CN105986041A - Porcine epizootic diarrhea virus nanometer PCR detection kit and testing method thereof - Google Patents
Porcine epizootic diarrhea virus nanometer PCR detection kit and testing method thereof Download PDFInfo
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- CN105986041A CN105986041A CN201510095287.5A CN201510095287A CN105986041A CN 105986041 A CN105986041 A CN 105986041A CN 201510095287 A CN201510095287 A CN 201510095287A CN 105986041 A CN105986041 A CN 105986041A
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- diarrhea virus
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Abstract
The invention discloses a porcine epizootic diarrhea virus nanometer PCR detection kit and a testing method thereof. The kit comprises a 5X reverse transcription buffer solution, a dNTP Mixture, a reverse transcriptase, an RNA inhibitor and a reverse transcription primer. The kit is characterized by further comprising a 2X Nano PCR mix, an upstream primer and a downstream primer, wherein the sequence of the upstream primer is shown in SEQ ID No.1, and the sequence of the downstream primer is shown in SEQ ID No.2. The kit can be used for detecting a porcine epizootic diarrhea virus. The invention further provides the method for detecting the porcine epizootic diarrhea virus by adopting the kit. According to the kit and the method, the porcine epizootic diarrhea virus can be quickly and specifically detected, the detection efficiency of the porcine epizootic diarrhea virus is greatly improved, and the specific amplification yield of the porcine epizootic diarrhea virus is greatly increased.
Description
Technical field
The present invention relates to the detection technique field of Porcine epidemic diarrhea virus.
Background technology
Pig epidemic diarrhea (porcine epidemic diarrhea virus, PED) is by Porcine Epidemic Diarrhea
The one of the pig that poison (porcine epidemic diarrhea virus, PEDV) causes, to suffer from diarrhoea, is vomitted and is taken off
Water is the high degree in contact sexually transmitted disease of main clinical characteristics.1971 starting in Britain, the eighties in 20th century
There is this disease successively in first China.Many main pig-raising countries have the report of this disease in the world at present.
The detection method of PEDV mainly includes the separation of traditional virus, Immunofluorescence assay, polymerization
PCRs (PCR) etc., these methods have played important function in Viral diagnosis.Wherein PCR inspection
Survey method is due to simple to operate, and the advantages such as sensitivity is high, reproducible are widely used.But in reality application
In, there is many limitation in round pcr, for example, there will be nonspecific products to the amplification of complex system and expand
The problem such as increase and amplification efficiency is high not.For solving problem above, it is specific that searching can improve PCR
Seem of crucial importance with the additive of efficiency.
Nano PCR technology is a kind of novel round pcr, and its principle is to be 1nm～100nm particle diameter
Solid phase nano-metal particle float on a liquid formation nano-fluid.Owing to nano material has good heat
Conductibility, therefore in the PCR cycle system that with the addition of nanogold particle, PCR reaction can reach quickly
To target temperature, decrease the time of staying in non-targeted temperature, and then shorten whole system and reach temperature and put down
The weighing apparatus time used, reduce the amplification of non-characteristic, improve specific amplification yield.Therefore, foundation is needed at present badly
A kind of can be quick, special detection Porcine epidemic diarrhea virus nano PCR detection means.
Content of the invention
The present invention is directed to above-mentioned the deficiencies in the prior art, provide a kind of for Porcine epidemic diarrhea virus detection
Nano PCR kit and application, the detection Porcine Epidemic Diarrhea that this kit can be quick, special
Poison.
The technical solution used in the present invention is: a kind of nano PCR for Porcine epidemic diarrhea virus detection
Kit, including 5 × reverse transcription buffer, dNTP Mixture, reverse transcriptase, RNase inhibitor and
Reverse transcription primer, it is characterised in that described kit also include 2 × NanoPCR Mix, upstream primer and under
Trip primer；The sequence of described upstream primer is that shown in SEQ ID No.1, the sequence of described downstream primer is SEQ
Shown in ID No.2.
Primer is the Porcine epidemic diarrhea virus sequence announced according to GenBank, at its N gene conserved sequence
A pair specific primer of region design.
Preferably, 2 × NanoPCR Mix is by archaeal dna polymerase, 2 × NanoPCR buffer and dNTP
Mixture forms.
Preferably, reverse transcriptase is M-MLV reverse transcriptase.
Preferably, the sequence of reverse transcription primer is shown in SEQ ID No.2.
Preferably, described kit also includes that RNA extracts reagent: TRIzol LS Reagent, chloroform, different
Propyl alcohol, 75% ethanol.
Preferably, described kit also includes positive control.
Wherein, after positive control can be for inoculating Vero cell by Porcine epidemic diarrhea virus, 72～96 are collected
Hour cell culture.
The invention allows for whether described kit contains pig epidemic in preparation detection testing sample
Application in the product of diarrhea virus.
