CN105256073A - Type-3 duck hepatitis A virus TaqMan fluorescent quantitation RT-PCR detection reagent kit and method - Google Patents
Type-3 duck hepatitis A virus TaqMan fluorescent quantitation RT-PCR detection reagent kit and method Download PDFInfo
- Publication number
- CN105256073A CN105256073A CN201510792207.1A CN201510792207A CN105256073A CN 105256073 A CN105256073 A CN 105256073A CN 201510792207 A CN201510792207 A CN 201510792207A CN 105256073 A CN105256073 A CN 105256073A
- Authority
- CN
- China
- Prior art keywords
- virus
- probe
- primer
- type
- duck
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
- C12Q1/706—Specific hybridization probes for hepatitis
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biophysics (AREA)
- Communicable Diseases (AREA)
- Virology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a type-3 duck hepatitis A virus TaqMan fluorescent quantitation RT-PCR detection reagent kit and method. The reagent kit comprises a primer pair composed of an upstream primer and a downstream primer and a probe composed of nucleotide sequences shown in the SEQ ID NO.5, wherein the upstream primer is composed of nucleotide sequences shown in the SEQ ID NO.3 and the downstream primer is composed of nucleotide sequences shown in the SEQ ID NO.4. The adopted reagent kit can be used for widely detecting the type-3 duck hepatitis A virus, the detection time is short, the detection cost is low, the repeatability is good, and the sensitivity is high. The detected target point has the single specificity, and the provided primer pair and the probe have no amplification signals for samples containing no target genes and have the good specificity; in addition, the sensitivity of the primer pair and the probe for detecting RNA can reach 48 copies/microliter and the good sensitivity is achieved.
Description
Technical field
The invention belongs to technical field of biological, be specifically related to 3 type duck hepatitis A virus (HAV) TaqMan fluorescence quantitative RT-PCR detecting kit and methods.
Background technology
Duck viral hepatitis is a kind of Important Infectious Diseases that duck industry is supported in harm, and its major virulent factor is picornavirus, i.e. the DHAV (duckhepatitisAvirus, DHAV) of Picornaviridae fowl hepatovirus.According to complete genome sequencing, DHAV can be divided into 3 independently genotype, Gene A type DHAV (DHAV-A), gene Type B DHAV (DHAV-B) and gene C type DHAV (DHAV-C); According to serology Neutralizing test, these 3 genotype serum-frees intersect, therefore the serotype that correspondence 3 is different respectively, serum 1 type duck hepatitis A virus (HAV) (DHAV-1), serum 2 type duck hepatitis A virus (HAV) (DHAV-2) and Serotype-3 duck hepatitis A virus (HAV) (DHAV-3).Wherein, DHAV-A represents traditional epidemic isolates, and DHAV-B represents Taiwan new virus strain, and DHAV-C represents the emerging epidemic isolates in the countries and regions such as China and Korea S in recent years, and DHAV-C strain is increasingly extensive at China's Epidemic Scope.Disease caused by 3 genotype viruses is comparatively similar in clinical symptom, pathological change, brings very large difficulty, so significant for the Prevention and controls quick and precisely detected for duck viral hepatitis of DHAV-3 to discriminating.
At present about the detection method of DHAV, what domestic and international report was more comprises serum neutralization test, agar diffusion test and ELISA etc., these traditional detection methods exist that length consuming time, susceptibility are lower, the not easily shortcoming such as stdn, there is certain limitation in actual applications, clinically usually because can not correct diagnosis and effective anti-system be made in time to it and cause huge financial loss.And the advantage such as real-time fluorescence quantitative RT-PCR relies on that it is simple to operate, visual result, susceptibility are high, high specificity, reproducible and cause of disease are quantitative, in animal epidemic detects, be able to widespread use in recent years.Therefore set up and optimize fluorescent quantitative RT-PCR method, not being only and determining that duck viral hepatitis paathogenic factor provides a kind of discriminating detection method, simultaneously also for the epidemiology survey of DHAV-3 provides a kind of important technical.
