CN108251558A - Real-time fluorescence quantitative RT-PCR kit and its application for chicken astrovirus detection - Google Patents
Real-time fluorescence quantitative RT-PCR kit and its application for chicken astrovirus detection Download PDFInfo
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Abstract
Real time fluorescent quantitative RT PCR kits and its application for chicken astrovirus detection, belong to biotechnology, the kit obtains cDNA by reverse transcription (RT) mRNA samples, in conjunction with real-time fluorescence quantitative PCR detection technique, the mrna expression amount of CAstV in sample can be detected with accurate quantification.The kit can be used for the expression analysis of CAstV in virus purification chicken embryo idiosome, and the CAstV viral genomic nucleic acids level in clinical inspection case that can be used for detects.The invention detects CAstV virus infection levels for clinical sample and provides a kind of quick, accurate, repeatable detection method.
Description
Technical field
The invention belongs to biotechnologies, are related to quantitative RT-PCR detecting kit technology.
Background technology
Fowl astrovirus belongs to Picornaviridae, no single-stranded underlying stock RNA virus of cyst membrane.Most early in 1975 in abdomen
It rushes down in paediatric faecal and finds, International Commission on Virus Classification（International Committee on Taxonomy of
Viruses, ICTV）According to the host range of virus infection, by Astroviridae（Astroviridae）It is divided into mammal star
Shape Tobamovirus（Mamastrovirus）With fowl Astrovirus (Avastrovirus), wherein fowl Astrovirus is divided into 3
Kind, respectively fowl astrovirus GI.A types, fowl astrovirus GI.B types and fowl astrovirus GII.A types.Fowl astrovirus GI.A
Type includes 1 type of turkey astrovirus（TAstV-1）, fowl astrovirus GI.B types include viral 1 type (ANV-1) of fowl ephritis, fowl kidney
2 types of scorching virus (ANV-2) and chicken astrovirus（CAstV）.And fowl astrovirus GII.A types include duck astrovirus（DAstV）
With 2 type of turkey astrovirus（TAstV-2）.In domestic fowl farming, chicken astrovirus and turkey astrovirus infection can lead to chicken, fire
The diarrhoeal enteritis of chicken and guinea fowl, development are slowly, productivity is low and chicken ephritis etc., and chick infringement is particularly acute.
Asia, Europe, America many countries and regions all once had the report of infection morbidity, poultry farming is caused certain
Economic loss.
The physicochemical property of astrovirus is extremely stable, chloroform, various aldehydes matters, acid, detergent, ammonia
The physical methods such as the chemical reagent and heating, room temperature of water, most of alcohols materials and Ester etc. can not make its inactivation.
Formaldehyde, beta-propiolactone, 90% methanol and the potassium peroxydisulfate comprising detergent can limit the infection of astrovirus.Astrovirus
Genome length is 6.5-7.9kb, including 5 ' end non-translational regions（UTR）, 3 ' end non-translational regions, Poly（A）Tail and three open
Put reading frame（ORF）.Three open reading frame are reading frame codes respectively without envelope protein（ORF1a）, viral RNA rely on
RNA polymerase（ORF1b）And capsid protein（ORF2）.The copy mode of astrovirus is different from other enteroviruses, in ORF1
There are a reverse transcriptions containing serine protease between ORF2 to move frame signal, and there are one mobile between ORF1a and ORF1b
Mount structure, ORF1 encode the RNA polymerase that a kind of protease and RNA are relied on, and ORF2 expression subgenomic RNAs encode virus
Capsid protein, the structure between different serotypes are different in size.
