CN107058624A - A kind of type nano PCR detection kit of duck hepatitis A virus 1 and its application - Google Patents
A kind of type nano PCR detection kit of duck hepatitis A virus 1 and its application Download PDFInfo
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- CN107058624A CN107058624A CN201710241150.5A CN201710241150A CN107058624A CN 107058624 A CN107058624 A CN 107058624A CN 201710241150 A CN201710241150 A CN 201710241150A CN 107058624 A CN107058624 A CN 107058624A
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Abstract
The invention provides a kind of nano PCR kit and its application for being used to detect the type of duck hepatitis A virus 1.The kit includes：Reverse transcriptase, 5 × reverse transcription buffer, RNase inhibitor, 0.1M DTT, dNTPs mixed liquor and reverse transcription primer, wherein 2 × NanoPCR Mix, sense primer and anti-sense primer, sequence are shown in SEQ ID No.1 2.The kit can be used for the detection type of duck hepatitis A virus 1.Present invention also offers a kind of method that use aforementioned agents box detects the type of duck hepatitis A virus 1, the type of duck hepatitis A virus 1 can specifically, be quickly detected using this kit, the detection efficiency and specific amplification yield of the type of duck hepatitis A virus 1 are substantially increased, detection time is shortened.This kit can detect extensive use in basic unit animal doctor, and the quick detection for infecting different cultivars duck and goose the type of duck hepatitis A virus 1 provides new method.
Description
Technical field
The present invention relates to the type nano PCR detection kit of duck hepatitis A virus 1 and its application, the invention belongs to Preventive Veterinary Medicine
Inspection field.
Background technology
Duck hepatitis A virus (duck hepatitis A virus, DHAV) is to cause duckling to occur duck viral liver
Scorching important infectiousness cause of disease, seriously endangers China's duck culturing industry and develops in a healthy way.According to viral genome molecules feature by the virus
It is divided into 3 kinds of genotype, the respectively type of duck hepatitis A virus 1（DHAV-1）, the duck hepatitis A virus that 2007 years occurs in TaiWan, China
2 types（DHAV-2）And there is the type of duck hepatitis A virus 3 in South Korea in 2007（DHAV-3）.Molecule Epidemiology Investigation shows, duck
The type of hepatitis A virus 1 is still to cause China duck group the main Virus Type of duck viral hepatitis occur.
Duck virus hepatitis is a kind of acute height lethal infectious diseases for endangering young age duckling, be characterized in morbidity it is anxious,
The course of disease is short, propagation is fast, the death rate is high, and this disease takes place mostly in duckling, is more easy to occur with the duckling in 3 week old especially, adult duck can
Infect but do not fall ill.After duckling morbidity, death steeply rises, can be dead after 3 ~ 5 days；Duckling age in days is smaller, and the death rate is higher,
Generally more than 30%, high person is up to 95% or even 100%.Die of illness and opisthotonos sample outward appearance is presented more duck, the change of its characteristic pathological
There are a large amount of being dispersed in property hemorrhagic focus for liver surface.
The detection method of the type of duck hepatitis A virus 1 mainly includes traditional pathogen separation, PCR（PCR）, ring
Mediated isothermal amplification technology, AGP test and Colloidal Gold Immunoelectron Microscopy Technique etc., the identification and epidemic disease of these methods in cause of disease
Important function has been played in making a definite diagnosis.PCR detection techniques because its is simple to operate, sensitivity is high, it is reproducible and the advantages of it is extensive
Using.But in actual applications, the technology has many limitations, there is in various degree non-specific in such as PCR reactions all the time
Property amplification, the problems such as nonspecific products amplification and not high enough amplification efficiency occurs in the amplification of especially complex system.For solution
Certainly problem above, the additives of PCR specificity and efficiencies can be improved and seem particularly important by finding.
