CN105039586A - Primer and kit for detecting duck type-II adenovirus - Google Patents
Primer and kit for detecting duck type-II adenovirus Download PDFInfo
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Abstract
The invention discloses a primer and a kit for detecting duck type-II adenovirus, wherein nucleic acid sequences of the primer pair are represented as the SEQ ID No.1-2. The primer and the kit can quickly identify and diagnose infection of the duck type-II adenovirus, are good in repeatability and are accurate in quantitation. The whole sample detection process, from DNA extraction to result, can be completed just in 2-3 h. The method is simple and can save time and cost, is increased in accuracy and allow high-throughput sample detection to be carried out at the same time.
Description
Technical field
The present invention relates to animal pathogenic technical field of microbial detection, be specifically related to a kind of primer and the test kit that detect duck 2 type adenovirus.
Background technology
Duck 2 type adenovirus (DuckAdenovirus2) finds at France Muscovy duck for 1977 first, mainly cause 15-35 Elderly kind of duck morbidities dead, infected duck only show as One's spirits are drooping, depressed, become thin, weak foot, draw yellow-white mucus loose stool, liver enlargement pale diffusivity yellow-white needle point sample necrosis region, the downright bad point of pancreas white, spleen enlargement is congested, the congested yellowly of kidney enlargement is streak, infect and have slight respiratory symptom in early days, onset peak period causes the mortality ratio of 1%-1.5% every day, and continues about 10 day time total death rate of the onset up to 15-25% not etc.Nineteen eighty-two, J.F.BOUQUET utilizes aquatic bird and duck source cell to be separated first and obtains this virus, to classify this virus and current 12 serotypes having found uncorrelated at that time, and be inferred as new Ana 1 aviadenovirus according to its host range, clinical symptom and serological method.Duck 2 type Adenovirus is in I subgroup adenovirus, and according to restriction enzyme fragment collection of illustrative plates and the temporary species indeterminata of nucleotide sequence equimolecular biologic criteria, viral nucleic acid is distrand DNA.
The adenovirus infection of duck 2 type in recent years in Guangdong, Zhejiang, the ground kind duck such as Fujian, cherry valley duck plant again and again break out and directly cause death rate of the onset up to 10%-30% not etc., support duck industry to China and cause larger financial loss.This investigator obtains duck 2 type adenovirus for domestic separation from the dead kind duck of morbidity first, obtains this viral aliquots genome sequence by molecular biology method simultaneously.Therefore set up a kind of accurate, quick, sensitive detection method to monitor it, ensure that supporting duck industry develops in a healthy way very necessary.In recent years, real-time fluorescence quantitative PCR, can be quantitative with its high specificity, highly sensitive, effectively solves PCR and pollute and level of automation advantages of higher, be widely applied in medical science, microbiology and animals and plants inspection and quarantine.Have no correlative study and the report of duck 2 type adenovirus temporarily.
Summary of the invention
The object of the invention is to for above-mentioned deficiency of the prior art, a kind of primer detecting duck 2 type adenovirus is provided; A kind of test kit detecting duck 2 type adenovirus is provided in addition.
The present invention is achieved through the following technical solutions above-mentioned purpose:
Detect a primer for Ana 1 aviadenovirus 2 type, its nucleotide sequence is as shown in SEQIDNO:1 ~ 2.
Upstream primer F:TGTTCCGGAATGTAACCTGTTTAA(SEQIDNO:1);
Upstream primer R:CATCGCGCTCGTAGTCGT(SEQIDNO:2).
A kind of fluorescent quantitation primer sets detecting Ana 1 aviadenovirus 2 type, the primer of above-mentioned detection Ana 1 aviadenovirus 2 type, and the probe that the nucleotide sequence shown in SEQIDNO:3 is formed, nucleotide sequence 5 ' end shown in SEQIDNO:3 is marked with luminous reporter fluorescence group, and 3 ' end is marked with non-luminous quenching group Eclipse.
Probe P:CTCCGAGAACCGAGGTGCCGC(SEQIDNO:3).
Preferably, in the fluorescent quantitation primer sets of above-mentioned detection Ana 1 aviadenovirus 2 type, described reporter fluorescence group is FAM(hydroxyl fluorescein).
Detect a fluorescence quantitative kit for Ana 1 aviadenovirus 2 type, the fluorescent quantitation primer sets of above-described detection Ana 1 aviadenovirus 2 type.
