CN106011315A - RT-PCR discriminating diagnosis primers for type 1 and new serotype duck hepatitis virus - Google Patents
RT-PCR discriminating diagnosis primers for type 1 and new serotype duck hepatitis virus Download PDFInfo
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Abstract
The invention discloses a group of RT-PCR discriminating diagnosis primers for discriminating type 1 and new serotype duck hepatitis virus. A primer sequence is shown as a SEQ ID NO. 1-2. The primer is designed according to existed difference of a 3'-terminal nucleotide sequence of a type 1 and new serotype duck hepatitis virus whole-genome sequence, The group of the primers performs discriminating diagnosis in a specific amplification area for amplifying the type 1 and new serotype duck hepatitis virus according to the length difference of the nucleotide sequences, wherein size of a type 1 duck hepatitis virus amplification fragment is about 258 bp, and the size of a new serotype duck hepatitis virus amplification fragment is about 321 bp. The identification method of the primer has the advantages of easy and rapid operation, and high accuracy, only one group of the primers can discriminate the type 1 and new serotype duck hepatitis virus types, and the primers provide technical guarantee for aquatic bird healthy culture.
Description
Technical field
The invention belongs to veterinary's infectious disease quick diagnosis technical field, be specifically related to one group distinguish 1 type DHV and
The RT-PCR Differential Diagnosis primer of new serotype DHV.
Background technology
1 type DHV (Duck hepatitis virus type 1, DHV-1) is caused by DHV
A kind of acute, high degree in contact sexually transmitted disease, duckling in main infringement 3 week old, with hepatitis as typical characteristic.The morbidity of this disease is anxious, sick
Journey is short, M & M is high, and serious threat duck culturing industry develops in a healthy way, and [Yang Weixing, Shi Shaohua, Chen Hongmei wait .3 strain duck liver
Scorching viral 1 type full gene sequencing and analysis [J]. China's agronomy circular, 2010,26(14): 8-12.].
Within 1999, the good grade of China Agricultural University Su Jing is separated to 2 strains and 1 type duck from the Beijing duck and cherry valley duck of morbidity
The DHV new serotype of hepatitis virus serum-free dependency (Duck hepatitis virus new serotype,
DHV-N).This team executes few China etc. and DHV new serotype DHV-N G strain has been carried out genome sequence determination, finds duck
Hepatitis virus new serotype G strain is compared with the 5 ' UTR and 3 ' UTR of DHV 1 type, there occurs that bigger insertion is existing with disappearance
As, and clear and definite DHV 1 type and new serotype exist larger difference, and [Shi Shaohua, Cheng Longfei, Fu Guanghua, etc. duck hepatitis
Poison new serotype genomic sequence analysis [J]. microorganism journal, 2009,49(3): 309-315.].
Currently, with respect to 1 type DHV and the RT-PCR Differential Diagnosis of new serotype DHV pathogeny detection
Method all has relevant report.But correlation technique is required to for 1 type DHV and new serotype DHV specificity
Nucleotide sequence separately designs specific primer, sets up double PCR differential diagnostic method, but double PCR condition optimizing is the most numerous
Trivial, operator is required higher.The method that the present invention provides only needs one group of primer (fewer than separately designing when specific primer detects
1 group of primer), this primer exists long according to 1 type DHV and new serotype DHV whole genome sequence at 3 '-end
Short difference designs primer；1 type DHV and new serotype DHV can effectively be expanded by this group primer simultaneously
Increase, and 1 type DHV of primer amplification and new serotype DHV Region Nucleotide sequence exist length difference
Making a distinction, wherein 1 type DHV amplified fragments size is 258bp, new serotype DHV amplified fragments size
For about 321bp, come 1 type and new serum according to the purpose clip size difference that 1 type and new serotype DHV expand
Type DHV infects and detects.The method of the present invention is simple to operate, it is not necessary to complicated loaded down with trivial details condition optimizing process, this
Bright quick accurate on 1 type DHV and new serotype DHV pathogeny detection, the most relevant neck can be filled up
Territory research blank.
