CN102634605B - Method for detecting egg drop syndrome viruses and kit for method - Google Patents
Method for detecting egg drop syndrome viruses and kit for method Download PDFInfo
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Abstract
The invention provides a method for detecting egg drop syndrome viruses and a kit for the method. By means of sequencing and comparing egg drop syndrome virus genes, a genetic marker for detecting the egg drop syndrome viruses, a real-time fluorescent quantitative PCR (polymerase chain reaction) primer and a probe are provided, and nucleotide sequences are respectively showed as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4. The invention further provides a real-time fluorescent quantitative PCR method for detecting the egg drop syndrome viruses and the detecting kit. The minimum detectable amount of the detection method reaches 10 copy/muL, the method has a good linear relation within a 1.0X102-1.0X108 copy/muL detection range, and R2 equals to 0.992. The method has the advantages of accurate detection, high sensitivity and specificity, simplicity, convenience and speediness, and has good specimen detection ability.
Description
Technical field
The present invention relates to biology field, particularly relate to target sequence, fluorescence quantification PCR primer and probe for detection of egg drop syndrome virus, the invention still further relates to and utilize this target sequence to carry out method and test kit that egg drop syndrome virus detects.
Background technology
Egg drop syndrome (Egg drop syndrome, EDS) is called again egg drop syndrome-76 or egg drop syndrome-76 (Egg drop syndrome-76, EDS-76).Egg drop syndrome virus (EDSV) belongs to Adenoviridae Aviadenovirus III group (Avian adenovirus Group III).This sick feature is that chicken is inapparent infection before sexual maturity, and chicken opens and just shows clinical symptom postpartum, mainly causes that egg drop reduction and chorion form not congruent clinical symptom, cause the massive losses of egg productivity.Egg drop syndrome is confined to Europe at first, subsequently national this disease that occurs such as Australia, Belgium, France, Britain.Li Gang equals to be separated to for 1991 EDSV, thereby confirms the existence of LiaoEDS China.
The method of the EDS of laboratory detection is at present mainly etiology method, serological method, viral nucleic acid detection method, wherein conventional method is hemagglutination test, conventional hemagglutination test object is often duck embryo allantoic liquid, serology detects high specificity, the features such as sensitivity height, but preliminary preparation is complicated, length consuming time, process of the test are loaded down with trivial details, and need to have corresponding antigen or antibody.Along with molecular biological fast development, round pcr is used widely, and Li Wengui etc. have reported that sleeve type PCR detects this virus, and its sensitivity is far away higher than conventional PCR.Raue etc. in order to six adjacent bodies be basic PCR in conjunction with restricted enzyme cutting analysis, successfully detect and differential diagnosis EDSV and 12 kinds of aviadenovirus.The use colloid gold particles such as Wang Aihua are marker, prepared EDSV colloidal gold strip and this virus is detected.Dong Chunna etc. use isothermal ring mediation amplification technique to detect EDSV, and its sensitivity reaches 60 copies, can accurately carry out the detection of egg drop syndrome virus.But traditional round pcr also exists deficiency in application, the one, can not accurate quantitative analysis, the 2nd, easy crossed contamination, produces false positive.
Real-time fluorescence quantitative PCR (real-time PCR) can and overcome deficiency in conjunction with the advantage of normal PCR, realizes detected result and detects the quantitative and qualitative analysis relation between sample, thereby can to sample, carry out accurate quantitative analysis as required.In addition, this technology not only realized to DNA profiling quantitatively, and there is highly sensitive, specificity and the feature such as reliability is stronger, can realize multiple reaction, level of automation is high, nonstaining property, tool real-time and accuracy, be widely used at present the fields such as molecular biology research and medical research.TaqMan fluorescence quantifying PCR method is expected to provide a kind of new method in the diagnosis of egg drop syndrome and prevention, simultaneously also for the Study on Pathogenicity of this virus in natural reservoir (of bird flu viruses) body provides a kind of new technique means.
Summary of the invention
The object of the present invention is to provide the fluorescence quantifying PCR method for detection of egg drop syndrome virus (EDSV), to improve the detectivity to egg drop syndrome virus.
The present invention also aims to provide a kind of test kit that detects egg drop syndrome virus.