CAstVs is distributed in the whole world, is a kind of popular enterovirus easily in 1-5 week old poults.Research shows that
Astrovirus has infected 86%~100% chicken group and nearly 100% turkey group, also often occurs with other enteroviruses mixed
Close infection.Chicken group's enterovirus infection situation is continued to monitor, the first positive sample for finding to detect is exactly often star
Shape virus or astrovirus and the mixed infection of other viruses.Astrovirus causes complexity jointly with most common enterovirus
Poult enteritis（PEC）, poult inflammatory bowel syndrome（PES）With the lethal poult inflammatory bowel syndrome of turkey（PEMS）, broiler chicken hair
Educate distress syndrome（RSS）Deng.However CAstVs can also be detached from the birds of clinical manifestation health and be obtained, this causes starlike
Virus also needed to further study on the problem of pathogenic.
Electronic Speculum is one of main means of early detection CAstV, but process is cumbersome, is not suitable for the detection of great amount of samples.No
Comprovincial chicken can infect a variety of different CAstV strains.Therefore diagnostic test should not can only differentiate astrovirus, should also
Distinguish different genotype.Some team develop ELISA kit using recombinant capsid protein and pass through poultry dung
Or intestinal contents detect CastV specific antibodies.
Before the appearance of Real time quantitative RT-PCR (Real-time PCR) technology, people are to PCR
Quantitative whether Direct PCR or the competitive PCR of template, being substantially all will pass through by PCR product electrophoresis, then by electrophoresis result
Computer Image Processing, determined according to the brightness of electrophoretic band final PCR product amount number or the PCR of tape label produced
Object is detected in a manner of ELISA, then thus speculates the amount of starting template, but these methods actually belong to sxemiquantitative water
Flat, because even PCR conditions have optimized, the unstability of the operation of electrophoresis and subsequent step still can bring shadow to interpretation of result
It rings, so as to influence quantitative this purpose.
At present for the research of CAstV also in initial period, the method for various detection CAstV expression is also short of very much.With
The rapid development and its extensive use in medical research for Protocols in Molecular Biology, are gathered by real time fluorescent quantitative reverse transcription
Synthase chain reaction (Real-time PCR) technology detection protein gene expression has been possibly realized.Real-time PCR methods have
The high sensitivity more incomparable than conventional methods such as ELISA and opposite high specific, and can be with accurate quantification.
Invention content
More than needle with problem of the existing technology, present invention aims at proposition one kind can easily detect CAstV tables
The kit of Real time quantitative RT-PCR detection chicken astrovirus reached.
Kit of the present invention includes：
1）Sense primer CAstV F：5’- TGCAGATCCCGACGTAAAGG -3’；
2）Downstream primer CAstV R：5’- CGGTCCATCCCTCTACCAGA -3’；
3）Standard items：Length is 132bp, and sequence is：
gcagatcccg acgtaaagga ttacttagat agacagatca attgcgtcga ggagtatgcc 60
gctgctgaag aaatacagtt accagaagtc gggcccgact tctttcagaa aatctggtag 120
agggatggac cg ；
4）Standard items template：102, 103, 104, 105, 106, 107, 108, 109The chicken astrovirus gene masculine matter of copy/uL
Grain；The chicken astrovirus gene masculine plasmid is using pGEM-T carriers as the carrier that sets out, in the EcoR I sites for the carrier that sets out
It is inserted into poultry chicken astrovirus genetic fragment；
5）Fluorescent dye and aseptic deionized water.
This kit is stored in -20 DEG C, reduces multigelation to the greatest extent.
The present invention establishes the method using the detection CAstV expression of Real-time round pcrs, and after testing tissue with it is thin
Born of the same parents' sample shows that this method is practical.Since this method employs Real-time PCR amplifications so that CAstV is expressed
Detection sensitivity greatly improve so that we can obtain enough information in few sample.Designed by the present invention
Primer and testing result can provide reliable foundation for the exploitation of fluorescent quantificationally PCR detecting kit.
The present invention is another object is that propose the application of mentioned reagent box.