Summary of the invention
In view of this, the object of the present invention is to provide that a kind of susceptibility is good, specificity is high, accurately and reliably, the single stage method fluorescence quantitative RT-PCR kit of fast and convenient detection Serotype-3 DHAV and method, described method utilizes TaqMan probe technology, can carry out accurate quantitative analysis detection to the nucleic acid of DHAV-3.
The technical scheme that the present invention takes is as follows:
1,3 type duck hepatitis A virus (HAV) TaqMan fluorescence quantitative RT-RCRs detect primer pair, probe, described primer pair is made up of the upstream primer of nucleotide sequence as shown in SEQIDNO.3 and the downstream primer of nucleotide sequence as shown in SEQIDNO.4, and the nucleotide sequence of described probe is as shown in SEQIDNO.5.
2, the test kit of primer pair, probe is detected containing described 3 type duck hepatitis A virus (HAV) TaqMan fluorescence quantitative RT-RCRs.
Preferably, described test kit reaction solution is by cumulative volume 20 μ L, composed of the following components: 2 × onestepRT-PCRBuffer10 μ L, the upstream primer 0.4 μ L of RTEnzymeMix0.4 μ L, E × TaqHSDNA polysaccharase 0.4 μ L, 20pmol/ μ L, the downstream primer 0.4 μ L of 20pmol/ μ L, the probe 0.8 μ L of 10pmol/ μ L, RNA template 2 μ L, finally uses RNaseFreedH
2o mends to 20 μ L.
Preferably, described test kit also comprises negative control and positive control, and described positive control is the RNA of DHAV-3, and described negative control is the RNA of healthy duck liver, healthy duck embryo allantoic liquid and idiosome.
3, utilize described test kit to detect the method for 3 type duck hepatitis A virus (HAV), the reaction conditions of fluorescence quantitative RT-RCR is 42 DEG C of 5min, 95 DEG C of 10s (circulation); 95 DEG C of 5s, 54 DEG C of 15s, 45 circulations are carried out in reaction, carry out fluorescence signal acquisition in 54 DEG C; Carry software automatic analysis with FQ-PCR instrument and read FQ-PCR detected result, to obtain amplification curve, if there is Ct<35 and the specific amplification curve increased in logarithm, then contain 3 type duck hepatitis A virus (HAV) in interpret sample; If there is not Ct<35 and the specific amplification curve increased in logarithm, then do not contain 3 type DHAVs in interpret sample.
Beneficial effect of the present invention is: adopt test kit of the present invention to can be used for extensively detecting 3 type duck hepatitis A virus (HAV), detection time is short, and testing cost is low, reproducible, highly sensitive; The target spot that the present invention detects has single specificity, and the primer pair provided and probe, to the sample not containing goal gene, all without amplified signal, have good specificity; In addition, primer pair and probe in detecting RNA sensitivity can reach 48copies/ μ L respectively, have good sensitivity.
Accompanying drawing explanation
In order to make object of the present invention, technical scheme and beneficial effect clearly, the invention provides following accompanying drawing:
The quantitation curves that Fig. 1 DHAV-3 detects, Y=-3.373X+44.261, R
2=0.996, amplification efficiency is 97.9%, shows that error is little, with a high credibility.
Fig. 2 detection method specific test result, curve 1 and 2 is DHAV-3 specific probe amplification curve, and other curve is the amplification curve of the pathogen nucleic acid such as duck plague virus (DPV), avian influenza virus (AIV) and negative control (healthy duck liver rna, healthy duck embryo allantoic liquid and idiosome RNA) and blank in table 2.
Fig. 3 is that the present invention detects repeated the result to DHAV-3 standard substance, and the template amount of curve 1 ~ 7 correspondence is respectively 4.72 × 10
9~ 4.72 × 10
3copies/ μ L, it is reproducible that result shows this detection method.