The invention provides the genetic marker for detection of egg drop syndrome virus, its nucleotide sequence is as shown in SEQ ID NO.1.In addition, it will be appreciated by those skilled in the art that the specific fragment of this sequence also can be used as the genetic marker of detection egg drop syndrome virus.
Further, the present invention is also provided for specificity and expands the primer levy above-mentioned target sequence, and the fluorescent probe being used in conjunction with described primer.Can use such as Primer Express Version 3 softwares such as grade and come designing probe and primer.Conventionally primer length is 15-30 base, and a primer sequence is identical with genetic marker sequence provided by the invention, and another primer sequence and this genetic marker sequence are complementary; Conventionally Taqman probe length is 20-50 base, and its sequence is identical with above-mentioned genetic marker or complementary, its 5 ' mark fluorescent group FAM, 3 ' mark quenching group BHQ1.Probe of the present invention and primer are not only applicable to the also detection of applicable domestic EDSV strain of external EDSV strain amplification of having reported.
In an embodiment of the invention, preferred primer sequence is:
Taq-EHFP：5’-GACCGGTCACAAAAACTTCATCT-3’(SEQ?ID?NO.2)，
Taq-EHRP：5’-CACAATCCTGTTATCACCCACATTT-3’(SEQ?ID?NO.3)。
Probe sequence is:
5’FAM-CGCATCGTCCCGATCCAAACAGA-BHQ3’(SEQ?ID?NO.4)。
The invention provides a kind of standard plasmid that detects egg drop syndrome virus for real time fluorescence quantifying PCR method, it contains EDSV specific fragment, and its nucleotide sequence, as shown in SEQ ID NO.5, for the Auele Specific Primer of this sequence that increases is:
EHF：5’-AAACGGTGTTACCACTGAC-3’(SEQ?ID?NO.6)，
EHR：5’-AGCTGCTACCCATATCCA-3’(SEQ?ID?NO.7)。
Above-mentioned EDSV specific fragment is selected from the region of high conservative in EDSV hexon gene order.The specific fragment amplifying is connected to after carrier, and sequencing result fits like a glove with expection fragment.Described carrier is preferably pEASY-T1 carrier.
The invention provides a kind of fluorescence quantifying PCR method that detects egg drop syndrome virus, to take above-mentioned standard plasmid as standard form, take sample total DNA as template, the above-mentioned genetic marker (SEQ ID NO.1) of take is target sequence, utilize primer and probe to carry out real-time fluorescence quantitative PCR, according to amplification curve result of determination; Described primer sequence is:
Taq-EHFP：5’-GACCGGTCACAAAAACTTCATCT-3’，
Taq-EHRP：5’-CACAATCCTGTTATCACCCACATTT-3’。
Probe sequence is:
5’FAM-CGCATCGTCCCGATCCAAACAGA-BHQ3’。
The real time fluorescence quantifying PCR method of detection egg drop syndrome virus provided by the invention, 20 μ l reaction systems of its real-time fluorescence quantitative PCR are: Premix Ex Taq2 * buffer 12.5 μ L, each 0.4 μ L of 10 μ mol/L upstream primer Taq-EHFP and 10 μ mol/L downstream primer Taq-EHRP, 10 μ M probe 0.8 μ L, ROX dyestuff 0.4 μ L, template (plasmid DNA) 2 μ L, mend ddH
2o to 20 μ L; The response procedures of described real-time fluorescence quantitative PCR is: 95 ℃ of denaturation 30s, 1 circulation; 95 ℃ of sex change 3s～5s, 58 ℃～60 ℃ annealing 30s～35s, 40 circulations.
The preferred reaction program of described real-time fluorescence quantitative PCR is: 95 ℃ of denaturation 30s, 1 circulation; 95 ℃ of sex change 5s, 60 ℃ of annealing 30s, 40 circulations.
When the present invention detects sample at every turn, must set up NTC contrast (without template contrast) and POS contrast (positive control), for result, interpretation plays a decisive role in two kinds of contrasts:
Effectively increase: NTC (-) AND POS (+)
Invalid amplification: NTC (+) AND POS (+) prompting system is polluted
Invalid amplification: NTC (-) AND POS (-) prompting system mistake or reagent lost efficacy.