Each standard items template is anti-with upstream and downstream primer, fluorescent dye and aseptic deionized water composition in kit respectively
System is answered to carry out PCR amplification, the wherein final concentration of sense primer and downstream primer in system is 0.5 μm of ol/L, and fluorescence contaminates
Expect final concentration of 0.5 μm of ol/L in system, the response procedures in PCR instrument are：95 DEG C of pre-degeneration 5min, then 95 DEG C 15
S, 60 DEG C of 1min, totally 40 recycle；The collection of fluorescence signal and the acquisition of data are scheduled on 60 DEG C, obtain real time fluorescent quantitative
RT-PCR standard curves；
Take upstream and downstream primer in the cDNA and kit of poultry organization's internal organs to be measured, fluorescent dye and aseptic deionized water composition
Reaction system carries out PCR amplification, and the final concentration of sense primer and downstream primer in system is 0.5 μm of ol/L, fluorescent dye
Final concentration of 0.5 μm of ol/L in system, the response procedures in PCR instrument are：95 DEG C of pre-degeneration 5min, then 95 DEG C of 15 s,
60 DEG C of 1min, totally 40 recycle；The collection of fluorescence signal and the acquisition of data are scheduled on 60 DEG C, obtain poultry organization's internal organs to be measured
Target gene amplification cycles number Ct values；By the real-time fluorescence quantitative RT-PCR standard curve, show that poultry organization to be measured is dirty
The copy number of device chicken astrovirus gene.
In this detection project, advanced real-time fluorescence quantitative PCR detection technique is employed, before hypersensitivity is kept
It puts and reduces false positive interference to the greatest extent.With the appearance of Real-Time Fluorescent Quantitative PCR Technique, people can veritably accomplish to PCR
The accurate quantification of template, this quantitative high sensitivity for not only maintaining Standard PCR, and also the specificity for detecting gene also obtains
It improves.
The present invention makes standard curve using the standard items of known starting copy number, as long as obtaining the CT values of detection sample,
The copy number of the sample can be calculated from standard curve.The characteristics of of the invention maximum and the advantage is that passing through a Real
Time PCR react, it is possible to the gene copy number of CAstV in accurate quantification detection sample.
Description of the drawings
Fig. 1 expands kinetic curve for real-time fluorescence quantitative RT-PCR standard items.
Fig. 2 is real-time fluorescence quantitative RT-PCR standard curve.
Specific embodiment
The present invention is described further with reference to the drawings and specific embodiments.It should be understood that these embodiments are merely to illustrate
Purpose rather than the limitation scope of the invention.
First, the standard curve of fluorescence quantitative RT-RCR method detection chicken astrovirus is obtained：
1st, material：
DNase I is purchased from Fermentas companies.Reverse transcription reagent box etc. is purchased from Dalian TaKaRa companies.Carrier T ligase is purchased from
Promega companies.DNTP is purchased from Shanghai Sheng Gong bioengineering Co., Ltd.Total RNA extraction reagent box is Hangzhou Axygen products.
SYBR® Premix Ex TaqTMII is TaKaRa Products.Other is domestic analytical reagents.
2nd, the design and synthesis of primer：
With chicken astrovirus ORF1b sequences (GenBank accession number JN582328.1) for template, Primer is used
ExpressTM(V3. 0, American AB I company) software analyze and design primer, and according to simultaneously consider CAstV genomic DNA sequences
Row situation therefrom selects best primer.
It detects with PCR upstream primer sequences and is：5 '-TGCAGATCCCGACGTAAAGG -3 ',
Downstream primer sequence is：5 '-CGGTCCATCCCTCTACCAGA-3 ', by Nanjing, Jin Sirui companies synthesize.
