CN106702026A - Real-time fluorescent quantitative PCR primers for distinguishing clade2.3.4 and clade7.2 H5 AIV - Google Patents
Real-time fluorescent quantitative PCR primers for distinguishing clade2.3.4 and clade7.2 H5 AIV Download PDFInfo
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Abstract
The invention provides a group of real-time fluorescent quantitative PCR primers for distinguishing clade2.3.4 from clade7.2 H5 AIV. The primers are designed according to a GC content difference between characteristic nucleotide sequences of hemagglutinin genes (HA) of clade2.3.4 and clade7.2 H5 AIV. When the group of primers is used for carrying out real-time fluorescent quantitative PCR on clade2.3.4 and clade7.2 H5 AIV, clade2.3.4 and clade7.2 H5 AIV can be effectively amplified, but the primers effectively distinguish clade2.3.4 from clade7.2 H5 AIV according to a difference of melting temperatures (TM values) on a melting curve generated after the real-time fluorescent quantitative PCR of clade2.3.4 and clade7.2 H5 AIV. By the cooperation with a computer connected with a real-time fluorescent quantitative PCR instrument and the use of analysis software of the instrument, only one group of primers can specifically distinguish and diagnose clade2.3.4 and clade7.2 H5 AIV infection situations. The distinguishing method is simple, and relatively high in efficiency and accuracy.
Description
Technical field
The invention belongs to epizootiology field, and in particular to group differentiation clade2.3.4 and clade7.2 a H5
The real-time fluorescence quantitative PCR primer of AIV.
Background technology
Bird flu（Avian influneza, AI）It is infringement poultry and wild bird caused by influenza A, main
A kind of acute, highly contagious disease.Highly pathogenic H5 subtype avian influenza virus（H5 subtype avian influenza
virus, H5 AIV）The infection for causing is at present by OIE（OIE）It is classified as the animal infection that must be reported
Disease, a class animal epidemic is listed in China.
Earliest record on influenza can trace back to the once report from Italy in 1878, then all over the world successively
There is breaking out and popular for influenza.1996, in first plant of H5N1 hypotype HPAIV [A/Goose/ that isolated in China is arrived
Guangdong/1/96（H5N1）, GS/GD/96], and then Hong Kong was directly felt in generation H5N1 subtype avian influenza virus in 1997
Dye people's event, this is first evidence of fowl source stream Influenza Virus direct infection people without intermediate host.Due to viral from ancestral source
GS/GD/96 evolve multiple pedigrees and name disunity, be this WHO, FAO and OIE combine formulated H5N1 HPAIV unification point
Class criterion divides for 0 ~ 9 amounts to 10 difference clade H5N1 HPAIV.Between wherein clade 2.3.4 are most earlier than 2003 ~ 2006 years
The Strain on the ground such as China, Vietnam, Thailand, Laos and Malaysia is popular in, clade 2.3.4 branches exist within 2006 ~ 2009 years
The many areas of China are advantage epidemic strains；Although and gradually being replaced by clade 2.3.2 branches after 2010, clade
2.3.4 branch is still popular in many areas of China.In recent years, the H5 subtype highly pathogenic avian influenza virus of clade 7.2
Also it is popular in some areas, discriminating is carried out to the H5 subtype highly pathogenic avian influenza virus of clade 2.3.4 and clade 7.2 and is examined
It is disconnected particularly important.For the vaccine of clade 2.3.4 H5 AIV（Re-5 plants）With the vaccine of the H5 AIV of clade 7.2（Re-7
Strain）It has been succeeded in developing that, carrying out antidiastole to the H5 AIV of clade 2.3.4 and clade 7.2 helps to instruct H5 AIV corresponding
The scientifical use of vaccine.
However, the HA gene order homologies therebetween of clade 2.3.4 and clade 7.2 are up to 92.7%（With classics
As a example by Reference strains compare）, it is difficult to pass through conventional molecular biology method be differentiated.
The real-time fluorescence quantitative PCR primer of group differentiation clade2.3.4 and clade7.2 H5 AIV of the invention only needs 1
Group primer pair（2 primers）Effectively clade2.3.4 and clade7.2 H5 AIV can effectively be distinguished.The primer according to
Clade2.3.4 and clade7.2 H5 AIV hemagglutinin genes（HA）Existing characteristic nucleotide sequence difference is designed；
The Tm values and the positively related feature of its G/C content of the melting curve generated after being reacted using real-time fluorescence quantitative PCR, by observation
Clade2.3.4 and clade7.2 H5 AIV carry out real-time fluorescence quantitative PCR amplified reaction through this group of primer after melting curve
Tm value differences are different, you can accurately carry out precise differential diagnosis to clade2.3.4 and clade7.2 H5 AIV infection conditions, correlation is ground
Study carefully there is not yet domestic and foreign literature reports that the present invention can fill up association area blank.