Fig. 4 detection method sensitivity test result, curve 1 ~ 4 is template amount is respectively 4.72 × 10
4~ 4.72 × 10
1according to amplification curve, the standard substance amplification curve of copies/ μ L, can find out that the sensitivity of the method lowest detection is 48copies/ μ L.
Embodiment
Below the preferred embodiments of the present invention are described in detail.The experimental technique of unreceipted actual conditions in embodiment, the usually conveniently conditioned disjunction condition of advising according to manufacturer.
Embodiment 1 detects the fluorescence quantitative RT-PCR detecting kit of 3 type DHAVs
According to DHAV-3 nucleotide sequence (accession number EU877916.1); sequence alignment is carried out by NCBI and MEGA5.2; utilize Primer5.0 to carry out primer and probe design, primer and probe synthesize by Jikang Biotechnology Co Ltd, Shanghai, primer and probe sequence as follows:
Standard substance primer:
Upstream primer F1 (DHAV-3-F1): 5 '-gatgtagttatggtgcttagacgc-3 ' (SEQIDNO.1),
Downstream primer R1 (DHAV-3-R1): 5 '-acgaaccagccattgacg-3 ' (SEQIDNO.2),
Quantitative probe and supporting primer:
Upstream primer (DHAV-3-F3): 5 '-gtgcttagacgctggcagatt-3 ' (SEQIDNO.3),
Downstream primer (DHAV-3-F4): 5 '-ttcgattgaaaactatctgaaaccta-3 ' (SEQIDNO.4),
Specific probe sequence (DHAV-3-FAM): 5 '-FAM-tcagtgggctaacacagtgacccctg-BHQ-3 ' (SEQIDNO.5).
The fluorescence quantitative RT-PCR detecting kit of described 3 type DHAVs comprises:
(1) Auele Specific Primer DHAV-3-F3 and DHAV-3-F4;
(2) specific probe DHAV-3-FAM;
(3) fluorescence quantitative RT-RCR reaction solution component (cumulative volume is 20 μ L): 2 × onestepRT-PCRBuffer III 10 μ L, RTEnzymeMix II 0.4 μ L, E × TaqHSDNA polysaccharase 0.4 μ L, the upstream primer 0.4 μ L of 20pmol/ μ L, the downstream primer 0.4 μ L of 20pmol/ μ L, the probe 0.8 μ L of 10pmol/ μ L, RNA template 2 μ L, finally uses RNaseFreedH
2o mends to 20 μ L;
(4) positive control: connect poison (DHAV-3) duck embryo, treats that the death of 24 ~ 48h duck embryo is collected allantoic fluid and extracted DHAV-3RNA;
(5) negative control: the RNA that healthy duck liver, healthy duck embryo allantoic liquid and idiosome extract.
The foundation of embodiment 23 type duck hepatitis A virus (HAV) fluorescence quantitative RT-PCR detecting method
1. material OneStepPrimerScript
tMrT-PCRKit (PerfectRealTime), RNAisoPlus are purchased from precious biological (Dalian company limited).
2. method and result
The preparation of 2.1RNA standard substance template
(1) transcription of viral RNA is cDNA according to the biological Reverse Transcription box of treasured by the clone of VP3: use precious biological RNAisoPlus to extract the RNA of 3 type duck hepatitis A virus (HAV) by test kit specification sheets.Application standard product primers F 1 and R1, carry out regular-PCR amplification with in-vitro transcription cDNA for template, obtains VP3 object fragment.Object fragment is connected construction recombination plasmid pGEM-T-VP3 with pGEM-T carrier.
(2) prepare RNA standard substance template: by the linearizing of recombinant plasmid pGEM-T-VP3 single endonuclease digestion, carry out in-vitro transcription according to the TranscriptAidT7HighYieldTranscriptionKit specification sheets step of ThermoScientific company and generate standard substance template DHAV-3-VP3-cRNA and extraction purification is carried out to it.Meanwhile, according to treasured biological DNA/RNA extraction agent box description of step, nucleic acid extraction is carried out to pathogenic agent such as duck plague viruses.
