diarrhea virus sequence that the present invention announces according to GenBank, devises a set of general RT-LAMP Auele Specific Primer in its N gene conserved sequence region, as shown in table 1.The RT-LAMP utilizing this primer to set up detects other 3 boar disease pathogens of transmissible gastro-enteritis virus, rotavirus and porcine reproductive and respiratory syndrome virus, result shows that this primer sets only can specific amplification Porcine epidemic diarrhea virus gene, and other virogenes that can not increase, there is good specificity.
One of technical problem solved by the invention there is provided a kind of RT-LAMP primer sets detected for Porcine epidemic diarrhea virus, it is characterized in that: described primer sets is made up of a pair outer primer, a pair inner primer and a pair ring primer; Described outer primer is to sequence for shown in SEQIDNo.1 and SEQIDNo.2, and described inner primer is to sequence for shown in SEQIDNo.3 and SEQIDNo.4, and described ring primer pair sequence is for shown in SEQIDNo.5 and SEQIDNo.6.
By called after N-F3, N-B3, N-FIP, N-BIP, N-LF, the N-LB respectively of the primer sequence corresponding to SEQIDNo.1, SEQIDNo.2, SEQIDNo.3, SEQIDNo.4, SEQIDNo.5, SEQIDNo.6.
The mol ratio of described primer N-F3, N-B3, N-FIP, N-BIP, N-LF, N-LB specifically can be 1:1:4:4:2:2.
In one embodiment of the invention, the final concentration of described a pair outer primer (N-F3 and N-B3) in RT-LAMP reaction system is 0.2 μM, the final concentration of described a pair inner primer (N-FIP and N-BIP) in RT-LAMP reaction system is 0.8 μM, and the final concentration of described a pair ring primer (N-LF and N-LB) in RT-LAMP reaction system is 0.4 μM.
Two of technical problem solved by the invention there is provided a kind of RT-LAMP test kit detected for Porcine epidemic diarrhea virus, comprises RT-LAMP reaction solution, strand displacement type archaeal dna polymerase and ThermoScript II, characterized by further comprising above-described primer sets.
Preferably, RT-LAMP reaction solution is by 10 × BstDNABuffer, MgCl
2, dNTPs, nuclease free water composition; Strand displacement type archaeal dna polymerase is BstDNA polysaccharase; ThermoScript II is made up of AMV ThermoScript II and RNA enzyme inhibitors.
Preferably, described test kit also comprises fluorescent color-developing agent, positive control and negative control.
Wherein, positive control can be Porcine epidemic diarrhea virus sample; Negative control can be sterilizing ultrapure water.
Preferably, fluorescent color-developing agent can be containing fluorexon and MnCl
2the aqueous solution.
Preferably, described test kit also comprises the reagent extracting RNA: TRIzolLSReagent, chloroform, Virahol, 75% ethanol, nuclease free water.
Following 1) or 2) application also belong to protection scope of the present invention:
1) application of primer sets as above in the described test kit of preparation;
2) whether described primer sets or described test kit detect in testing sample containing the application in the product of Porcine epidemic diarrhea virus in preparation.
Apply the method whether containing Porcine epidemic diarrhea virus in described primer sets or described test kit detection testing sample, comprise the steps:
1, the extraction of viral RNA
Guanidinium isothiocyanate-phenol-chloroform single stage method is adopted to extract the RNA of Porcine epidemic diarrhea virus.Concrete steps are as follows:
(1) add in 750 μ LTRIzolLSReagent by 250 μ L samples, thermal agitation 2min, room temperature places 5min.Add 250 μ L chloroforms, thermal agitation 1min, after room temperature places 5min, 4 DEG C of 12,000rpm centrifugal 15min, proceeds to new centrifuge tube by upper strata aqueous phase.
(2) add with aqueous phase isopyknic Virahol, mixing, room temperature leaves standstill 15min, and 4 DEG C of 12,000rpm centrifugal 15min, removes supernatant gently, and then add 75% ethanol 800 μ L of DEPC process, 4 DEG C of 12,000rpm centrifugal 5min, abandons supernatant.
(3) dry by precipitation natural air drying or in 50 DEG C of loft drier, carry out RT-LAMP after fully dissolving with the water of appropriate nuclease free immediately or be stored in-80 DEG C for subsequent use.