The content of the invention
It is an object of the invention to provide the real time fluorescent quantitative of a group differentiation clade2.3.4 and clade7.2 H5 AIV
PCR primer and its application, the primer can effectively distinguish clade2.3.4 and clade7.2 H5 AIV infection（Or coinfection）, it is
Science bridle H5 AIV provide technology and ensure.
The present invention is according to clade2.3.4 and clade7.2 H5 AIV hemagglutinin genes（HA）The nucleotides of existing characteristics
Sequence difference designs one group of real-time fluorescence quantitative PCR primer.Using amplification region clade2.3.4 and clade7.2 H5
AIV hemagglutinin genes（HA）There is nucleotides G/C content difference, by setting up the real-time fluorescence quantitative PCR based on Eva Green
Method is to clade2.3.4 and clade7.2 H5 AIV hemagglutinin genes（HA）Real-time fluorescence quantitative PCR amplified reaction is carried out, is obtained
There are different melting temperatures in the melting curve for obtaining（Tm values）Difference, according to melting curve Tm value differences are different can be directly right
Clade2.3.4 and clade7.2 H5 AIV infection conditions carry out antidiastole.
To achieve the above object, the present invention uses following technical scheme：
The real-time fluorescence quantitative PCR primer of one group differentiation clade2.3.4 and clade7.2 H5 AIV, its nucleotides sequence is classified as：
Sense primer F1：5’- CAACATACACCCTCTCACCAT -3’；
Anti-sense primer R1：5’- CATCTACCATTCCCTGCCAT -3’.
Real time fluorescence quantifying PCR method based on Eva Green is set up by the primer, according to using primer amplification
Clade2.3.4 and clade7.2 H5 AIV hemagglutinin genes（HA）Existing nucleotides feature sex differernce --- G/C content
There are different melting temperatures in difference, the melting curve that real-time fluorescence quantitative PCR can be caused to react the generation after amplified reaction（Tm
Value）, directly clade2.3.4 and clade7.2 H5 AIV infection can be differentiated.
Specifically include following steps：
（1）Above-mentioned specific primer is designed and synthesized to F1/R1；
（2）Viral RNA is extracted：Extract the RNA of clade2.3.4 and clade7.2 H5 AIV respectively from detection sample；
（3）RT-PCR is expanded：H5 AIV hemagglutinin gene HA specific primers are designed and synthesized to H5-HAF/H5-HAR to extracting
Viral RNA carry out RT-PCR amplifications, obtain its HA gene order, the purpose fragment that RT-PCR is expanded is carried out into glue reclaim and pure
After change, it is cloned into carrier T, obtains the positive weight of the hemagglutinin gene HA containing clade2.3.4 and clade7.2 H5 AIV
Group plasmid（T-clade2.3.4-1 and T-clade7.2-1）, determine after its OD value calculates copy number, continuous doubling dilution,
As the standard items that real-time fluorescence quantitative PCR reacts.
H5 AIV hemagglutinin gene HA specific primers used are classified as to the nucleotides sequence of H5-HAF/H5-HAR：
H5-HAF: 5’- TGGTTACCATGCAAACAAC -3’；
H5-HAR: 5’- TTACACTTTCCATACATTCATTATC -3’。
（4）Real-time fluorescence quantitative PCR：With the real time fluorescent quantitative primer pair F1/R1 to positive recombinant plasmid（T-
Clade2.3.4-1 and T-clade7.2-1）The amplification of Eva Green real-time fluorescence quantitative PCRs is carried out, phase is obtained after the completion of reaction
The amplification curve answered, and generate the melting curve of Eva Green real-time fluorescence quantitative PCRs amplification.
（5）Virus Infection judges：After real-time fluorescence quantitative PCR reaction terminates, it is by observing amplification curve judgement
It is no to there is clade2.3.4 and clade7.2 H5 AIV infection；Can be right by observing that melting curve its Tm value difference is different
Clade2.3.4 and clade7.2 H5 AIV are differentiated.Wherein, the real-time fluorescence quantitative PCR primer pair designed by the present invention
F1/R1 meets following requirement：
（1）High specificity：The real-time fluorescence quantitative PCR product need to select clade2.3.4 and clade7.2 H5 AIV hemagglutinin
The region design that gene HA nucleotide sequences are guarded after being analyzed, high specific is respectively provided with to the two, is reached and only need one group and draw
Thing just can be expanded to clade2.3.4 and clade7.2 H5 AIV, that is, observe the reacted amplification of real-time fluorescence quantitative PCR
Curve is that can determine whether infection clade2.3.4 or clade7.2 H5 AIV.