Summary of the invention
The RT-PCR that it is an object of the invention to provide a kind of difference 1 type DHV and new serotype DHV draws
Thing.This primer can effectively distinguish 1 type DHV and new serotype DHV infects (or coinfection), is good for for aquatic bird industry
Health cultivation offer technology ensures.
The purpose of the present invention is achieved through the following technical solutions:
One group of RT-PCR Differential Diagnosis primer distinguishing 1 type DHV and new serotype DHV, described primer
Sequence is as follows:
Forward primer P1:5 '-AGGCCCAGAAGCATTCAAACA-3 '；
Downstream primer P2:5 '-TGGGTGTTTTACGTGTACTC-3 '.
Described primer needs 1 type DHV and new serotype DHV all can be carried out RT-PCR amplification,
And there is nucleotide fragments length difference, wherein 1 type at this amplification region 1 type DHV and new serotype DHV
DHV amplified fragments size is about 258bp, and new serotype DHV amplified fragments size is about 321bp.
As it is shown in figure 1, the forward primer of present invention design is guarded at 1 type DHV and new serotype DHV
District's design；As in figure 2 it is shown, the downstream primer of present invention design is protected at 1 type DHV and new serotype DHV
Defending zone design.1 type DHV and new serotype DHV can be entered by the upstream and downstream primer of present invention design simultaneously
Row amplification.As in figure 2 it is shown, 1 type DHV and new serotype DHV are entered by the upstream and downstream primer of present invention design
During row amplification, amplification region exists nucleotide difference, and (see Fig. 2 difference section, wherein 1 type DHV amplified fragments size is
About 258bp, new serotype DHV amplified fragments size is about 321bp).
From shown in Fig. 3,1 type DHV (DHV-1) that Fig. 1 and Fig. 2 chooses and new serotype DHV (DHV-
N) all meet the requirements, the feature of correlated virus in GenBank can be represented.
Distinguish 1 type DHV and the RT-PCR detection method of new serotype DHV, utilize upstream to draw for one group
Thing P1:5 '-AGGCCCAGAAGCATTCAAACA-3 ' and downstream primer P2:5 '-TGGGTGTTTTACGTGTACTC-3 ' builds
Be based on 1 type DHV and the difference section design of new serotype DHV genome 3 '-end, and primer is scorching to 1 type duck liver
The product 1 type DHV amplified fragments that virus and new serotype DHV nucleic acid RNA are carried out after RT-PCR amplification is big
Little for about 258bp, new serotype DHV amplified fragments size is about 321bp (as shown in Figure 4).
Specifically include following steps:
1) the nucleic acid RNA of 1 type DHV and new serotype DHV is conventionally extracted；
2) utilize forward primer P1:5 '-AGGCCCAGAAGCATTCAAACA-3 ' and downstream primer P2:5 '-
TGGGTGTTTTACGTGTACTC-3 ', carries out RT-PCR inspection to 1 type DHV and new serotype DHV simultaneously
Survey, after having reacted, carry out conventional agarose gel electroresis appraisal；
3) according to RT-PCR amplification the big I of product directly 1 type DHV and new serotype DHV are infected into
Row Differential Diagnosis, wherein 1 type DHV amplified fragments size is about 258bp, new serotype DHV amplification sheet
Duan great little is about 321bp.
Described primer preparation detection 1 type DHV and new serotype DHV reagent in application.
For prior art, it is an advantage of the current invention that: authentication method of the present invention is simple, quick and accuracy rate height:
The hepatitis symptom death duck pathological material of disease of 12 parts of clinical acquisitions is detected by the present invention, carries out RT-PCR detection after extracting nucleic acid RNA,
Through agarose gel electrophoresis it can be seen that there is 1 type DHV positive pathological material of disease 2 parts, positive rate is 16.67%(2/12)；Fresh blood
Clear type DHV positive pathological material of disease 5 parts, positive rate is 41.67%(5/12), it is not detected by 12 parts of samples and there is coinfection now
As；The present invention only needs this group primer of forward primer P1 and downstream primer P2 can be to 1 type DHV and new serotype duck
Hepatites virus infections carries out Differential Diagnosis, and without empty complicated loaded down with trivial details condition optimizing process, the present invention is at 1 type DHV and new
The detection of serotype DHV rapid differential diagnosis is upper quick accurate, can fill up domestic and international association area research blank.