Preferably, in the fluorescence quantitative kit of above-mentioned detection Ana 1 aviadenovirus 2 type, following component is also comprised: PCR reaction buffer, Mg
2+, aseptic double-distilled water, the mixture of dNTP and Taq enzyme.
Preferably, in the fluorescence quantitative kit of above-mentioned detection Ana 1 aviadenovirus 2 type, also comprise standard positive reference substance, for inserting the pMD18-T plasmid of nucleotide fragments shown in duck 2 type adenovirus 52K genes of SEQ IDNO:4.This pMD18-T plasmid can be bred in E. coli TOP10, and 3.55 × 10
10copy number/μ L ,-20 DEG C of preservations.Nucleotide fragments shown in this duck 2 type adenovirus 52K genes of SEQ IDNO:4 is that this research is obtained by design primer specificity amplification order-checking.
Compared with prior art, the present invention has following beneficial effect:
The present invention is according to the duck 2 type adenovirus (GenBank accession number is KJ469653.1) logged in NCBI and other I subgroup aviadenovirus 52K gene sequence information, through compare of analysis, the primed probe of design specific detection duck 2 type adenovirus, establish fluorescence quantifying PCR method, provide a kind of duck 2 type adenovirus detection kit, rapid differential diagnosis can be carried out to the adenovirus infection of duck 2 type, save time and cost, improve accuracy.
Duck 2 type adenovirus Tagman real-time fluorescence quantitative PCR detection kit of the present invention is reproducible in duck 2 type adenovirus Pathogen test; quantitatively accurately; sample detection only needs to complete for 2-3 hour; use test kit to carry out disease diagnosis and greatly can shorten detection time; be easy to quantitatively, the feature such as false positive is low, be conducive to mass-producing and Aulomatizeted Detect analysis.
Accompanying drawing explanation
Fig. 1: TaqMan quantitative fluorescent PCR curve, wherein a is standard positive template, and b is the dependency that typical curve analyzes plasmid copy number and Ct value, and X-coordinate represents plasmid template copy number (x), and ordinate zou is Ct value;
Fig. 2: the specific detection experimental result of primer of the present invention and probe;
Fig. 3: the sensitivity technique experimental result of primer of the present invention and probe;
Fig. 4: the repeated test experience result of primer of the present invention and probe.
Embodiment
For ease of understanding technical scheme of the present invention, set forth the present invention further below in conjunction with specific embodiment.
Standard positive product are the plasmid pMD18-52K formed containing duck 2 type adenovirus 52K gene 1298 nucleotide fragments, carrier pMD18-T is purchased from TAKARA company, and this carrier can be bred in E. coli TOP10, extraction purification is carried out through plasmid extraction test kit, the optical density(OD) of positive plasmid spectrophotometric determination A260(institute's test sample product under absorbing wavelength is 260nm uviolizing) quantitatively, standard positive plasmid is 3.55 × 10
10copy number/μ L ,-20 DEG C of preservations.
Fluorescent quantitation reaction solution is by forward primer (SEQIDNO:1,10 μMs) and reverse primer (SEQIDNO:2,10 μMs) and fluorescent probe (the nucleotide sequence 5 ' end shown in SEQIDNO:3 is marked with luminous reporter fluorescence group FAM, 3 ' end is marked with non-luminous quenching group Eclipse, 10 μMs) composition.Premix enzyme reaction solution is LifeTechnologies Products Platinum
?quantitative PCR SuperMix-UDG.It is as follows that the PCR system of 20 μ L contains composition: premix enzyme reaction solution 10 μ L, fluorescent quantitation reaction solution 1 μ L (forward primer 0.4 μ L, reverse primer 0.4 μ L, fluorescent probe 0.2 μ L), ROX reference dye 0.04 μ L, aseptic double-distilled water 6.96 μ L, template (genomic dna of 100pg to 1 μ g, 10-10
7the plasmid DNA of copy or the cDNA by the acquisition of 10pg to 1 μ g total serum IgE) 2 μ L.After application of sample, PCR reagent pipe is put into quantitative real time PCR Instrument and is increased, and response procedures is: 50 DEG C of 2min(UDG are hatched), 95 DEG C of 2min; 95 DEG C, 3s, 60 DEG C, 30s, 40 circulations.Quantitative fluorescent PCR instrument gathers fluorescent signal automatically, as calculated machine Software Create negative control and positive control point.Wherein amplification curve CT value is less than 35 and is judged as the positive, and CT value is greater than 35 and is judged to feminine gender.Positive control CT value should be less than 30, otherwise reforms.