（2）Two kinds of amplification region G/C contents of virus are different：The hemagglutinin gene HA of this group of primer amplification exists
There is nucleotide sequence G/C content difference in clade2.3.4 and clade7.2 H5 AIV.Based on real-time fluorescence quantitative PCR reaction
The Tm value differences of the melting curve of generation are different（The G/C content difference positive correlation of the difference and nucleotide sequence）, you can it is real by observing
When the reacted melting curve of quantitative fluorescent PCR to clade2.3.4 and clade7.2 H5 AIV infection judge（Can be effective
Differentiation is clade2.3.4 H5 AIV or clade7.2 H5 AIV）.
Wherein, described step（5）Virus Infection determination methods it is as follows：
If in Tm=（82.04±0.09）Single specificity T m peaks DEG C occur, to be judged to clade2.3.4 H5 AIV positive；
If in Tm=（83.06±0.05）Single specificity T m peaks DEG C occur, to be judged to clade7.2 H5 AIV positive；
If in Tm=（82.04±0.09）DEG C and Tm=（83.06±0.05）DEG C appearance is bimodal, then be judged as clade2.3.4 and
Clade7.2 H5 AIV double infections；
Other situations are judged to feminine gender.
Real-time fluorescence quantitative PCR primer pair F1/R1 designed by the present invention can be used for prepare distinguish clade2.3.4 and
Application in terms of clade7.2 H5 AIV infection conditions.
Beneficial effects of the present invention：Authentication method is simple, and efficiency and accuracy rate are higher.The one group of reality provided using this research
When fluorescence quantification PCR primer 2 plants of clade2.3.4 H5 AIV to early stage separate and 3 plants of clade7.2 H5 AIV carry out in real time
Fluorescence quantitative PCR detection, is consistent with expection.And only need 1 group of primer pair（2 primers）Can effectively to clade2.3.4 and
Clade7.2 H5 AIV are effectively distinguished.
Brief description of the drawings
Fig. 1 is clade 2.3.4 and clade7.2 H5 AIV hemagglutinin genes HA design of primers areas and nucleotide sequence
Characteristic differences figure.
Amplification curves of Fig. 2 primer pairs F1/R1 to clade 2.3.4 and clade7.2 H5 AIV virus result of infection.
The judgement must be made under the premise of amplification successfully, if amplification failure, can not make infection type according to melting curve and judge；
If copy number is identical, clade 2.3.4 are consistent with clade7.2 H5 AIV amplification curves.
Fig. 3 primer pairs F1/R1 carries out real-time fluorescence quantitative PCR reaction to clade 2.3.4 and clade7.2 H5 AIV
Melting curve.
Specific embodiment
With reference to embodiment, the present invention will be further described.
Embodiment 1
1st, strain：
H5 subtype influenza virus clade 2.3.4 branches（CX1 plants）With clade7.2 branches（DK7 plants）By Fujian Agricultural section
Animal and veterinary research institute of institute preserves.
2nd, design of primers and synthesis
Hemagglutinin gene HA nucleotides feature sex differernces area design according to clade 2.3.4 and clade7.2 H5 AIV is real-time
The primers F 1 and R1 of quantitative fluorescent PCR reaction, wherein F1 and R1 primer sequences are：
Sense primer F1：5 '-CAACATACACCCTCTCACCAT -3 ',
Anti-sense primer R1：5’- CATCTACCATTCCCTGCCAT -3’.
3rd, viral RNA is extracted and RT-PCR amplifications：
Clade 2.3.4 branches are extracted in conventional manner（CX1 plants）With clade7.2 branches（DK7 plants）The virus of H5 AIV
RNA.Using for H5 AIV hemagglutinin gene HA genes, specific primer is designed（ H5-HAF: 5’-
TGGTTACCATGCAAACAAC -3 ' and H5-HAR: 5’- TTACACTTTCCATACATTCATTATC -3’）To the core for extracting
Sour RNA carries out RT-PCR amplifications, obtains its HA gene order；Purified after the purpose fragment that RT-PCR is expanded is carried out into glue reclaim
Afterwards, it is cloned into carrier T, obtains the positive restructuring of the hemagglutinin gene HA containing clade2.3.4 and clade7.2 H5 AIV
Plasmid（T-clade2.3.4-1 and T-clade7.2-1）, determine after its OD value calculates copy number, continuous doubling dilution, as
The standard items of real-time fluorescence quantitative PCR reaction.