Accompanying drawing explanation
Fig. 1 is that the forward primer that designs of the present invention is at 1 type DHV and new serotype DHV conserved region base
Because of analysis chart.
Fig. 2 is that the downstream primer that designs of the present invention is at 1 type DHV and new serotype DHV conserved region base
Because the difference section gene of analysis chart and the primer amplification 1 type DHV of this experimental design and new serotype DHV divides
Analysis figure.
Fig. 3 is Fig. 1 and the 1 type DHV chosen of Fig. 2 and new serotype DHV phylogenetic analysis figure.From
Result is visible, and the representative strains that the present invention chooses is representative.The specificity upstream and downstream primer symbol designed according to above-mentioned representative strains
Close experiment expection.
Fig. 4 is to utilize primer P1 and P2 to carry out 1 type DHV and new serotype DHV RT-PCR to expand
Electrophoretogram.Wherein: 1:DHV-1；2:DHV-N；3: blank；M:DL2000 DNA molecular amount standard.
Fig. 5 is primer P1 and the electrophoretogram of P2 specific detection.Wherein: 1: bird flu virus；2: paramyxovirus type 1；
3: reovirus；4: fowl tembusu virus；5: duck circovirus；6: duck plague virus；7: Muscovy duck parvovirus；8: new serotype
DHV: 9:1 type DHV；M:DL2000 DNA molecular amount standard.
Detailed description of the invention
Below in conjunction with Figure of description and embodiment, present invention is described in detail:
One group of RT-PCR Differential Diagnosis primer distinguishing 1 type DHV and new serotype DHV and detection method,
The sequence of described primer is as follows:
Forward primer P1:5 '-AGGCCCAGAAGCATTCAAACA-3 '；
Downstream primer P2:5 '-TGGGTGTTTTACGTGTACTC-3 '.
Distinguishing 1 type DHV and the RT-PCR detection method of new serotype DHV for one group, it includes following
Step:
1) the nucleic acid RNA of 1 type DHV and new serotype DHV is extracted；
2) utilize forward primer P1:5 '-AGGCCCAGAAGCATTCAAACA-3 ' and downstream primer P2:5 '-
TGGGTGTTTTACGTGTACTC-3 ', carries out RT-PCR inspection to 1 type DHV and new serotype DHV simultaneously
Survey, after having reacted, carry out conventional agarose gel electroresis appraisal；
3) according to RT-PCR amplification the big I of product directly 1 type DHV and new serotype DHV are infected into
Row Differential Diagnosis, wherein 1 type DHV amplified fragments size is about 258bp, new serotype DHV amplification sheet
Duan great little is about 321bp.
Described one group distinguish 1 type DHV and new serotype DHV RT-PCR Differential Diagnosis primer and
Detection method, it is characterised in that: the reaction system of described RT-PCR detection is:
Addition following component in 20 μ L systems:
Step 2) described in the reaction condition that arranges of base RT-PCR detection be: the reaction of 45 DEG C of reverse transcription 30min laggard performing PCRs,
94 DEG C of denaturations 5min；Carry out 94 DEG C of 50s, 54 DEG C of 30s, 72 DEG C of 30s subsequently, carry out 40 and circulate rear 72 DEG C of extensions
10min.After reaction terminates, identify through conventional agarose gel electrophoresis.
Embodiment 1
1 materials and methods:
1.1 experiment reagents and consumptive material: viral nucleic acid RNA extracts test kit (EasyPure Viral DNA/RNA Kit), RT-
PCR kit (EasyScript One-Step RT-PCR SuperMix) is purchased from Beijing Quanshijin Biotechnology Co., Ltd,
PCR is purchased from AXYGEN company in real time；
1.2 experiment strains:
1 type DHV (DHV-1) (GenBank JX390983) and new serotype DHV (DHV-N)
(GenBank EU755009), bird flu virus, paramyxovirus type 1, reovirus, fowl tembusu virus, duck annulus disease
Poison, duck plague virus and Muscovy duck parvovirus are by Fujian Province Academy Of Agricultural Sciences Animal Husbandry And Veterinary Medicine Institute isolation identification and preservation.