Embodiment 1, utilization duck 2 type adenovirus Tagman PCR kit for fluorescence quantitative detect duck 2 type adenovirus
(1) measuring samples DNA extraction: 100 μ L cell culture fluids (infect autonomous strain isolated ZLY, through being accredited as duck 2 type adenovirus, also can use other duck 2 type adenovirus strain isolateds), the clinical pathological material of disease liver organization of 100mg (the unknown whether infected duck 2 type adenovirus), a clinical onset duck brush,throat, cloacal swab and DMEM(cell culture fluid, not containing DADV2, for negative control) as measuring samples, carry out following operation respectively:
Pathological material of disease tissue adds 1mLPBS damping fluid and fully grinds, and after-20 DEG C of multigelations 3 times, the centrifugal 10min of 8000rpm gets supernatant liquor nucleic acid to be extracted.Brush,throat adds 1mLPBS damping fluid and fully shakes mixing, gets supernatant liquor nucleic acid to be extracted.Nucleic acid extraction adopts taco nucleic acid automatic extraction appearance and matched reagent to carry out.Step is as follows:
A. 96 special orifice plates are prepared;
B. at the 1#(7# of 96 orifice plates) in add 200 μ L95% alcohol and 500 μ LLysisBuffer;
C. at 2#, 3#(8#, 9# of 96 orifice plates) in add 750 μ LBufferA;
D. at 4#, 5#(10#, 11# of 96 orifice plates) in add 750 μ LBufferB;
E. at the 6#(12# of 96 orifice plates) in add 100 μ LElutionBuffer;
F. at the 2#(8# of 96 orifice plates) in add 50 μ L magnetic beads;
G. at the 1#(7# of 96 orifice plates) in add 100 μ L measuring samples or yin and yang attribute contrast;
H. above-mentioned 96 orifice plates are put into taco nucleic acid automatic extraction appearance and carry out nucleic acid extraction;
The nucleic acid DNA extracted is in-80 DEG C of preservations.
(2) being diluted by positive criteria moulding plate series is 3.55 × 10
8, 3.55 × 10
7, 3.55 × 10
6, 3.55 × 10
5, 3.55 × 10
4the reference liquid of copy/microlitre.
(3) premix enzyme reaction solution 10 μ L is got respectively, fluorescent quantitation reaction solution 1 μ L, ROX reference dye 0.04 μ L, aseptic double-distilled water 6.96 μ L, the nucleic acid DNA template extracted and each 2 μ L of standard positive template of dilution, and set up negative control, add in different PCR reaction tubess respectively, configure 20 μ L systems, and mix and be placed at the bottom of pipe.Primed probe is synthesized by the precious biotech firm in Dalian, and probe 5 ' is held and 3 ' difference flag F AM and Eclipse.
(4) PCR pipe of step (3) be positioned on quantitative real time PCR Instrument and do Parallel testing, amplification condition is: 50 DEG C of 2min(UDG are hatched); 95 DEG C of 2min; 40 circulations: 95 DEG C, 3s, 60 DEG C, 30s, collects fluorescent signal.Quantitative fluorescent PCR instrument gathers fluorescent signal automatically, and machine Software Create is with starting template number logarithm for X-axis as calculated, and Ct value is the typical curve of Y-axis.
(5) result judges: reporter gene FAM has amplification curve to occur, and amplification curve Ct value is less than 35 is judged as the positive, and Ct value is greater than 35 and is judged to feminine gender.Standard positive control Ct value should be less than 30, otherwise reforms.Concrete outcome is in table 1.
Table 1 measuring samples detected result
5 dilution point of standard positive template shown in Fig. 1 a, all on same straight line, show when gene copy number is 7.1 × 10
8, 7.1 × 10
7, 7.1 × 10
6, 7.1 × 10
5, 7.1 × 10
4during scope, in good linear relationship between detection threshold and copy number.Analyze the dependency of plasmid copy number and Ct value according to Fig. 1 b typical curve, X-coordinate represents plasmid template copy number (x), and ordinate zou is Ct value, and copy number is (x) Ct=-3.335x+42.61, R with the linear relationship of Ct value
2=1, amplification efficiency is 99.46%, and the primed probe amplification efficiency of visible the present invention's design is good.
Embodiment 2, specific assay
Virus liquid is cultivated for positive control with oneself Isolation of duck 2 type adenovirus infected cells, Duck parvovirus, Goose Parvovirus, muscovy duck reovirus, the novel reovirus of kind duck, duck hepatitis virus, kind duck C type Pneumovirinae, a kind duck tembusu virus carry out specific detection, the method of embodiment 1 and primed probe is used to carry out fluorescent quantitative PCR, result except positive control other virus all without amplification curve, the better (see figure 2) of visible the method specificity.