The establishment of 2.2 fluorescence quantitative RT-RCRs (rRT-PCR) optimum reaction condition
For determining optimum reaction condition, carried out corresponding optimization to temperature of reaction, primer and concentration and probe concentration, reaction conditions is: 42 DEG C of 5min, 95 DEG C of 10s (circulation); 95 DEG C of 5s, 54 DEG C of 15s, 45 circulations are carried out in reaction, carry out fluorescence signal acquisition in 54 DEG C.20 μ L reaction systems are as table 1:
Table 1 optimal reaction system
The foundation of 2.3 typical curves
Carrying out 10 times of concentration gradient dilutions to concentration known template is: 4.72 × 10
10~ 4.72 × 10
1copies/ μ L, in Application Example 2,2.2 joint optimum experimental conditions react.By fluorescent quantitation instrument BIO-RADCFXConnect
tMreal-TimeSystem draws corresponding amplification curve, typical curve and formula thereof: Y=-3.373X+44.261.See Fig. 1.
2.4 specific test
Step one, nucleic acid-templated preparation
According to the present embodiment 2.1 amplifying nucleic acid extracting method, extract 3 type duck hepatitis A virus (HAV) CH strains respectively, duck plague virus, riemerella anatipestifer, duck source pathogenic colon bacillus, escherichia coli DH5a, duck source Salmonella enteritidis, duck source Salmonella typhimurium, duck source pasteurella multocida, DHAV-1H strain, Avian pneumo-encephalitis virus, Muscovy duck parvovirus, duck viral swell head haemorrhagic virus, avian influenza virus, bursa of Fabricius virus, Gosling new type viral enteritis virus, healthy duck embryo allantoic liquid, healthy duck embryo idiosome, healthy duck liver homogenate nucleic acid as template.
Step 2, rRT-PCR detection method Evaluation on specificity
RRT-PCR reaction system is saved according to the present embodiment 2.2, getting in step one the DNA/RNA template 2 μ L preparing each strain and bacterial strain adds in rRT-PCR reaction system as rRT-PCR reaction template respectively, negative control makes template with healthy duck liver, healthy duck embryo allantoic liquid and healthy duck embryo idiosome RNA, blank then with 1 ‰ of 2 μ L the water of DEPC process for template, then carry out amplified reaction according to 2.2 joint rRT-PCR loop parameters in embodiment 2.
Step 3, result is differentiated and is judged
Carry software automatic analysis with FQ-PCR instrument and read FQ-PCR detected result, to obtain amplification curve, if there is Ct<35 and the specific amplification curve increased in logarithm, then contain 3 type duck hepatitis A virus (HAV) in interpret sample; If there is not Ct<35 and the specific amplification curve increased in logarithm, then do not contain 3 type duck hepatitis A virus (HAV) in interpret sample.
Figure 2 shows that the detected result that the method Evaluation on specificity is tested, strain and Strain type refer to table 2 and illustrate.
The detected result of table 2 Evaluation on specificity test
In table 2: "+" FQ-PCR result is negative; "-" FQ-PCR result is positive.
Can find out from Fig. 2 and table 2 with 3 type duck hepatitis virus RNA for template, Ct<35 can be obtained and the specific amplification curve increased in logarithm, and duck plague virus of increasing, riemerella anatipestifer, duck source pathogenic colon bacillus, escherichia coli DH5a, duck source Salmonella enteritidis, duck source Salmonella typhimurium, pasteurellosis bacillus, Avian pneumo-encephalitis virus, DHAV-1H strain, Muscovy duck parvovirus, duck viral swells head haemorrhagic virus, avian influenza virus, bursa of Fabricius virus, Gosling new type viral enteritis virus, healthy duck embryo allantoic liquid, healthy duck embryo idiosome, the DNA/RNA of healthy duck liver homogenate and blank all do not obtain Ct<35 and the specific amplification curve increased in logarithm.These results suggest that the specificity of primer pair and probe is good, the rRT-PCR method of foundation specificly can detect 3 type duck hepatitis virus.