1.3 methods:
1.3.1 the design of primers of RT-PCR:
Utilize Oligo7 that it is carried out design of primers, need when this is to design of primers DHV-1 and DHV-N all can be expanded simultaneously
Increasing, and there is nucleotide sequence length difference in this amplification region DHV-1 and DHV-N, described primer is:
Forward primer P1:5 '-AGGCCCAGAAGCATTCAAACA-3 '；
Downstream primer P2:5 '-TGGGTGTTTTACGTGTACTC-3 '.
Wherein 1 type DHV amplified fragments size is about 258bp, and new serotype DHV amplified fragments is big
Little for about 321bp.
1.3.2 the reaction system of the detection of RT-PCR and the optimization of reaction condition:
The reaction system of described RT-PCR detection is:
Addition following component in 20 μ L systems:
Step 2) described in the reaction condition that arranges of base RT-PCR detection be: the reaction of 45 DEG C of reverse transcription 30min laggard performing PCRs,
94 DEG C of denaturations 5min；Carry out 94 DEG C of 50s, 54 DEG C of 30s, 72 DEG C of 30s subsequently, carry out 40 and circulate rear 72 DEG C of extensions
10min.After reaction terminates, identify through conventional agarose gel electrophoresis.As shown in Figure 4: as can be seen from Figure 4: only 1 type DHV
(DHV-1) (GenBank JX390983) and new serotype DHV (DHV-N) (GenBank EU755009) are permissible
RT-PCR expands.As seen from Figure 4,1 type DHV amplified fragments size is about 258bp, new serotype DHV
Amplified fragments size is about 321bp.
1.3.3 specificity experiments
Sick such as bird flu virus, fowl to duck group's common transmittable with the primer (forward primer P1 and downstream primer P2) of this research design
1 type paramyxovirus, reovirus, fowl tembusu virus, duck circovirus, duck plague virus and Muscovy duck parvovirus nucleic acid are carried out
Detection, does not all occur that specific band expands, and testing result is not the most negative, and only 1 type DHV and new serotype duck liver are scorching
There is specific amplification (see figure 5) in viral nucleic acid, shows that the primer of this research design has extremely strong specificity.
1.3.4 clinical practice
Utilize the present invention to provide one group distinguishes RT-PCR Differential Diagnosis primer and the detection of 1 type and new serotype DHV
The hepatitis symptom death duck pathological material of disease of 12 parts of clinical acquisitions is detected by method, carries out RT-PCR detection, warp after extracting nucleic acid RNA
Agarose gel electrophoresis is it can be seen that there is 1 type DHV positive pathological material of disease 2 parts, and positive rate is 16.67%(2/12)；New serum
Type DHV positive pathological material of disease 5 parts, positive rate is 41.67%(5/12), it is not detected by 12 parts of samples and there is coinfection phenomenon；
The present invention only needs forward primer P1 and this group of downstream primer P2 can be to 1 type DHV and new serotype DHV
Differentiating, without empty complicated loaded down with trivial details condition optimizing process, the present invention is at 1 type DHV and new serotype DHV
Rapid differential diagnosis detection is upper quick accurate, can fill up domestic and international association area research blank.
1.3.5 result verification
It is accredited as 1 type DHV positive pathological material of disease 2 parts, new serotype DHV positive pathological material of disease 5 parts by above-mentioned, uses document
The RT-PCR method used carries out duplicate detection checking.1 type DHV positive pathological material of disease 2 parts uses document, and [Yang Weixing executes few
China, Chen Hongmei, waits .3 strain DHV 1 type full gene sequencing and analysis [J]. China's agronomy circular, 2010,26(14): 8-
12.] detecting, result is consistent with the method for this research, and coincidence rate is 100%.New serotype DHV positive pathological material of disease 5
Part, the RT-PCR method used with document carries out duplicate detection checking.New serotype DHV positive pathological material of disease 5 parts uses literary composition
Offer [Shi Shaohua, Cheng Longfei, Fu Guanghua, etc. DHV new serotype genomic sequence analysis [J]. microorganism journal,
2009,49(3): 309-315.] detecting, result is consistent with the method for this research, and coincidence rate is 100%.