Apply the method whether containing Porcine epidemic diarrhea virus in described kit detection testing sample, including
Following steps:
1st, the extraction of viral RNA
(1) 250 μ L are added 750 μ L TRIzol LS Reagent through the testing sample of pretreatment
In, acutely vibrating 2min, room temperature places 5min.Add 250 μ L chloroforms, acutely vibrate 1min,
After room temperature places 5min, 4 DEG C 12,000rpm centrifuges 15min, and upper strata aqueous phase is proceeded to new centrifuge tube.
(2) add with the isopyknic isopropanol of aqueous phase, mix, room temperature stand 15min, 4 DEG C 12,000rpm from
Heart 15min, removes supernatant gently, be subsequently adding DEPC process 75% ethanol 700～800 μ L, 4 DEG C
12,000rpm centrifuge 5min, abandon supernatant.(3) dry by precipitation natural air drying or in 50 DEG C of drying boxes,
Enter immediately after fully dissolving with appropriate nuclease free water performing PCR or be stored in-80 DEG C standby.
Testing sample can be ight soil, diarrhoea pig stomach and intestine and content thereof.Wherein, the side of testing sample pretreatment
Method is: after shredding testing sample, adds physiological saline according to the mass volume ratio of 1:5 and grinds uniformly,
3000～5000rpm takes supernatant after centrifuging 5～10 minutes, carries out the extraction of RNA afterwards again.If treating test sample
Product are ight soil or gastrointestinal contents, and 3000～5000rpm takes supernatant after centrifuging 5～10 minutes can extracting directly
RNA。
2nd, reverse transcription reaction
Take the virus total RNA 11 μ L of above-mentioned steps gained, add the reverse transcription primer of 2 μ L 10 μM
(SEQ ID No.2), 4 μ L 5 × reverse transcription buffer, 0.5 μ L reverse transcriptase, 0.5 μ L RNase press down
Preparation, 2 μ L dNTP Mixture to 20 μ L, 42 DEG C of water-bath 1～2h, i.e. obtain cDNA solution.
3rd, nano PCR reaction
10 μ L 2 × NanoPCR Mix, the 1 above-mentioned cDNA solution of μ L, 0.5 μ L is added in reaction tube
The upstream primer (SEQ ID No.1) of 10 μM, the downstream primer (SEQ ID No.2) of 0.5 μ L 10 μM,
Being eventually adding nuclease free water to cumulative volume is 20 μ L.
Carry out by following response procedures after mixing: 94 DEG C of 3min, 1 circulation；94 DEG C of 10s, 55 DEG C of 30s,
72 DEG C of 25s, totally 30 circulations；1 circulation of 72 DEG C of 5min.After reaction terminates, coagulate through 1% agarose
Gel electrophoresis checks pcr amplification product, if there is the band of 477bp size, it is popular that testing sample contains pig
Property diarrhea virus.
The invention provides the specific primer of a pair detection Porcine epidemic diarrhea virus, utilize what it made to receive
Rice PCR kit, detection Porcine epidemic diarrhea virus that can be quick, special.The present invention can make PCR
Reaction reaches target temperature quickly, decreases the time of staying in non-targeted temperature, and then shortens whole body
System reaches the time used by equalized temperature, substantially increases the detection efficiency of Porcine epidemic diarrhea virus, and has
Effect improves the specific amplification yield of Porcine epidemic diarrhea virus, decreases non-specific amplification, has very
Good application prospect.
Brief description
Fig. 1 is Porcine epidemic diarrhea virus nano PCR testing result；
M:DL2000Marker；1: positive control；
Fig. 2 is Porcine epidemic diarrhea virus nano PCR sensitivity Detection result；
M:DL2000Marker；1～6 is followed successively by 100～10-5The amplified production of doubling dilution viral nucleic acid；
7: negative control；
Fig. 3 is Porcine epidemic diarrhea virus nano PCR specific detection result；
M:DL2000Marker；1: Porcine epidemic diarrhea virus；2: porcine reproductive and respiratory syndrome virus；3:
Pig bocavirus；4: transmissible gastro-enteritis virus；5: rotavirus；6: negative control.
Detailed description of the invention
It is described in further detail with reference to the accompanying drawings and in conjunction with the embodiments to the present invention.But the present invention does not limits
In given example.Experimental technique used in following embodiment, if no special instructions, is routine
Method.Material used in following embodiment, reagent etc., if no special instructions, all commercially
Arrive.