Nano PCR technology is a kind of new round pcr, and its principle is the solid phase nanometer a diameter of 1 nm -100 nm
Metallic particles floats on a liquid to form nano-fluid.Because nanogold particle has good heat-conductive characteristic, therefore adding
In the PCR reaction systems for having added nanogold particle, reaction mixture can reach target temperature faster, reduce whole system and reach
To the time used in equalized temperature, and then shorten in the residence time of non-targeted temperature, reduce non-specific amplification, improve special
Specific amplification yield.Therefore, it is badly in need of setting up a kind of nano PCR inspection of the type of detection duck hepatitis A virus 1 that can quickly, specifically at present
Survey technology.
The content of the invention
There is provided a kind of nano PCR detected for the type of duck hepatitis A virus 1 for above-mentioned the deficiencies in the prior art by the present invention
Kit and its application, the type of detection duck hepatitis A virus 1 that the kit can be quick, special.
The technical solution adopted in the present invention is：
A kind of nano PCR kit detected for the type of duck hepatitis A virus 1, including reverse transcriptase, 5 × reverse transcription buffer, RNA
Enzyme inhibitor, 0.1 M DTT, dNTPs mixed liquor, reverse transcription primer, 2 × NanoPCR Mix, sense primer and anti-sense primer,
Wherein, the sequence of sense primer is as shown in SEQ ID No.1, and the sequence of anti-sense primer is as shown in SEQ ID No.2.
Primer is the type sequence of duck hepatitis A virus 1 announced according to GenBank, in the design of its VP1 genes conserved sequence region
A pair of specific primers.
Described 2 × NanoPCR Mix are made up of archaeal dna polymerase, dNTPs mixed liquors and 2 × NanoPCR buffer solutions.
Described reverse transcriptase is M-MLV reverse transcriptase.
The sequence of described reverse transcription primer is shown in SEQ ID No.2.
Described kit also includes RNA extracts reagents：Trizol, chloroform, isopropanol and 75% ethanol.
Described kit also includes positive control.
Wherein, after positive control can be for the type of duck hepatitis A virus 1 inoculation duck embryos, the allantoic fluid of 60 ~ 96 hours dead duck embryos.
The invention also provides whether described kit contains the type of duck hepatitis A virus 1 in detection measuring samples are prepared
Application in product.
The method that the type of duck hepatitis A virus 1 is detected using the kit, comprises the following steps：
1st, the processing of measuring samples
Measuring samples are duck, the liver of goose, pancreas, spleen and brain tissue or throat cotton swab etc., and its processing method is：Tissue is dirty
Device etc. shreds rear and isotonic phosphate buffer liquid（0.01M PBS）By 1:3~1:5 mass volume ratio is ground even after mixing
Slurry, 6 000 ~ 8 000 rpm are centrifuged 5 minutes, and supernatant is used for the extraction of total serum IgE.Throat cotton swab etc. is carried out after vibration mixing, and 6
000 ~ 8 000 rpm are centrifuged 5 minutes, and the supernatant of acquisition can be used for extracting RNA.
2nd, the extraction of viral RNA
（1）Take measuring samples of the 250 μ L by pretreatment to add in 750 μ L RNA extracts reagents Trizol, fully mix 15
After s, the chloroform for adding 200 μ L precoolings, fully it is inverted and mixes.
（2）Be stored at room temperature after 10 min, 15 min centrifuged in 4 DEG C of 12 000 rpm, it is careful draw 600 μ L of supernatant liquid with
The isopropanol of -20 DEG C of precoolings in equal volume.Overturn repeatedly after mixing, 15 min are centrifuged in 12 000 rpm at 4 DEG C.
（3）Gently topple over supernatant, cleaned 2 times with the ethanol of 800 μ L 75%, be inverted in 5 min on blotting paper, room temperature is dried in the air
It is dry.
（4）Nucleic acid precipitation is fully dissolved with nuclease-free water, the RNA of acquisition should carry out RT-PCR amplifications or guarantor in 2 h
- 80 DEG C are stored in save backup.