2.5 replica test
4.72 × 10 are carried out to standard substance
9~ 4.72 × 10
3copies/ μ L ten times of concentration gradient dilutions, every gradient carries out repeating fluorescence quantitative RT-RCR reaction for 3 times, result shows each concentration gradient example reaction Ct value and is respectively: 10.16/9.99/10.14,13.72/14.06/13.90,17.89/17.65/17.63,21.43/21.41/21.22,25.08/25.00/24.96,28.54/28.30/28.29 and 31.63/31.96/31.96, between parallel sample, Ct value difference is different little, shows each concentration gradient sample detection result reproducible (see Fig. 3).
2.6 susceptibility tests
Standard substance are done 4.72 × 10
4copies/ μ L, 4.72 × 10
3copies/ μ L, 4.72 × 10
2copies/ μ L, 4.72 × 10
1copies/ μ L concentration dilution, corresponding Ct value difference: 28.16,31.47,34.17,36.41, the results are shown in Figure 4, and the sensitivity of result display lowest detection is 48copies/ μ L.
Embodiment 3 fluorescence quantitative RT-RCR detects the clinical pathological material of disease of DHAV-3
Collect 2005 ~ 2015 years from 20 parts of duck hepatitis pathological material of diseases of domestic various places, doubtful duck hepatitis pathological material of disease and 10 parts of DHAV-3 artificial challenge duck embryo pathological material of diseases, extract its RNA, fluorescence quantitative RT-RCR reaction is carried out with test kit of the present invention, set up negative control simultaneously, utilize above-mentioned optimal reaction system and program thereof to react.Result shows 14 parts for positive, and 16 parts of detected results are negative, consistent with the result of Virus Isolation.
What finally illustrate is, above preferred embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although by above preferred embodiment to invention has been detailed description, but those skilled in the art are to be understood that, various change can be made to it in the form and details, and not depart from claims of the present invention limited range.
Claims (5)
1.3 type duck hepatitis A virus (HAV) TaqMan fluorescence quantitative RT-RCRs detect primer pair, probe, it is characterized in that, described primer pair is made up of the upstream primer of nucleotide sequence as shown in SEQIDNO.3 and the downstream primer of nucleotide sequence as shown in SEQIDNO.4, and the nucleotide sequence of described probe is as shown in SEQIDNO.5.
2. detect the test kit of primer pair, probe containing 3 type duck hepatitis A virus (HAV) TaqMan fluorescence quantitative RT-RCRs described in claim 1.
3. test kit according to claim 2, it is characterized in that, described test kit reaction solution is by cumulative volume 20 μ L, composed of the following components: 2 × onestepRT-PCRBuffer10 μ L, RTEnzymeMix0.4 μ L, E × TaqHSDNA polysaccharase 0.4 μ L, the probe 0.8 μ L of the downstream primer 0.4 μ L of the upstream primer 0.4 μ L of 20pmol/ μ L, 20pmol/ μ L, 10pmol/ μ L, RNA template 2 μ L, finally uses RNaseFreedH
2o mends to 20 μ L.
4. test kit according to claim 3, is characterized in that, described test kit also comprises negative control and positive control, and described positive control is the RNA of DHAV-3, and described negative control is the RNA of healthy duck liver, healthy duck embryo allantoic liquid and idiosome.