Embodiment 1 one kinds detects the nano PCR kit most preferred embodiment of Porcine epidemic diarrhea virus
The present embodiment kit includes: (1) Porcine epidemic diarrhea virus RNA extracts reagent: TRIzol LS
Reagent, chloroform, isopropanol, 75% ethanol；(2) Reverse Transcription: 5 × reverse transcription buffer, dense
Spend the dNTPMixture for 2.5mM, concentration is the M-MLV reverse transcriptase of 2.5U/ μ L, concentration is
The RNase inhibitor of 30U/ μ L and reverse transcription primer；(3) nano PCR reaction reagent: 2 ×
NanoPCR Mix, upstream primer, downstream primer；(4) other: positive control and nuclease free water.Its
In, 2 × NanoPCR Mix is by archaeal dna polymerase, 2 × NanoPCR buffer and dNTP Mixture
Composition, after positive control is for inoculating Vero cell by Porcine epidemic diarrhea virus, within 72～96 hours, results are thin
Born of the same parents' culture, primer is freeze-dried powder, and HPLC is pure.The primer sequence that the present embodiment kit comprises is shown in Table 1.
Table 1 primer sequence
Embodiment 2 non-diagnostic purpose is with the method for nano PCR method detection Porcine epidemic diarrhea virus
The present embodiment detection method uses the kit in embodiment 1.Taking diarrhoea swine excrement is testing sample.
In Shi Ji, testing sample can be diarrhoea pig stomach and intestine and content thereof.
The present embodiment detection method comprises the following steps:
1st, viral RNA extracts and specifically comprises the following steps that (1) by testing sample swine excrement, 3000～5000rpm
Centrifugal 5～10 minutes, take supernatant.(2) testing sample and the positive after the 250 above-mentioned process of μ L is taken respectively
Comparison, adds 750 μ L TRIzol LS Reagent, acutely vibrates 2min, and room temperature places 10min.Add
250 μ L chloroforms, acutely vibrate 15s, and after room temperature places 10min, 4 DEG C 12,000g centrifuges 10min,
600 μ L upper strata aqueous phases are proceeded to new centrifuge tube.(3) in aqueous phase, isopyknic isopropanol is added ,-20 DEG C
After precipitation 30min, 4 DEG C 12,000g centrifuges 10min, inhales and abandons supernatant, adds the 75% of DEPC process
After ethanol 800 μ L shakes gently, 4 DEG C 7,500g centrifuges 5min.(4) after natural air drying being precipitated, point
The water not being dissolved in 11 μ L nuclease free i.e. obtains RNA template.
2nd, reverse transcription reaction: add 2 μ L concentration to be 10 μM in above-mentioned 11 μ L RNA templates respectively
Downstream primer (SEQ ID No.2), 4 μ L 5 × reverse transcription buffer, 0.5 μ L reverse transcriptase, 0.5 μ L
RNase inhibitor, 2 μ L dNTP Mixture to 20 μ L, 42 DEG C of water-bath 1～2h, i.e. obtain cDNA
Solution.
3rd, nano PCR reaction: add 10 μ L 2 × NanoPCR Mix, 1 μ respectively in reaction tube
L above-mentioned cDNA solution, 0.5 μ L upstream primer (10 μM), 0.5 μ L downstream primer (10 μM),
Finally adding nuclease free water to volume is 20 μ L, mixes.
Response procedures is as follows: 94 DEG C of 3min, 1 circulation；94 DEG C of 10s, 55 DEG C of 30s, 72 DEG C of 25s,
Totally 30 circulations；1 circulation of 72 DEG C of 5min.
4th, electrophoresis detection: after reaction terminates, checks pcr amplification product, pig through 1% agarose gel electrophoresis
The stripe size of the positive control amplified production of epidemic diarrhea virus is 477bp, and result is shown in Fig. 1.
If the band of 477bp size occurs after testing sample electrophoresis detection, containing Porcine epidemic diarrhea virus, otherwise
Then do not contain Porcine epidemic diarrhea virus.
The sensitivity tests of embodiment 3 Porcine epidemic diarrhea virus nano PCR detection method
Being measured by ultramicrospectrophotometer, taking the Porcine epidemic diarrhea virus total rna concentration recording is
The nucleic acid-templated of 2.7ng/ μ L carries out sensitivity tests.Use nano PCR and regular-PCR method pair simultaneously
The nucleic acid-templated detection of Porcine epidemic diarrhea virus of 10 times of dilutions.
The nano PCR of Porcine epidemic diarrhea virus and regular-PCR operating process are carried out according to embodiment 2,
Wherein, common PCR reaction changes the 2 × NanoPCR Mix in step 3 into 2 × PCR reactant liquor, its
Forming as follows: archaeal dna polymerase, 2 × PCRbuffer, dNTP Mixture, remaining operating process keeps not
Become.
With 1% agarose gel electrophoresis detection pcr amplification product, result is as shown in Figure 2.Visible, commonly
PCR low energy detects that 0.27pg Porcine epidemic diarrhea virus is nucleic acid-templated, and the inspection of nano PCR low energy
Measure 0.0027pg Porcine epidemic diarrhea virus nucleic acid-templated.Nano PCR reaction is common PCR reaction
100 times of sensitiveness.