3rd, prepared by examination criteria product：
The amplification of chicken astrovirus gene is carried out using freshly prepared cDNA as template, reaction system is 50 μ L：CDNA templates 1
μ L, each 1 μ L of upstream and downstream primer（10 nM）, dNTP 4 μ L, 10 × buffer（Containing MgCl2）5 μ L, LA Taq DNA polymerize
0.5 37.5 μ L of μ L, ddw of enzyme.The loop parameter of PCR reactions：95℃ 5 min、95℃ 30 s、60℃ 30 s、72℃ 30
S, through 30 cycles；72 DEG C of 10 min of extension.Electrophoresis, 1 % applied sample amounts of gel strength, 5 μ L are carried out after reaction.Glue recycles
50 μ L electrophoresis of loading recycles PCR product to get chicken astrovirus target gene fragment, in -20 DEG C according to plastic recovery kit
It saves backup.
By chicken astrovirus target gene fragment and pGEM-T Easy carriers（It is purchased from Promega companies of the U.S.）Illustratively
Book is required with 3:1 molar ratio connects overnight in 16 DEG C of water-baths（10 μ L of linked system：5×ligation buffer 2 μ
3 μ L of L, pGEM-T carrier, 3 μ L, T4 ligase of target fragment, 11 μ L of μ L, ddw）, connection product is obtained, connection product is
Chicken astrovirus target gene fragment is inserted in the EcoR I sites of pGEM-T Easy carriers.
5 μ L of connection product is taken to add in 5 α competent escherichia coli cells of DH, the competent cell after conversion is uniformly applied
It is distributed in LB-Amp tablets（Add X-Gal and IPTG）, 37 DEG C are incubated overnight.Picking white colony is cultivated, according to a conventional method
Plasmid is extracted, with carrying out EcoR single endonuclease digestion identifications（10 μ L of digestion system：1 0.5 μ L of μ L, EcoR of Buffer, plasmid 3
5.5 μ L of μ L, ddw）, screen positive plasmid.
By the way of screening, filtered out from material recombination material DNA sequence dna band length for 132bp as glimmering
The chicken astrovirus standard items plasmid of Fluorescent Quantitative PCR uses：
gcagatcccg acgtaaagga ttacttagat agacagatca attgcgtcga ggagtatgcc 60
gctgctgaag aaatacagtt accagaagtc gggcccgact tctttcagaa aatctggtag 120
agggatggac cg。
And the positive colony of the positive recombinant plasmid with more than DNA sequence dna feature is expanded and freezes strain.Together
When to chicken astrovirus standard items plasmid with ultraviolet specrophotometer measure DNA concentration.
4th, the making of standard items curve：
More than chicken astrovirus standard items plasmid is diluted to 10 with aseptic deionized water respectively2, 103, 104, 105, 106, 107,
108, 109Copy/μ L carry out the amplification of standard items curve as standard items template.
It is as follows：Reaction system into the standard items template of Real time PCR instruments is 25 μ L：Wherein YBR
® Premix Ex TaqTMII Mix additions are 12.5 μ L, and standard items template addition is 3 μ L, and sense primer and downstream are drawn
Final concentration of the object in system is 0.5 μm of ol/L, remaining is aseptic deionized water；Wherein sense primer CAstV F：5’-
TGCAGATCCCGACGTAAAGG -3 ' (SEQ ID N0. 1), downstream primer CAstV R：5’-
CGGTCCATCCCTCTACCAGA -3’ (SEQ ID N0. 2).Response procedures in PCR instrument are：95 DEG C of pre-degeneration 5min, so
95 DEG C of 15 s afterwards, 60 DEG C of 1min, totally 40 recycle；The collection of fluorescence signal and the acquisition of data are scheduled on 60 DEG C.
Detection terminates, and baseline and threshold value are set and adjusted according to noise situation and is analyzed using accompanying software, is established
Standard curve.Obtained standard items amplification kinetic curve is shown in Fig. 1, and the examination criteria curve expanded is shown in Fig. 2.
As seen from Figure 1：Standard items expand kinetic curve and the standard items of gradient dilution correspond, in good multiple
Relationship.
As seen from Figure 2：The amplification of different copy number plasmids and Ct values are there are good linear correlation, available for purpose base
Because of the quantitative analysis of segment.