3rd, reverse transcription reaction
The 10 μM of reverse transcription primers of total serum IgE and 1 μ L (SEQ ID No.2) for taking 11 μ L above-mentioned steps to obtain mix rearmounted 65 DEG C
The min of water-bath 5, the min of ice bath 2, then sequentially add the M DTT of 1 μ L 0.1,4 μ L 5X reverse transcriptase buffer solutions, 1 μ L
DNTPs mixtures (10 mM each), 0.5 μ L M-MLV reverse transcriptase (200 U/ μ L) and 0.5 μ L RNase inhibitors
(20 U/ μ L), 42 DEG C of water-baths 40 ~ 60 min, 85 DEG C of 5 min terminate reaction, and the reverse transcription product of acquisition is directly used in nanometer
PCR is expanded or is placed in -20 DEG C and saves backup.
4th, nano PCR is expanded
Take 2 μ L reverse transcription products to be placed in PCR reaction tubes, sequentially add 10 μ 2 × NanoPCR of L Mix, 10 μM of upstream and downstream are drawn
Thing（SEQ ID No.1 and SEQ ID No.2）Each 0.5 μ L, are finally mended reaction system to 20 μ L with nuclease-free water.
It is placed in after above-mentioned reaction mixture is mixed in Standard PCR instrument, performing PCR amplification is entered by following program：The first step 95
DEG C pre-degeneration 3min, then carries out 35 PCR cycles（95 DEG C of 15s, 53 DEG C of 15s, 72 DEG C of 30s）, the 3rd 72 DEG C 7 of step min
After terminate reaction.Amplified production is analyzed through 1% agarose gel electrophoresis, if visible 340bp nucleic acid bands are to show to contain in sample
The type of duck hepatitis A virus 1.
The advantage of the invention is that：The invention provides the specific primer of a pair of detection types of duck hepatitis A virus 1, it is utilized
The nano PCR kit being assembled into, quick, the special type of detection duck hepatitis A virus 1 of energy.The present invention can make PCR reactions quickly
Target temperature is reached, whole system is reduced and reaches time used in equalized temperature, and then shorten the stop in non-targeted temperature
Time, and the specific amplification yield of the type of duck hepatitis A virus 1 is effectively increased, non-specific amplification is reduced, with good
Application prospect.
Brief description of the drawings
Fig. 1 is that the type nano PCR of duck hepatitis A virus 1 expands electrophoresis result；M：DNA Marker (DL2000); 1：It is positive right
According to；2：Negative control.
Fig. 2 is the type nano PCR sensitivity Detection result of duck hepatitis A virus 1；M：DNA Marker (DL2000);1~6:100
~105Dilute viral nucleic acid amplified production；7：Negative control.A：Nano PCR amplified production electrophoretogram；B：Standard PCR amplification product
Electrophoretogram.
Fig. 3 is the type nano PCR specific detection result of duck hepatitis A virus 1；M：DNA Marker (DL2000); 1：Duck first
The type of hepatovirus 1；2：The type hepatitis A virus of duck 3；3：H9 subtype avian influenza virus；4：Fowl tembusu virus；5：Avian paramyxovirus 1；
6：Muscovy duck reovirus；7：Negative control.
Embodiment
The present invention is described in further detail with reference to the accompanying drawings and in conjunction with the embodiments.But the invention is not restricted to given
The example gone out.Experimental method used in following embodiments, is conventional method, material therefor and examination unless otherwise instructed
Agent etc., unless otherwise instructed, commercially purchase is obtained.