The specific test of embodiment 4 Porcine epidemic diarrhea virus nano PCR detection method
According to the method for embodiment 2, respectively to Porcine epidemic diarrhea virus (porcine epidemic diarrhea
Virus, PEDV), transmissible gastro-enteritis virus (transmissible gastroenteritis virus, TGEV),
The breeding of rotavirus (rotavirus, RV), pig bocavirus (pig bocavirus, PBoV) and pig and breathing
The mark of syndrome virus (porcine reproductive and respiratory syndrome virus, PRRSV)
Quasi-Reference strains, and the negative control of sterilizing ultra-pure water carries out nano PCR reaction, detects nano PCR
Method specific.Above amplified production is all analyzed by the agarose gel electrophoresis of 1%.
Result is as it is shown on figure 3, Porcine epidemic diarrhea virus nucleic acid amplification has gone out the 477bp being consistent with expected size
Band, and negative control and other 4 kinds of swine disease nucleic acid all do not amplify any band.The above results table
Bright nano PCR detection method energy specific amplification Porcine epidemic diarrhea virus nucleic acid of the present invention, and not with other
Poultry diease viral nucleic acid generation cross reaction, nano PCR detection method of the present invention has well specific.
The present invention can make PCR reaction reach target temperature quickly, decreases when the stop of non-targeted temperature
Between, and then shorten whole system and reach the time used by equalized temperature, substantially increase Porcine Epidemic Diarrhea
The detection efficiency of poison, and effectively increase the specific amplification yield of Porcine epidemic diarrhea virus, there is detection
Quickly, specifically high advantage.
Claims (7)
1. the nano PCR kit for Porcine epidemic diarrhea virus detection, including 5 × reverse transcription buffer, dNTP Mixture, reverse transcriptase, RNase inhibitor and reverse transcription primer, it is characterised in that described kit also includes 2 × NanoPCR Mix, upstream primer and downstream primer；The sequence of described upstream primer is shown in SEQ ID No.1, and the sequence of described downstream primer is shown in SEQ ID No.2.
2. the nano PCR kit for Porcine epidemic diarrhea virus detection according to claim 1, it is characterised in that described 2 × NanoPCR Mix is by archaeal dna polymerase, 2 × NanoPCR
Buffer and dNTP Mixture forms.
3. the nano PCR kit for Porcine epidemic diarrhea virus detection according to claim 1, it is characterised in that described reverse transcriptase is M-MLV reverse transcriptase.
4. the nano PCR kit for Porcine epidemic diarrhea virus detection according to claim 1, it is characterised in that the sequence of described reverse transcription primer is shown in SEQ ID No.2.
5. according to claim 1 for Porcine epidemic diarrhea virus detection nano PCR kit, it is characterised in that described kit also include RNA extract reagent: TRIzol LS Reagent, chloroform, isopropanol, 75% ethanol.
6. the nano PCR kit for Porcine epidemic diarrhea virus detection according to claim 1, it is characterised in that described kit also includes positive control.
7. whether the kit as described in any one of claim 1～6 contains the application in the product of Porcine epidemic diarrhea virus in preparation detection testing sample.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107058620A (en) * | 2017-03-10 | 2017-08-18 | 中国农业科学院特产研究所 | Infectious bovine rhinotrachetis virus nano PCR detection kits and preparation method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103224995A (en) * | 2013-04-10 | 2013-07-31 | 中国农业科学院哈尔滨兽医研究所 | Nanometer PCR detection kit for rapidly identifying and diagnosing virulent virus and attenuated virus of porcine pseudorabies virus, and applications thereof |
| CN103409558A (en) * | 2013-07-10 | 2013-11-27 | 东北农业大学 | Multiplex PCR primer group for detecting porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus and porcine rotavirus simultaneously |
-
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- 2015-03-04 CN CN201510095287.5A patent/CN105986041A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103224995A (en) * | 2013-04-10 | 2013-07-31 | 中国农业科学院哈尔滨兽医研究所 | Nanometer PCR detection kit for rapidly identifying and diagnosing virulent virus and attenuated virus of porcine pseudorabies virus, and applications thereof |
| CN103409558A (en) * | 2013-07-10 | 2013-11-27 | 东北农业大学 | Multiplex PCR primer group for detecting porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus and porcine rotavirus simultaneously |
Non-Patent Citations (1)
| Title |
|---|
| 刘清岱等: "纳米粒子PCR研究进展", 《生物学通报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107058620A (en) * | 2017-03-10 | 2017-08-18 | 中国农业科学院特产研究所 | Infectious bovine rhinotrachetis virus nano PCR detection kits and preparation method thereof |
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