2nd, it applies：
1st, Samples detection：
After known CAstV positives intestinal samples historrhexis, aseptic process allantoic fluid approach is inoculated with instar chicken embryo on the 9th.Treat chicken embryo
To chicken embryo idiosome is collected during 11 age in days, cell total rna is extracted with Axgen total RNA extraction reagents box after liquid nitrogen grinding.Take 1 μ g
RNA, the Oligo (dT) in 20 μ L total reaction volumes15-18For primer pair, it carries out reverse transcription, obtains each cDNA samples to be tested.
Using upstream and downstream primer in kit and SYBR Premix Ex TaqTMII fluorescent dye, in ABI companies
PCR amplification is carried out on 7500 type fluorescence quantitative PCR instruments, reaction system is 25 μ L：Wherein SYBR Premix Ex TaqTMⅡ
Fluorescent dye addition is that 12.5 μ L, cDNA samples to be tested additions are 3 μ L, and wherein sense primer and downstream primer is in system
In final concentration be 0.5 μm of ol/L, remaining is aseptic deionized water.Condition be 95 DEG C of pre-degeneration 5min, then 95 DEG C 15
S, 60 DEG C of 1min, totally 40 recycle；The collection of fluorescence signal and the acquisition of data are scheduled on 60 DEG C.The quasi- product examine of mark-on simultaneously is surveyed
Make standard curve.The collection of fluorescence signal and the acquisition of data are scheduled on 60 DEG C.
Measurement result handles through software and calculates chicken astrovirus gene expression amount in detection sample according to standard curve.
2nd, fluorescence quantitative RT-RCR method detects the expression of results of chicken astrovirus gene in inoculated into chick embryo idiosome：
On the real-time fluorescence quantitative RT-PCR canonical plotting of Fig. 1, can correspondingly it be searched by the recurring number Ct values measured
The copy number of CAstV in corresponding measuring samples.
11 Japanese instar chickling embryo idiosome tissue specimen testing results are as follows：
| Chicken embryo is numbered | Copy/ μ L |
| 1-1 | 2.6530×104 |
| 1-2 | 6.009×103 |
| 2-1 | 1.9303×104 |
| 2-2 | 4.5833×104 |
| 3-1 | 1.20659×105 |
| 3-2 | 1.58977×105 |
Present invention combination most preferred embodiment is described, however after the above for having read the present invention, this field skill
Art personnel can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims institute
The range of restriction.
Sequence table
<110>Yangzhou University
<120>Real-time fluorescence quantitative RT-PCR kit and its application for chicken astrovirus detection
<141> 2017-12-21
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>Astrovirus (Astrovirus)
<220>
<221> gene
<222> (（1）)..(.（20）)
<223>The sense primer of chicken astrovirus open reading frame ORF1b
<400> 1
tgcagatccc gacgtaaagg 20
<210> 2
<211> 20
<212> DNA
<213>Astrovirus (Astrovirus)
<220>
<221> gene
Claims (2)
1. for the real-time fluorescence quantitative RT-PCR kit of chicken astrovirus detection, it is characterised in that kit includes：
1）Sense primer CAstV F：5’- TGCAGATCCCGACGTAAAGG -3’；
2）Downstream primer CAstV R：5’- CGGTCCATCCCTCTACCAGA -3’；
3）Standard items：Length is 132bp, and sequence is：
gcagatcccg acgtaaagga ttacttagat agacagatca attgcgtcga ggagtatgcc 60
gctgctgaag aaatacagtt accagaagtc gggcccgact tctttcagaa aatctggtag 120
agggatggac cg ；
4）Standard items template：102, 103, 104, 105, 106, 107, 108, 109The chicken astrovirus gene masculine plasmid of copy/uL；
The chicken astrovirus gene masculine plasmid is using pGEM-T carriers as the carrier that sets out, and is inserted into the EcoR I sites for the carrier that sets out
Poultry chicken astrovirus genetic fragment；
5）Fluorescent dye and aseptic deionized water.