A kind of type nano PCR detection kit most preferred embodiment of duck hepatitis A virus 1 of embodiment 1
The present embodiment kit includes:（1）The type RNA extracts reagents of duck hepatitis A virus 1：Trizol, chloroform, isopropanol and
75% ethanol；（2）Reverse Transcription：M-MLV reverse transcriptase（200 U/μL）, 5 × reverse transcription buffer, 0.1 M DTT, dNTPs
Mixture（10 mM）, RNase inhibitor（20 U/μL）And reverse transcription primer；（3）Nano PCR reaction reagent：2×NanoPCR
Mix, sense primer and anti-sense primer；（4）Other；Positive control and nuclease-free water.Wherein 2 × NanoPCR Mix are gathered by DNA
Synthase, 2 × NanoPCR buffer solutions and dNTP mixed liquors composition, positive control are that the type of duck hepatitis A virus 1 is inoculated with after duck embryos, 60 ~ 96
The allantoic fluid of hour dead duck embryos, primer is HPLC grades of pure 2OD freeze-dried powders.Primer sequence is shown in Table 1.
The primer sequence of table 1
The non-diagnostic purpose of embodiment 2 is in the method for the type of nano PCR technology for detection duck hepatitis A virus 1
The present embodiment detection method uses the kit in embodiment 1.The liver for taking clinically dead duckling is measuring samples.It is actual
In, measuring samples are duck, the liver of goose, pancreas, spleen and brain tissue or throat cotton swab etc..
The present embodiment detection method comprises the following steps：
1st, viral RNA is extracted：
（1）Duckling liver shreds rear and isotonic PBS by 1:3~1:5 mass volume ratio grinds homogenate, 6 000 ~ 8 000 after mixing
Rpm is centrifuged 5 minutes；
（2）Take 250 μ L homogenates supernatants to add in 750 μ L RNA extracts reagents Trizol, fully mix 15 s, add
After the chloroform of 200 μ L precoolings, fully it is inverted and mixes；
（3）Be stored at room temperature after 10 min, 15 min centrifuged in 4 DEG C of 12 000 rpm, draw 600 μ L of supernatant liquid with it is isometric-
The isopropanol of 20 DEG C of precoolings.Overturn repeatedly after mixing, 15 min are centrifuged in 12 000 rpm at 4 DEG C；
（4）Topple over supernatant, cleaned 2 times, after room temperature is dried with the ethanol of 800 μ L 75%, fully dissolved with 30 μ L nuclease-free waters
Obtain viral RNA.
2nd, reverse transcription reaction：Take RNA and 1 μ L reverse transcription primers (SEQ ID No.2) that 11 μ L above-mentioned steps are obtained mixed
Even rearmounted 65 DEG C of water-baths 5 min, the subsequent min of ice bath 2, sequentially add the M DTT of 1 μ L 0.1,4 μ L 5X reverse transcriptase buffering
Liquid, 1 μ L dNTPs mixtures, 1 μ L M-MLV reverse transcriptase and 1 μ L RNase inhibitors, 42 DEG C of water-bath 40 ~ 60 min, 85
DEG C 5 min terminate after reaction the i.e. reverse transcription product of acquisition.
3rd, nano PCR is expanded：Take 2 μ L reverse transcription products to be placed in PCR reaction tubes, then sequentially add 10 μ L 2 ×
NanoPCR Mix, 10 μM of upstream and downstream primers（SEQ ID No.1 and SEQ ID No.2）Each 0.5 μ L, finally use nuclease-free water
Reaction system is mended to 20 μ L and mixed.
Nano PCR amplification is carried out by following program：95 DEG C of pre-degeneration 3min of the first step, then carry out 35 PCR cycles（95
DEG C 15s, 53 DEG C of 15s, 72 DEG C of 30s）, terminate reaction after the 3rd 72 DEG C 7 of step min.
4th, electrophoresis detection：After nano PCR amplification terminates, take 5 μ LPCR amplified productions to put and electricity is carried out in 1% Ago-Gel
Swimming, the stripe size of the positive control sample amplified production of the type of duck hepatitis A virus 1 is 340bp, as a result sees Fig. 1.If measuring samples
Occur 340bp nucleic acid bands after pcr amplification product electrophoresis, show in sample contain the type of duck hepatitis A virus 1, it is on the contrary then be free of duck first
The type of hepatovirus 1.