Only have the sample detection result in the effective amplification situation of contrast just credible, no person's test needs repetition.
When in detection, two kinds of contrasts are effectively amplification, sample results judging criterion is as follows:
Ct value is less than or equal to 38 the positive result of sample;
The negative result of sample that Ct value is greater than 40;
The sample of Ct value between 38-40 needs repetition, and revision test, as Ct value is still judged to be positive amplification lower than 40, surpasses 40 and is judged to be negative amplification.
The invention provides a kind of test kit detecting for egg drop syndrome virus, it comprises above-mentioned Taq Auele Specific Primer and the Taqman probe that coordinates primer to use.
The primer that test kit of the present invention contains, its sequence is:
Taq-EHFP：5’-GACCGGTCACAAAAACTTCATCT-3’，
Taq-EHRP：5’-CACAATCCTGTTATCACCCACATTT-3’。
The probe that test kit of the present invention contains, its sequence is:
5’FAM-CGCATCGTCCCGATCCAAACAGA-BHQ3’。
Test kit provided by the invention, also comprises fluorescent quantitation reaction solution, negative template and positive template, and described negative template is stoning sour water, the total DNA of described positive template EDSV, positive template is preferably the standard plasmid making for the present invention.
The present invention also provides above-mentioned Taq Auele Specific Primer and has coordinated the Taqman probe that primer is used to detect the application in egg drop syndrome virus test kit or detection reagent in preparation.
The probe that quantitative fluorescent PCR of the present invention is used and supporting primer specificity are strong and highly stable, in reaction process, can collect good signal, and there is no the situation of cross reaction with other viruses, have brought into play well " double insurance " effect.In group and between group, in replica test, result display density is 1 * 10
8copies/ μ L and 1 * 10
1the template CV value of copies/ μ L is relatively higher, but still lower than 2.5%, the CV value of other concentration template is all less than 1.2% the method minimum detectable activity and reaches 10 copies/μ L, 1.0 * 10
2～1.0 * 10
8between copy/μ L sensing range, there is good linear relationship, R
2=0.992.The template concentrations that the inventive method detects all samples all, in the dynamics range of establishment method, needn't be done sample to do any dilution and concentrated.EDSV artificial infection chicken group tests the detected result demonstration of separating sample, and the positive rate of fluorescence quantifying PCR method reaches 90.6%, and conventional PCR recall rate is 56.3%.The inventive method has detection accurately, highly sensitive, high specificity, and simple and rapid advantage, has good Samples detection ability.The foundation of the inventive method can solve the problem of egg drop syndrome early diagnosis, and can reflect in real time the amplification situation of viral DNA, dynamically reflects the distribution situation of virus in body tissue.
Accompanying drawing explanation
Fig. 1 is the amplification kinetic curve of EDSV quantitative fluorescent PCR, wherein N: negative control; 1～8:1.0 * 10
1～1.0 * 10
8the amplification curve of copies/ μ L standard plasmid.
Fig. 2 is the typical curve R of fluorescence quantitative PCR detection EDSV
2=0.992.
Fig. 3 is the sensitivity test result of conventional PCR, 1:DNA molecular mass standard DNA 2000plus Marker; 2～7:1.0 * 10
8～1.0 * 10
3the amplification of copies/ μ L standard form; 8: negative control.
The specificity experimental result of Fig. 4 EDSV quantitative fluorescent PCR, 1～3: the amplification curve of standard form; 4～6:1.0 * 10
2the amplification curve of copies/ μ L standard form; The amplification curve of 7～9:CIAV, DEV, MDV; 10: negative control; 11: positive control.
Embodiment
Following examples further illustrate content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, the modification that the inventive method, step or condition are done or replacement, all belong to scope of the present invention.
If do not specialize, the conventional means that in embodiment, technique means used is well known to those skilled in the art.
The structure of embodiment 1 EDSV standard plasmid
1 experiment material
1.1 bacterial strains, seed culture of viruses and carrier
Egg drop syndrome virus (EDSV) NE4 strain, Chicken Anemia Virus (CAV) (CIAV) CUX-1 strain, duck plague virus (DEV) CV strain, Mareks disease virus (MDV) 814 strains are for preserving in this laboratory; DH5 α competent cell is prepared and is preserved by this laboratory; PEASY-T1 clone test kit is purchased from Beijing Quanshijin Biotechnology Co., Ltd.