2. the application of kit as described in claim 1, it is characterised in that：
By each standard items template respectively with upstream and downstream primer, fluorescent dye and aseptic deionized water anabolic reaction body in kit
System carries out PCR amplification, and the wherein final concentration of sense primer and downstream primer in system is 0.5 μm of ol/L, and fluorescent dye exists
Final concentration of 0.5 μm of ol/L in system, the response procedures in PCR instrument are：95 DEG C of pre-degeneration 5min, then 95 DEG C of 15 s,
60 DEG C of 1min, totally 40 recycle；The collection of fluorescence signal and the acquisition of data are scheduled on 60 DEG C, obtain real time fluorescent quantitative RT-
PCR standard curves；
Take upstream and downstream primer in the cDNA and kit of poultry organization's internal organs to be measured, fluorescent dye and aseptic deionized water composition
Reaction system carries out PCR amplification, and the final concentration of sense primer and downstream primer in system is 0.5 μm of ol/L, fluorescent dye
Final concentration of 0.5 μm of ol/L in system, the response procedures in PCR instrument are：95 DEG C of pre-degeneration 5min, then 95 DEG C of 15 s,
60 DEG C of 1min, totally 40 recycle；The collection of fluorescence signal and the acquisition of data are scheduled on 60 DEG C, obtain poultry organization's internal organs to be measured
Target gene amplification cycles number Ct values；By the real-time fluorescence quantitative RT-PCR standard curve, show that poultry organization to be measured is dirty
The copy number of device chicken astrovirus gene.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108929919A (en) * | 2018-08-27 | 2018-12-04 | 扬州大学 | II type astrovirus ring mediated isothermal amplification detection primer group of chicken, kit and its detection method |
| CN109055613A (en) * | 2018-08-27 | 2018-12-21 | 扬州大学 | I type astrovirus ring mediated isothermal amplification detection primer group of chicken, kit and its detection method |
| CN111041128A (en) * | 2020-01-16 | 2020-04-21 | 福建省农业科学院畜牧兽医研究所 | Primer probe set, kit and detection method for detecting duck astrovirus type 3 based on real-time fluorescent quantitative PCR |
| CN111676323A (en) * | 2020-07-01 | 2020-09-18 | 广西壮族自治区兽医研究所 | LAMP primer group and kit for detecting chicken astrovirus, detection method and application |
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| CN1681919A (en) * | 2002-09-18 | 2005-10-12 | 阿克佐诺贝尔公司 | Chicken astrovirus type 2 |
| WO2011010168A1 (en) * | 2009-07-24 | 2011-01-27 | Agri-Food Biosciences Institute | Oligonucleotides for detecting chicken astrovirus |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108929919A (en) * | 2018-08-27 | 2018-12-04 | 扬州大学 | II type astrovirus ring mediated isothermal amplification detection primer group of chicken, kit and its detection method |
| CN109055613A (en) * | 2018-08-27 | 2018-12-21 | 扬州大学 | I type astrovirus ring mediated isothermal amplification detection primer group of chicken, kit and its detection method |
| CN111041128A (en) * | 2020-01-16 | 2020-04-21 | 福建省农业科学院畜牧兽医研究所 | Primer probe set, kit and detection method for detecting duck astrovirus type 3 based on real-time fluorescent quantitative PCR |
| CN111041128B (en) * | 2020-01-16 | 2022-05-17 | 福建省农业科学院畜牧兽医研究所 | Primer probe set, kit and detection method for detecting duck astrovirus type 3 based on real-time fluorescent quantitative PCR |
| CN111676323A (en) * | 2020-07-01 | 2020-09-18 | 广西壮族自治区兽医研究所 | LAMP primer group and kit for detecting chicken astrovirus, detection method and application |
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