5. utilize test kit described in any one of claim 2 ~ 4 to detect the method for 3 type duck hepatitis A virus (HAV), it is characterized in that, the reaction conditions of fluorescence quantitative RT-RCR is 42 DEG C of 5min, 95 DEG C of 10s (circulation); 95 DEG C of 5s, 54 DEG C of 15s, 45 circulations are carried out in reaction, carry out fluorescence signal acquisition in 54 DEG C; Carry software automatic analysis with FQ-PCR instrument and read FQ-PCR detected result, to obtain amplification curve, if there is Ct<35 and the specific amplification curve increased in logarithm, then contain 3 type duck hepatitis A virus (HAV) in interpret sample; If there is not Ct<35 and the specific amplification curve increased in logarithm, then do not contain 3 type DHAVs in interpret sample.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510792207.1A CN105256073A (en) | 2015-11-17 | 2015-11-17 | Type-3 duck hepatitis A virus TaqMan fluorescent quantitation RT-PCR detection reagent kit and method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510792207.1A CN105256073A (en) | 2015-11-17 | 2015-11-17 | Type-3 duck hepatitis A virus TaqMan fluorescent quantitation RT-PCR detection reagent kit and method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105256073A true CN105256073A (en) | 2016-01-20 |
Family
ID=55095998
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510792207.1A Pending CN105256073A (en) | 2015-11-17 | 2015-11-17 | Type-3 duck hepatitis A virus TaqMan fluorescent quantitation RT-PCR detection reagent kit and method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105256073A (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109487015A (en) * | 2019-01-12 | 2019-03-19 | 福建省农业科学院畜牧兽医研究所 | The real-time fluorescence quantitative PCR detection primer and its kit of 2 type hepatitis A virus of duck |
| CN109576398A (en) * | 2019-01-12 | 2019-04-05 | 福建省农业科学院畜牧兽医研究所 | A kind of 2 type hepatitis A virus detection kit of duck and its detection method |
| CN109576399A (en) * | 2019-01-12 | 2019-04-05 | 福建省农业科学院畜牧兽医研究所 | 2 type hepatitis A virus real-time fluorescence quantitative PCR detection primer of duck and probe |
| CN109852733A (en) * | 2019-04-02 | 2019-06-07 | 深圳华大智造科技有限公司 | A kind of method detecting duck astrovirus and its kit used |
| CN111690778A (en) * | 2020-07-16 | 2020-09-22 | 四川农业大学 | PCR primer, probe and identification method of APPV virus |
| CN111733289A (en) * | 2020-07-16 | 2020-10-02 | 四川农业大学 | Primer group, probe and method for detecting APPV, CSFV, PCV3 and PTV1 |
| CN114438266A (en) * | 2022-04-11 | 2022-05-06 | 潍坊华卓生物科技有限公司 | Kit and method for detecting multiple common duck-origin viruses |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103060478A (en) * | 2013-01-30 | 2013-04-24 | 山东农业大学 | Dual RT-PCR (reverse transcription-polymerase chain reaction) method for quickly identifying duck hepatitis A virus serotype |
| CN103088165A (en) * | 2013-01-30 | 2013-05-08 | 山东农业大学 | Multi-RT-PCR (Reverse Transcription-Polymerase Chain Reaction) method for rapidly identifying virus serotype of duck viral hepatitis |
| 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 |
| CN104046704A (en) * | 2014-07-04 | 2014-09-17 | 山东农业大学 | Dual fluorescence quantitative method for quickly identifying type 1 and type 3 duck hepatitis A viruses |
| CN104232798A (en) * | 2014-09-28 | 2014-12-24 | 武汉中博生物股份有限公司 | Multi-fluorogenic quantitative PCR (polymerase chain reaction) method for detecting duck hepatitis A virus (DHAV) and identifying DHAV-A and DHAV-C and kit |
| CN104789700A (en) * | 2015-04-03 | 2015-07-22 | 中国农业科学院上海兽医研究所 | DHAV (duck hepatitis A virus) typing detection method based on fluorescent quantitative PCR (polymerase chain reaction) melting curve method |