2, RT-LAMP reaction
12 μ LRT-LAMP reaction solutions, 7 μ L primer sets, the above-mentioned RNA template of 2 μ L, 1.5 μ L ThermoScript II, 1.5 μ LBstDNA polysaccharases and 1 μ L fluorescent color-developing agent is added successively in reaction tubes, make 25 μ LRT-LAMP reaction systems, the N-LF that the N-BIP that the N-FIP that the N-B3 that N-F3,0.5 μ L concentration that wherein 7 μ L primer sets are 10 μMs by 0.5 μ L concentration are 10 μMs, 2 μ L concentration are 10 μMs, 2 μ L concentration are 10 μMs, 1 μ L concentration are 10 μMs, 1 μ L concentration are that the N-LB of 10 μMs forms.After mixing, reaction tubes is placed in water-bath, 62 DEG C of insulation 60min, 80 DEG C of 10min termination reactions.
3, detect
(1) directly observe: observing response liquid colour-change immediately after having reacted.Positive reaction solution becomes green, and negative sample reaction solution keeps orange-yellow constant.
(2) centrifugal detection: after positive reaction, reaction tubes is muddy, reaction tubes clarification after negative sample reaction.After the centrifugal 5min of 4000rpm, the magnesium pyrophosphate precipitation of positive reaction tubes visible white, negative sample reaction tubes is without precipitation.
(3) electrophoresis detection: check RT-LAMP amplified production with 1% agarose gel electrophoresis.Positive reaction presents distinctive scalariform band, and negative reaction then occurs without band.
The present invention uses fluorescent color-developing agent as indicator, directly can judge detected result by visual inspection reaction solution color relation, avoids and causes environmental pollution after opening reaction tubes lid and cause next false positive results of testing.The present invention only needs the water-bath of a temperature controllable to complete total overall reaction, does not need valuable thermal cycler.RT-LAMP amplification rate is fast, can obtain result in 60min; Detection sensitivity is high, minimumly detects 0.7pg viral RNA.
The present invention can carry out Porcine epidemic diarrhea virus detection to diarrhoea swine excrement, enteron aisle and content, can be wide for the object range detected, and suitability is strong.
The Porcine epidemic diarrhea virus sequence that the present invention announces according to GenBank, devises a set of general RT-LAMP Auele Specific Primer in its N gene conserved sequence region, has ubiquity and representativeness.The RT-LAMP utilizing this primer to set up detects other 3 boar disease pathogens such as transmissible gastroenteritis of swine, and result shows that this primer sets only can specific amplification Porcine epidemic diarrhea virus gene, and other virogenes that can not increase, there is good specificity.
High specificity of the present invention, susceptibility are high, plant and instrument and simple to operate, be easy to observations, be particluarly suitable for the rapid detection at basic unit and scene, have broad application prospects.
Accompanying drawing explanation
Fig. 1 is Porcine epidemic diarrhea virus RT-LAMP specific detection result;
1: Porcine epidemic diarrhea virus; 2: transmissible gastro-enteritis virus; 3: rotavirus; 4 porcine reproductive and respiratory syndrome virus; 5: nuclease free water;
Fig. 2 is Porcine epidemic diarrhea virus RT-LAMP susceptibility detected result;
A: fluorescence isothermal duplication instrument amplification curve; B: directly observe;
1:7.08 × 10
1ng/ μ l2:7.08 × 10
0ng/ μ l3:7.08 × 10
-1ng/ μ l4:7.08 × 10
-2ng/ μ l5:7.08 × 10
-3ng/ μ l6:7.08 × 10
-4ng/ μ l7:7.08 × 10
-5ng/ μ l8: nuclease free water.
Embodiment
With reference to the accompanying drawings and the present invention is described in further detail in conjunction with the embodiments.But the invention is not restricted to given example.The experimental technique used in following embodiment, if no special instructions, is ordinary method.Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Embodiment 1 detects the test kit most preferred embodiment of Porcine epidemic diarrhea virus with RT-LAMP method
The present embodiment test kit comprises following RT-LAMP primer sets, in table 1.Test kit is divided into A box and B box, and A box is that RNA extracts test kit, and its test kit composition and preservation condition are in table 2.B box is Porcine epidemic diarrhea virus reverse transcription loop-mediated isothermal amplification (RT-LAMP) test kit, and its test kit composition and preservation condition are in table 3.