The foregoing is only presently preferred embodiments of the present invention, all impartial changes done according to scope of the present invention patent with
Modify, all should belong to the covering scope of the present invention.
SEQUENCE LISTING
<110>Fujian Province Academy Of Agricultural Sciences Animal Husbandry And Veterinary Medicine Institute
<120>one groups of 1 types and the RT-PCR Differential Diagnosis primer of new serotype DHV
<130> 2
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213>artificial sequence
<400> 1
aggcccagaa gcattcaaac a 21
<210> 2
<211> 20
<212> DNA
<213>artificial sequence
<400> 2
tgggtgtttt acgtgtactc 20
Claims (2)
1. one group of RT-PCR Differential Diagnosis primer distinguishing 1 type DHV and new serotype DHV, its feature exists
In: the sequence of described primer is as follows:
Forward primer P1:5 '-AGGCCCAGAAGCATTCAAACA-3 '；
Downstream primer P2:5 '-TGGGTGTTTTACGTGTACTC-3 '.
2. as claimed in claim 1 primer in preparation detection 1 type and new serotype DHV reagent application.
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Cited By (6)
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| CN108486280A (en) * | 2018-03-06 | 2018-09-04 | 福建省农业科学院畜牧兽医研究所 | A kind of PCR primer for the short beak runting syndrome goose parvovirus of 5`-UTR genetic tests |
| CN108913813A (en) * | 2018-07-23 | 2018-11-30 | 福建省农业科学院畜牧兽医研究所 | For identifying the primer sets of DAdV-2 and DAdV-3 |
| CN109750125A (en) * | 2019-03-22 | 2019-05-14 | 福建省农业科学院畜牧兽医研究所 | One group of primer and kit for quickly identifying PCV1 and PCV2 |
| CN110358850A (en) * | 2019-08-14 | 2019-10-22 | 扬州大学 | A kind of primer sets and kit detecting accessory mamma streptococcus serum type |
| CN111500784A (en) * | 2020-05-22 | 2020-08-07 | 福建省农业科学院畜牧兽医研究所 | Primer group for differential diagnosis of different types of duck hepatitis viruses |
| CN111500783A (en) * | 2020-05-22 | 2020-08-07 | 福建省农业科学院畜牧兽医研究所 | Primer group for differential diagnosis of DHAV-1, DHAV-N and DHBV |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108486280A (en) * | 2018-03-06 | 2018-09-04 | 福建省农业科学院畜牧兽医研究所 | A kind of PCR primer for the short beak runting syndrome goose parvovirus of 5`-UTR genetic tests |
| CN108486280B (en) * | 2018-03-06 | 2021-09-17 | 福建省农业科学院畜牧兽医研究所 | PCR primer for detecting short beak dwarf syndrome goose parvovirus by aiming at 5' -UTR gene |
| CN108913813A (en) * | 2018-07-23 | 2018-11-30 | 福建省农业科学院畜牧兽医研究所 | For identifying the primer sets of DAdV-2 and DAdV-3 |
| CN108913813B (en) * | 2018-07-23 | 2022-04-12 | 福建省农业科学院畜牧兽医研究所 | Primer set for identifying DAdV-2 and DAdV-3 |
| CN109750125A (en) * | 2019-03-22 | 2019-05-14 | 福建省农业科学院畜牧兽医研究所 | One group of primer and kit for quickly identifying PCV1 and PCV2 |
| CN110358850A (en) * | 2019-08-14 | 2019-10-22 | 扬州大学 | A kind of primer sets and kit detecting accessory mamma streptococcus serum type |
| CN111500784A (en) * | 2020-05-22 | 2020-08-07 | 福建省农业科学院畜牧兽医研究所 | Primer group for differential diagnosis of different types of duck hepatitis viruses |
| CN111500783A (en) * | 2020-05-22 | 2020-08-07 | 福建省农业科学院畜牧兽医研究所 | Primer group for differential diagnosis of DHAV-1, DHAV-N and DHBV |
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