The sensitivity testing of the type nano PCR detection kit of 3 duck hepatitis A virus of embodiment 1
It is 73 ng/ μ L that the type RNA concentration of duck hepatitis A virus 1 obtained is determined with ultraviolet specrophotometer, and RNA is carried out into 10 times of ladders
Degree dilution, takes 100~105The RNA samples of dilution carry out nano PCR sensitivity testing again.Simultaneously using regular-PCR method to these
The viral nucleic acid of 10 times of gradient dilutions is detected, compares the sensitiveness of two methods.
The operating process of the type nano PCR of duck hepatitis A virus 1 and regular-PCR detection is carried out with reference to embodiment 2, wherein, it is general
2 × NanoPCR Mix in step 3 are replaced with 2 × PCR Mix by logical PCR detections, and it is included：Archaeal dna polymerase, 2 × PCR delay
Fliud flushing and dNTPs mixed liquors, remaining operating procedure are constant.
Pcr amplification product is analyzed through 1% agarose gel electrophoresis, as a result as shown in Figure 2.As a result show, nano PCR is most
Low energy detects 104Dilute again（That is 7.3pg）The type RNA samples of duck hepatitis A virus 1, and regular-PCR most low energy detects 103Times
Dilution（That is 73 pg）The type RNA samples of duck hepatitis A virus 1.Nano PCR detection sensitivity is the 10 of regular-PCR detection sensitivity
Times.
The specific assay of the type nano PCR detection kit of 4 duck hepatitis A virus of embodiment 1
According to the method for embodiment 2, respectively to the type of duck hepatitis A virus 1, the type of duck hepatitis A virus 3, H9 subtype avian influenzas, fowl Tan Busu
The Reference strains of virus, avian paramyxovirus 1 and muscovy duck reovirus, and the negative control of sterilizing ultra-pure water carry out nanometer
PCR is expanded, and detects the specificity of nano PCR method.Above amplified production is analyzed by 1% agarose gel electrophoresis.
As a result as shown in figure 3, the type nucleic acid amplification of duck hepatitis A virus 1 obtains the band for the 340bp being consistent with expected size, and
Negative control and other 5 kinds of duck common virus nucleic acid fail to amplify any band.The above results show that what the present invention was provided receives
Rice PCR detection techniques specific can expand the type of duck hepatitis A virus 1, non-specific without occurring with the nucleic acid of other duck common virus
Property amplification, the nano PCR detection technique that provides of the present invention has good specificity.
The present invention is reduced in the residence time of non-targeted temperature, and then contract but PCR reactions reach target temperature faster
The time required to short whole system reaches equalized temperature, the detection efficiency of the type of duck hepatitis A virus 1 is substantially increased, and effectively increase
The specific amplification yield of the type of duck hepatitis A virus 1, has the advantages that detection is quick, specific high.
SEQUENCE LISTING
<110>Fujian Province Academy Of Agricultural Sciences Animal Husbandry And Veterinary Medicine Institute
<120>A kind of type nano PCR detection kit of duck hepatitis A virus 1 and its application
<130> 2
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 22
<212> DNA
<213>Artificial sequence
<400> 1
actctgccat ttacatcaac ca 22
<210> 2
<211> 21
<212> DNA
<213>Artificial sequence
<400> 2
gcacagtcct aaccaaccac t 21
Claims (8)
1. a kind of nano PCR kit detected for the type of duck hepatitis A virus 1, it is characterised in that：The kit include 2 ×
NanoPCR Mix, sense primer and anti-sense primer, the sequence of sense primer is as shown in SEQ ID No.1, the sequence of anti-sense primer
As shown in SEQ ID No.2.
2. the nano PCR kit according to claim 1 detected for the type of duck hepatitis A virus 1, it is characterised in that：It is described
2 × NanoPCR Mix are made up of archaeal dna polymerase, dNTPs mixed liquors and 2 × NanoPCR buffer solutions.
3. the nano PCR kit according to claim 1 detected for the type of duck hepatitis A virus 1, it is characterised in that：It is described
Kit also includes reverse transcriptase, 5 × reverse transcription buffer, RNase inhibitor, 0.1M DTT, dNTPs mixed liquor and reverse transcription
Primer.