| CN104862425A (en) * | 2015-06-12 | 2015-08-26 | 广西壮族自治区兽医研究所 | Double two-temperature RT-PCR (reverse transcription-polymerase chain reaction) detection kit for duck hepatitis viruses I and III |
| CN104878124A (en) * | 2015-06-12 | 2015-09-02 | 广西壮族自治区兽医研究所 | Multiple PCR (Polymerase Chain Reaction) detection kit for duck hepatitis A virus 1 and 3 as well as MDPV (Muscovy Duck Parvovirus) |
-
2015
- 2015-11-17 CN CN201510792207.1A patent/CN105256073A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103060478A (en) * | 2013-01-30 | 2013-04-24 | 山东农业大学 | Dual RT-PCR (reverse transcription-polymerase chain reaction) method for quickly identifying duck hepatitis A virus serotype |
| CN103088165A (en) * | 2013-01-30 | 2013-05-08 | 山东农业大学 | Multi-RT-PCR (Reverse Transcription-Polymerase Chain Reaction) method for rapidly identifying virus serotype of duck viral hepatitis |
| 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 |
| CN104046704A (en) * | 2014-07-04 | 2014-09-17 | 山东农业大学 | Dual fluorescence quantitative method for quickly identifying type 1 and type 3 duck hepatitis A viruses |
| CN104232798A (en) * | 2014-09-28 | 2014-12-24 | 武汉中博生物股份有限公司 | Multi-fluorogenic quantitative PCR (polymerase chain reaction) method for detecting duck hepatitis A virus (DHAV) and identifying DHAV-A and DHAV-C and kit |
| CN104789700A (en) * | 2015-04-03 | 2015-07-22 | 中国农业科学院上海兽医研究所 | DHAV (duck hepatitis A virus) typing detection method based on fluorescent quantitative PCR (polymerase chain reaction) melting curve method |
| CN104862425A (en) * | 2015-06-12 | 2015-08-26 | 广西壮族自治区兽医研究所 | Double two-temperature RT-PCR (reverse transcription-polymerase chain reaction) detection kit for duck hepatitis viruses I and III |
| CN104878124A (en) * | 2015-06-12 | 2015-09-02 | 广西壮族自治区兽医研究所 | Multiple PCR (Polymerase Chain Reaction) detection kit for duck hepatitis A virus 1 and 3 as well as MDPV (Muscovy Duck Parvovirus) |
Non-Patent Citations (4)
| Title |
|---|
| LIN-LIN CHEN等: "Improved duplex RT-PCR assay for differential diagnosis of mixed infection of duck hepatitis A virus type 1 and type 3 in ducklings", 《JOURNAL OF VIROLOGICAL METHODS》 * |
| LINLIN CHEN等: "Simultaneous detection of duck hepatitis A virus types 1 and 3,and of duck astrovirus type 1, by multiplex RT-PCR", 《VIROLOGICA SINICA》 * |
| 李井新: "李井新", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
| 谢小冬主编: "《现代生物技术概论》", 31 January 2007, 军事医学科学出版社 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109487015A (en) * | 2019-01-12 | 2019-03-19 | 福建省农业科学院畜牧兽医研究所 | The real-time fluorescence quantitative PCR detection primer and its kit of 2 type hepatitis A virus of duck |
| CN109576398A (en) * | 2019-01-12 | 2019-04-05 | 福建省农业科学院畜牧兽医研究所 | A kind of 2 type hepatitis A virus detection kit of duck and its detection method |
| CN109576399A (en) * | 2019-01-12 | 2019-04-05 | 福建省农业科学院畜牧兽医研究所 | 2 type hepatitis A virus real-time fluorescence quantitative PCR detection primer of duck and probe |
| CN109852733A (en) * | 2019-04-02 | 2019-06-07 | 深圳华大智造科技有限公司 | A kind of method detecting duck astrovirus and its kit used |
| CN111690778A (en) * | 2020-07-16 | 2020-09-22 | 四川农业大学 | PCR primer, probe and identification method of APPV virus |
| CN111733289A (en) * | 2020-07-16 | 2020-10-02 | 四川农业大学 | Primer group, probe and method for detecting APPV, CSFV, PCV3 and PTV1 |
| CN111733289B (en) * | 2020-07-16 | 2022-04-05 | 四川农业大学 | Primer group, probe and method for detecting APPV, CSFV, PCV3 and PTV1 |
| CN111690778B (en) * | 2020-07-16 | 2022-06-03 | 四川农业大学 | PCR primer, probe and identification method of APPV virus |