4th, real-time fluorescence quantitative PCR reaction：
20 μ L optimal reaction systems of optimization are system：The μ L of Eva Green 10, concentration is that F1, R1 of 10 μm of ol/L are each
0.2 μ L, the μ L of template 2, water complement to 20 μ L.Optimum reaction condition is：95 DEG C, 2 min predegenerations；95 DEG C of 10 s, 60 DEG C
15 s, totally 40 circulations, after circulation terminates, corresponding melting curve are made according to condition.
5th, Virus Infection judges：
After real-time fluorescence quantitative PCR reaction terminates, amplification curve is observed, judge the method set up whether there is specific amplification（No
Amplification curve, the testing result of melting curve is nonsensical）.The melting curve that real-time fluorescence quantitative PCR reaction is made after terminating,
Directly on the computer connected with real-time fluorescence quantitative PCR instrument, it is analyzed using the corresponding software of real-time fluorescence quantitative PCR
（Can directly visually observe）, determination methods are to be judged to clade 2.3.4 and clade7.2 H5 AIV infection conditions：
If in Tm=（82.04±0.09）Single specificity T m peaks DEG C occur, to be judged to clade2.3.4 H5 AIV positive；
If in Tm=（83.06±0.05）Single specificity T m peaks DEG C occur, to be judged to clade7.2 H5 AIV positive；
If in Tm=（82.04±0.09）DEG C and Tm=（83.06±0.05）DEG C appearance is bimodal, then be judged as clade2.3.4 and
Clade7.2 H5 AIV double infections；
Other situations are judged to feminine gender.
From figure 3, it can be seen that primer pair F1/R1 is to two strain virus agglutinin of blood genes（HA）Fragment amplification high specificity,
And the Tm values of the two have differences, two strain virus can be effectively distinguished.Result of the test shows, real-time using one group of this research offer
2 plants of clade2.3.4 H5 AIV that fluorescence quantification PCR primer to early stage separate and 3 plants of clade7.2 H5 AIV carry out glimmering in real time
Fluorescent Quantitative PCR is detected, is consistent with expection.And only need 1 group of primer pair（2 primers）Can effectively to clade2.3.4 and
Clade7.2 H5 AIV are effectively distinguished.
Embodiment 2
Found after real-time fluorescence quantitative PCR detection is carried out after treatment to 54 parts of clinical censorship dead duck pathological material of diseases,
The clade2.3.4 H5 AIV infection positive has 3 plants, and positive rate is 5.55%（3/54）；The clade7.2 H5 AIV infection positive has 4
Strain, positive rate is 7.40%（4/54）；, it is not detected by 54 parts of clinical censorship dead ducks and there is clade2.3.4 and clade7.2
H5 AIV coinfection phenomenons.
The foregoing is only presently preferred embodiments of the present invention, all impartial changes done according to scope of the present invention patent with
Modification, should all belong to covering scope of the invention.
SEQUENCE LISTING
<110>Fujian Province Academy Of Agricultural Sciences Animal Husbandry And Veterinary Medicine Institute
<120>Distinguish the real-time fluorescence quantitative PCR primer of clade2.3.4 and clade7.2 H5 AIV
<130> 4
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213> F1
<400> 1
caacatacac cctctcacca t 21
<210> 2
<211> 20
<212> DNA
<213> R1
<400> 2
catctaccat tccctgccat 20
<210> 3
<211> 19
<212> DNA
<213> H5-HAF
<400> 3
tggttaccat gcaaacaac 19
<210> 4
<211> 25
<212> DNA
<213> H5-HAR
<400> 4
ttacactttc catacattca ttatc 25
Claims (2)
1. the real-time fluorescence quantitative PCR primer of a group differentiation clade2.3.4 and clade7.2 H5 AIV, it is characterised in that：Institute
Stating PCR primer sequence is：
Sense primer F1：5 '-CAACATACACCCTCTCACCAT -3 ',
Anti-sense primer R1：5’- CATCTACCATTCCCTGCCAT -3’.
2. drawn using the real-time fluorescence quantitative PCR of a group differentiation clade2.3.4 and clade7.2 H5 AIV described in claim 1
Thing is preparing the application distinguished on clade2.3.4 and clade7.2 H5 AIV kits.
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| CN105969914A (en) * | 2016-07-26 | 2016-09-28 | 福建省农业科学院畜牧兽医研究所 | Group of real-time fluorescence quantification PCR primers for distinguishing duck circovirus genotypes |
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