Wherein, RT-LAMP reaction solution is the MgCl2 solution of 25mM by 50uL10 × BstDNABuffer, 50uL concentration, and 60uL concentration is the dNTPs solution of 2.5mM, and 120uL nuclease free water forms.
ThermoScript II is made up of the RNA enzyme inhibitors of 20uL concentration to be the AMV ThermoScript II of 200U/ μ L and 10uL concentration be 50U/ μ L.
Fluorescent color-developing agent is for containing fluorexon and MnCl
2the aqueous solution.
Positive control is Porcine epidemic diarrhea virus sample, and negative control is sterilizing ultrapure water.
Primer is the aqueous solution, and the N-BIP primer, the 40uL concentration that are 10 μMs by 40uL concentration to be N-FIP primer, the 10uL concentration of 10 μMs the be M-F3 primer of 10 μMs and 10uL concentration are that the M-B3 primer of 10 μMs forms, 20uL concentration is the N-LF primer of 10 μMs and 20uL concentration is that the N-LB primer of 10 μMs forms.
Table 1 Porcine epidemic diarrhea virus RT-LAMP primer
Table 2:RNA extracts test kit composition, volume and preservation condition
| Numbering | Test kit forms | Volume (mL) | Preservation condition |
| 1 | TRIzol LS Reagent | 12.5 | 4℃ |
| 2 | Chloroform | 5 | 4℃ |
| 3 | Virahol | 10 | 4℃ |
| 4 | 75% ethanol | 12.5 | 4℃ |
| 5 | Nuclease free water | 0.2 | 4℃ |
Table 3: Porcine epidemic diarrhea virus RT-LAMP test kit composition, volume and preservation condition
| Numbering | Test kit forms | Volume (μ L) | Preservation condition |
| 1 | RT-LAMP reaction solution | 280 | -20℃ |
| 2 | Bst DNA polysaccharase | 25 | -20℃ |
| 3 | ThermoScript II | 30 | -20℃ |
| 4 | Primer | 140 | -20℃ |
| 5 | Fluorescent color-developing agent | 13 | -20 DEG C (lucifuge) |
| 6 | Positive control | 750 | -20℃ |
| 7 | Negative control | 750 | -20℃ |
Embodiment 2 non-diagnostic object detects the detection method of Porcine epidemic diarrhea virus with RT-LAMP method
The present embodiment detection method adopts embodiment 1 test kit to detect.The present embodiment detection method comprises the following steps:
1, viral RNA extracts and extracts Porcine epidemic diarrhea virus RNA by guanidinium isothiocyanate-phenol-chloroform single stage method.Concrete steps are as follows: (1) sex change.Respectively 250 μ L testing samples, positive control and negative control are added 750 μ LTRIzolLSReagent, thermal agitation 2min, room temperature places 10min.Add 250 μ L chloroforms, thermal agitation 15s, after room temperature places 10min, 4 DEG C of 12,000g centrifugal 10min, proceeds to new centrifuge tube by upper strata aqueous phase (about 500 ~ 600 μ L).Wherein, testing sample can be diarrhoea swine excrement, enteron aisle and its content.
(2) RNA precipitation and cleaning.In aqueous phase, add isopyknic Virahol (about 500 ~ 600 μ L), after-20 DEG C of precipitation 30min, 4 DEG C of 12,000g centrifugal 10min, inhales and abandons supernatant, and the 75% ethanol 1mL adding DEPC process is gently after jolting, 4 DEG C of 7,500g centrifugal 5min.
(3) RNA dissolves and preserves.After will precipitating natural air drying, be dissolved in the water of 10 μ L nuclease free and carry out reverse transcription reaction immediately.
2, RT-LAMP reaction
Respectively the sample to be tested of said extracted, positive control and negative control RNA template are carried out RT-LAMP reaction.According to the number order in table 4, add in reaction tubes successively by each component, mixing, makes 25 μ LRT-LAMP reaction systems.Reaction tubes is placed in water-bath, 62 DEG C of insulation 60min, 80 DEG C of 10min termination reactions.
Table 4RT-LAMP reaction system
Observing response liquid colour-change immediately after having reacted.Positive reaction reaction solution becomes green, and negative reaction reaction solution keeps orange-yellow constant.