| CN114438266A (en) * | 2022-04-11 | 2022-05-06 | 潍坊华卓生物科技有限公司 | Kit and method for detecting multiple common duck-origin viruses |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105256073A (en) | Type-3 duck hepatitis A virus TaqMan fluorescent quantitation RT-PCR detection reagent kit and method | |
| CN106947838B (en) | African swine fever virus non-structural gene real-time fluorescence LAMP (loop-mediated isothermal amplification) detection primer group, kit and detection method | |
| Chen et al. | Detection of shrimp hemocyte iridescent virus by recombinase polymerase amplification assay | |
| CN103382507B (en) | 1 type and 3 type duck hepatitis A virus (HAV) single stage method dual RT-PCR detection kit, primer pair and method | |
| CN104232798B (en) | The multiple fluorescence quantitative PCR method of DHAV detection and Gene A type and the discriminating of C type and test kit | |
| CN103757134A (en) | Fluorescent quantitative PCR (Polymerase Chain Reaction) detection reagent, kit and detection method for African swine fever virus (ASFV) | |
| CN104046704A (en) | Dual fluorescence quantitative method for quickly identifying type 1 and type 3 duck hepatitis A viruses | |
| CN105256074A (en) | Kit and method for detecting duck hepatitis A viruses through dual TaqMan fluorescent quantitation RT-PCR | |
| CN105039586A (en) | Primer and kit for detecting duck type-II adenovirus | |
| CN104032034A (en) | Method for simultaneously detecting influenza A virus, influenza B virus and influenza C virus and kit | |
| CN104789700A (en) | DHAV (duck hepatitis A virus) typing detection method based on fluorescent quantitative PCR (polymerase chain reaction) melting curve method | |
| CN105256072A (en) | Type-1 duck hepatitis A virus TaqMan fluorescent quantitation RT-PCR detection reagent kit and method | |
| CN108977582B (en) | A kind of real-time fluorescence quantitative RT-PCR detection method of avian infectious bronchitis virus | |
| CN105886667A (en) | Detection kit for porcine epidemic diarrhea virus and detection method thereof | |
| CN103773896B (en) | The fluorescence quantitative RT-PCR detecting agent of west nile virus, test kit and detection method thereof | |
| CN103993101A (en) | Method and kit for simultaneous detection of human coronavirus 229E, OC43 and NL63 | |
| CN112094944A (en) | Kit for quantitatively detecting copy number of novel coronavirus | |
| CN101962689B (en) | Identification and detection method for hepatitis E virus by utilizing quadruple fluorescence quantitative PCR (Polymerase Chain Reaction) | |
| CN103725793A (en) | Multiple fluorescent quantitative RT-PCR (reverse transcriptase-polymerase chain reaction) method for detecting PRRSV (porcine reproductive and respiratory syndrome virus) and application thereof | |
| CN108251558A (en) | Real-time fluorescence quantitative RT-PCR kit and its application for chicken astrovirus detection | |
| CN105567874A (en) | Porcine delta coronavirus fluorogenic quantitative PCR detection kit and non-diagnostic detection method | |
| CN105525038A (en) | Newcastle disease virus strong/weak virulent one-step real-time fluorescence RT-PCR detection kit | |
| CN103103289B (en) | Fluorescent quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) kit applied to avian pneumovirus C-subgroup specificity detection, and application of fluorescent quantitative RT-PCR kit | |
| CN109468411A (en) | A kind of real-time fluorescence quantitative RT-PCR detection method of H5 subtype avian influenza virus | |
| CN108384894A (en) | Taqman real-time fluorescence quantitative RT-PCRs kit for detecting Sai Nika paddy viruses and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20160120 |
|
| RJ01 | Rejection of invention patent application after publication |