4. the nano PCR kit according to claim 3 detected for the type of duck hepatitis A virus 1, it is characterised in that：It is described
Reverse transcriptase is M-MLV reverse transcriptase.
5. the nano PCR kit according to claim 3 detected for the type of duck hepatitis A virus 1, it is characterised in that：It is described
The sequence of reverse transcription primer is shown in SEQ ID No.2.
6. the nano PCR kit according to claim 1 detected for the type of duck hepatitis A virus 1, it is characterised in that：It is described
Kit also includes RNA extracts reagents：Trizol, chloroform, isopropanol and 75% ethanol.
7. the nano PCR kit according to claim 1 detected for the type of duck hepatitis A virus 1, it is characterised in that：It is described
Kit also includes positive control.
8. application of the kit in the product for preparing the detection type of duck hepatitis A virus 1 as described in claim any one of 1-7.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109609698A (en) * | 2019-01-07 | 2019-04-12 | 福建省农业科学院畜牧兽医研究所 | It is a kind of for detecting the nano PCR kit of aviadenovirus IV type |
| CN111518956A (en) * | 2020-05-20 | 2020-08-11 | 广西壮族自治区兽医研究所 | Nano PCR method, kit and application for detecting avian reovirus |
| CN113881811A (en) * | 2021-11-12 | 2022-01-04 | 渤海大学 | Nano PCR (polymerase chain reaction) rapid detection kit for hepatitis A virus in shellfish aquatic products and application of kit |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103382507A (en) * | 2013-06-08 | 2013-11-06 | 四川农业大学 | One-step dual RT-PCR detection kit of 1-type and 3-type duck hepatitis A virus, primer pairs and method thereof |
| CN104673937A (en) * | 2015-03-05 | 2015-06-03 | 河北农业大学 | Porcine encephalomyocarditis virus nano PCR detection kit and detection method thereof |
| CN104711371A (en) * | 2015-03-23 | 2015-06-17 | 河北农业大学 | Tembusu virus nano PCR detection kit and detection method thereof |
-
2017
- 2017-04-13 CN CN201710241150.5A patent/CN107058624A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103382507A (en) * | 2013-06-08 | 2013-11-06 | 四川农业大学 | One-step dual RT-PCR detection kit of 1-type and 3-type duck hepatitis A virus, primer pairs and method thereof |
| CN104673937A (en) * | 2015-03-05 | 2015-06-03 | 河北农业大学 | Porcine encephalomyocarditis virus nano PCR detection kit and detection method thereof |
| CN104711371A (en) * | 2015-03-23 | 2015-06-17 | 河北农业大学 | Tembusu virus nano PCR detection kit and detection method thereof |
Non-Patent Citations (2)
| Title |
|---|
| X. J. WEN等: ""Detection, differentiation, and VP1 sequencing of duck hepatitis A virus type 1 and type 3 by a 1-step duplex reverse-transcription PCR assay"", 《POULTRY SCIENCE》 * |
| 刘清岱等: ""纳米粒子PCR研究进展"", 《生物学通报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109609698A (en) * | 2019-01-07 | 2019-04-12 | 福建省农业科学院畜牧兽医研究所 | It is a kind of for detecting the nano PCR kit of aviadenovirus IV type |
| CN109609698B (en) * | 2019-01-07 | 2022-08-16 | 福建省农业科学院畜牧兽医研究所 | Nano PCR kit for detecting avian adenovirus IV |
| CN111518956A (en) * | 2020-05-20 | 2020-08-11 | 广西壮族自治区兽医研究所 | Nano PCR method, kit and application for detecting avian reovirus |
| CN113881811A (en) * | 2021-11-12 | 2022-01-04 | 渤海大学 | Nano PCR (polymerase chain reaction) rapid detection kit for hepatitis A virus in shellfish aquatic products and application of kit |
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