Embodiment 3, sensitivity test
The sterilizing distilled water that standard positive plasmid pMD18-52K containing 52K gene configures is carried out 10 times of gradient dilutions, each extent of dilution respectively gets 2 μ L as template, fluorescent quantitative PCR is carried out by embodiment 1 method, observe amplification curve, to occur that the most high dilution of the template used amount of positive expecting curve calculates its susceptibility.In Fig. 3, amplification curve is from left to right followed successively by 10
-5, 10
-6, 10
-7, 10
-8with 10
-9the standard positive template amplification curve of extension rate, shows that minimum detectable activity is 71 copy (see figure 3)s.
Embodiment 4, Repeatability checking
Use fluorescence quantifying PCR method in embodiment 1,10 times of gradient dilutions are carried out to the sterilizing distilled water that standard positive plasmid pMD18-52K configures, chooses through 10
-3standard positive plasmid replication 3 times in same reaction of dilution, calculates the variation coefficient (CV) of repeat samples.The variation coefficient (CV) of result display repeat samples Ct value is 0.23%, shows PCR kit for fluorescence quantitative of the present invention repeatability good, sees Fig. 4.
Claims (6)
1. detect a primer for Ana 1 aviadenovirus 2 type, it is characterized in that, nucleotide sequence is as shown in SEQIDNO:1 ~ 2.
2. one kind is detected the fluorescent quantitation primer sets of Ana 1 aviadenovirus 2 type, it is characterized in that, comprise the primer of detection Ana 1 aviadenovirus 2 type according to claim 1, and the probe that the nucleotide sequence shown in SEQIDNO:3 is formed, nucleotide sequence 5 ' end shown in SEQIDNO:3 is marked with luminous reporter fluorescence group, and 3 ' end is marked with non-luminous quenching group Eclipse.
3. the fluorescent quantitation primer sets of detection Ana 1 aviadenovirus 2 type according to claim 2, is characterized in that, described reporter fluorescence group is FAM.
4. detect a fluorescence quantitative kit for Ana 1 aviadenovirus 2 type, it is characterized in that, comprise the fluorescent quantitation primer sets of detection Ana 1 aviadenovirus 2 type described in Claims 2 or 3.
5. the fluorescence quantitative kit of detection Ana 1 aviadenovirus 2 type according to claim 4, is characterized in that, also comprise following component: PCR reaction buffer, Mg
2+, aseptic double-distilled water, the mixture of dNTP and Taq enzyme.
6. the fluorescence quantitative kit of detection Ana 1 aviadenovirus 2 type according to claim 5, is characterized in that, also comprise standard positive reference substance, for inserting the pMD18-T plasmid of nucleotide fragments shown in duck 2 type adenovirus 52K genes of SEQ IDNO:4.
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106868226A (en) * | 2017-04-28 | 2017-06-20 | 福建省农业科学院畜牧兽医研究所 | A kind of primer and probe of the detection of duck New-type adenovirus real-time fluorescence quantitative PCR |
| CN106978510A (en) * | 2017-04-28 | 2017-07-25 | 福建省农业科学院畜牧兽医研究所 | A kind of primer of duck New-type adenovirus Eva Green real-time fluorescence quantitative PCRs detection |
| CN107012261A (en) * | 2017-06-13 | 2017-08-04 | 福建省农业科学院畜牧兽医研究所 | Ana 1 aviadenovirus A types and the dual EvaGreen real-time fluorescence quantitative PCRs detection primer of 2 types |
| CN107043831A (en) * | 2017-06-13 | 2017-08-15 | 福建省农业科学院畜牧兽医研究所 | Ana 1 aviadenovirus A types and 2 type Real time PCR detection primers, probe and kit |
| CN107142248A (en) * | 2016-08-20 | 2017-09-08 | 山东信得科技股份有限公司 | A kind of type adenopathy strain of duck 2 |
| CN107137704A (en) * | 2016-08-20 | 2017-09-08 | 山东信得科技股份有限公司 | A kind of type adenovirus inactivated vaccine of duck 2 |