The specific test of the RT-LAMP of embodiment 3 Porcine epidemic diarrhea virus
According to the process of embodiment 2, respectively RT-LAMP reaction is carried out to Porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus (PRRSV), transmissible gastro-enteritis virus (TGEV), rotavirus (RV), detect the specificity of RT-LAMP method.As shown in Figure 1, Porcine epidemic diarrhea virus nucleic acid amplification has gone out distinctive " S " type curve to result, and other 3 kinds of swine disease nucleic acid all do not amplify " S " type curve.The above results shows RT-LAMP detection method energy specific amplification Porcine epidemic diarrhea virus of the present invention, and not with other swine disease viral nucleic acid generation cross reaction, RT-LAMP method of the present invention has good specificity.
The sensitivity test of the RT-LAMP of embodiment 4 Porcine epidemic diarrhea virus
Measured by ultramicrospectrophotometer, get nucleic acid-templated mensuration that the Porcine epidemic diarrhea virus total rna concentration recorded is 70ng/ μ L.The Porcine epidemic diarrhea virus of employing RT-LAMP to 10 times of dilutions is nucleic acid-templated to be detected.
The RT-LAMP operation process of Porcine epidemic diarrhea virus is with embodiment 2, and as shown in Figure 2, the most low energy of RT-LAMP detects that 0.7pg Porcine epidemic diarrhea virus is nucleic acid-templated to result.
The experimental result of integrated embodiment 3 and 4 can be found out, the present invention is sensitive, special, convenient and swift, is adapted at rapid detection and early screening that Porcine epidemic diarrhea virus is carried out at basic unit or scene.
Claims (7)
1., for the RT-LAMP primer sets that Porcine epidemic diarrhea virus detects, it is characterized in that: described primer sets is made up of a pair outer primer, a pair inner primer and a pair ring primer; Described outer primer is to sequence for shown in SEQIDNo.1 and SEQIDNo.2, and described inner primer is to sequence for shown in SEQIDNo.3 and SEQIDNo.4, and described ring primer pair sequence is for shown in SEQIDNo.5 and SEQIDNo.6.
2., for the RT-LAMP test kit that Porcine epidemic diarrhea virus detects, comprise RT-LAMP reaction solution, strand displacement type archaeal dna polymerase and ThermoScript II, characterized by further comprising primer sets according to claim 1.
3. the RT-LAMP test kit detected for Porcine epidemic diarrhea virus according to claim 2, is characterized in that described RT-LAMP reaction solution is by 10 × BstDNABuffer, MgCl
2, dNTPs, nuclease free water composition; Described strand displacement type archaeal dna polymerase is BstDNA polysaccharase; Described ThermoScript II is made up of AMV ThermoScript II and RNA enzyme inhibitors.
4. the RT-LAMP test kit detected for Porcine epidemic diarrhea virus according to Claims 2 or 3, is characterized in that: described test kit also comprises fluorescent color-developing agent, positive control and negative control.
5. the RT-LAMP test kit detected for Porcine epidemic diarrhea virus according to claim 4, is characterized in that: described fluorescent color-developing agent is for containing fluorexon and MnCl
2the aqueous solution.
6. the RT-LAMP test kit detected for Porcine epidemic diarrhea virus according to Claims 2 or 3, is characterized in that: described test kit also comprises the reagent extracting RNA: TRIzolLSReagent, chloroform, Virahol, 75% ethanol, nuclease free water.
7. following 1) or 2) application:
1) application of primer sets according to claim 1 in test kit described in preparation claim 2;
2) whether primer sets according to claim 1 or test kit according to claim 2 detect in testing sample containing the application in the product of Porcine epidemic diarrhea virus in preparation.
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| CN107475456A (en) * | 2017-09-27 | 2017-12-15 | 中国农业科学院兰州兽医研究所 | PEDV quick determination methods and its kit based on isothermal reverse transcription recombinase polymeric enzymatic amplification method |
| CN108411040A (en) * | 2018-05-21 | 2018-08-17 | 浙江大学 | Pig acute diarrhea syndrome coronavirus Primer composition and its kit and method |
| CN108950065A (en) * | 2018-06-28 | 2018-12-07 | 暨南大学 | Primer and probe and its kit and method based on digital pcr technology detection Porcine epidemic diarrhea virus |
| CN108950072A (en) * | 2018-07-30 | 2018-12-07 | 珠海出入境检验检疫局检验检疫技术中心 | A kind of Porcine epidemic diarrhea virus fluorescence LAMP primer group, kit and detection method |
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