| CN107400736A (en) * | 2017-09-26 | 2017-11-28 | 福建省农业科学院畜牧兽医研究所 | The type adenovirus ring mediated isothermal amplification detection primer group of duck 2 and kit |
| CN108842004A (en) * | 2018-07-28 | 2018-11-20 | 福建省农业科学院畜牧兽医研究所 | Distinguish real-time fluorescence quantitative PCR primer and its differentiating method and the application of Ana 1 aviadenovirus c-type and Ana 1 aviadenovirus Type B |
| CN108842005A (en) * | 2018-07-28 | 2018-11-20 | 福建省农业科学院畜牧兽医研究所 | The MGB probe and its mating primer and method of Ana 1 aviadenovirus c-type and Ana 1 aviadenovirus Type B are detected simultaneously |
| CN108950070A (en) * | 2018-07-28 | 2018-12-07 | 福建省农业科学院畜牧兽医研究所 | Identify the PCR primer and its discrimination method and application of Ana 1 aviadenovirus c-type and Ana 1 aviadenovirus Type B |
| CN112575122A (en) * | 2020-12-29 | 2021-03-30 | 商丘美兰生物工程有限公司 | Dual PCR primer group for rapidly detecting duck type 2 adenovirus and duck circovirus as well as detection method and application thereof |
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Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107137704B (en) * | 2016-08-20 | 2020-02-21 | 山东信得科技股份有限公司 | Duck type 2 adenovirus inactivated vaccine |
| CN107142248A (en) * | 2016-08-20 | 2017-09-08 | 山东信得科技股份有限公司 | A kind of type adenopathy strain of duck 2 |
| CN107137704A (en) * | 2016-08-20 | 2017-09-08 | 山东信得科技股份有限公司 | A kind of type adenovirus inactivated vaccine of duck 2 |
| CN107142248B (en) * | 2016-08-20 | 2020-08-14 | 山东信得科技股份有限公司 | Duck type 2 adenovirus strain |
| CN106978510A (en) * | 2017-04-28 | 2017-07-25 | 福建省农业科学院畜牧兽医研究所 | A kind of primer of duck New-type adenovirus Eva Green real-time fluorescence quantitative PCRs detection |
| CN106868226A (en) * | 2017-04-28 | 2017-06-20 | 福建省农业科学院畜牧兽医研究所 | A kind of primer and probe of the detection of duck New-type adenovirus real-time fluorescence quantitative PCR |
| CN107012261A (en) * | 2017-06-13 | 2017-08-04 | 福建省农业科学院畜牧兽医研究所 | Ana 1 aviadenovirus A types and the dual EvaGreen real-time fluorescence quantitative PCRs detection primer of 2 types |
| CN107043831A (en) * | 2017-06-13 | 2017-08-15 | 福建省农业科学院畜牧兽医研究所 | Ana 1 aviadenovirus A types and 2 type Real time PCR detection primers, probe and kit |
| CN107012261B (en) * | 2017-06-13 | 2020-07-24 | 福建省农业科学院畜牧兽医研究所 | Duck adenovirus type A and type 2 dual EvaGreen real-time fluorescent quantitative PCR detection primers |
| CN107400736A (en) * | 2017-09-26 | 2017-11-28 | 福建省农业科学院畜牧兽医研究所 | The type adenovirus ring mediated isothermal amplification detection primer group of duck 2 and kit |
| CN108950070A (en) * | 2018-07-28 | 2018-12-07 | 福建省农业科学院畜牧兽医研究所 | Identify the PCR primer and its discrimination method and application of Ana 1 aviadenovirus c-type and Ana 1 aviadenovirus Type B |
| CN108842005A (en) * | 2018-07-28 | 2018-11-20 | 福建省农业科学院畜牧兽医研究所 | The MGB probe and its mating primer and method of Ana 1 aviadenovirus c-type and Ana 1 aviadenovirus Type B are detected simultaneously |
| CN108842004A (en) * | 2018-07-28 | 2018-11-20 | 福建省农业科学院畜牧兽医研究所 | Distinguish real-time fluorescence quantitative PCR primer and its differentiating method and the application of Ana 1 aviadenovirus c-type and Ana 1 aviadenovirus Type B |
| CN112575122A (en) * | 2020-12-29 | 2021-03-30 | 商丘美兰生物工程有限公司 | Dual PCR primer group for rapidly detecting duck type 2 adenovirus and duck circovirus as well as detection method and application thereof |
| CN112575122B (en) * | 2020-12-29 | 2024-04-09 | 商丘美兰生物工程有限公司 | Dual PCR primer set for rapidly detecting duck type 2 adenovirus and duck circovirus, and detection method and application thereof |
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