1.2 key instruments and reagent
ABI7900HT type quantitative real time PCR Instrument is purchased from Applied biosystems (ABI); Quantitative fluorescent PCR 2 * Ex premix Taq test kit, DNA fragmentation reclaim test kit and are all purchased from the precious biotechnology company limited (TaKaRa) in Dalian; The little extraction reagent kit of plasmid is purchased from TIANGEN Biotech (Beijing) Co., Ltd..
2 methods and result
2.1 primers and probe design
According to EDSV AV-127 pnca gene (Accession No.Y09598.1) sequence of GenBank login, by sequence alignment, select the hexon coding region gene of high conservative as amplification region, 1 pair of special primer of application Primer 5.0 designs, and synthetic by Shanghai Sheng Gong company, EHF:5 '-AAACGGTGTTACCACTGAC-3 ', EHR:5 '-AGCTGCTACCCATATCCA-3 ', its pcr amplification product is 140bp, nucleotide sequence is as shown in SEQ ID No.5, and above-mentioned primer is used for building standard plasmid.
The propagation of 2.2 viruses and evaluation
1: 100 times of dilution of EDSVNE4 strain venom that this experiment is preserved is inoculated in 10 age in days duck embryos, and 0.2mL/ only, collects allantoic fluid after 120h, with this method, connect and passed for 3 generations, the fresh allantoic fluid obtaining is for hemagglutination test, choose hemagglutinative titer and be stored in-70 ℃ at more than 214 allantoic fluids, standby.
The extraction of 2.3 viral DNAs
With reference to [the Zhou Jinping such as Li Gang [16], Li Gang, Zheng Mingqiu, Deng. Ji Yuan and duck source egg drop syndrome viral DNA Cleavage Map [J]. Agricultural University Of Nanjing's journal, 1998,22 (2): 71～75] extract the method for allantoic fluid viral DNA, the SDS for virus (10%) of purifying (final concentration is 1%)-Proteinase K (20mg/mL) is digested, saturated phenol/chloroform/primary isoamyl alcohol (25: 24: 1) extracting, precipitation absolute ethanol washing, and with TE damping fluid dissolving DNA, be stored in-20 ℃ of preservations, standby.
The foundation of 2.4 standard forms
The DNA of EDSV of take is template, with Auele Specific Primer EHF and EHR, carries out pcr amplification.PCR reaction system is 25 μ L, wherein ddH
2o 16.25 μ L, 10 * PCR buffer2.5 μ L, dNTP (2.5mmol/L) 2 μ L, primer EHF and primer EHR (being 10 μ mol/L) they are respectively 1 μ L, EasyTaq archaeal dna polymerase (5U/ μ L) 0.25 μ L, DNA profiling 2 μ L.PCR reaction conditions: 94 ℃ of 3min of denaturation; 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 30s, totally 30 circulations; 72 ℃ of 7min.After PCR product is identified with 1.5.% agarose gel electrophoresis, with primer EHF and EHR, carrying out the specific fragment that PCR reaction amplifies is 140bp, conforms to the object clip size of expection.With glue, reclaiming test kit reclaims and purified genes fragment.
The good gene fragment of purifying is connected on pEASY-T1 carrier, be converted in E.coli DH5 α, after PCR identifies, the order-checking of picking positive colony, through order-checking, identify, the goal gene sequence of insertion reaches 100% with the consistence of hexon gene conserved sequence of having announced the AV-127 strain of EDSV.With extraction of plasmid DNA test kit, extract plasmid, by uv-spectrophotometric instrument, measuring pEASY-T1-edsv-h plasmid concentration is 0.17 μ g/ μ L, A
260/ A
280=1.75, according to formula copy number=plasmid concentration (g/ μ L) * application of sample volume * A Fujiadeluo constant/(molecular-weight average of plasmid total length * mono-base pair), A Fujiadeluo constant (particle number of every mol) is 6.02 * 10
23copy/mol, obtaining plasmid sample copy number is 3.94 * 10
10/ μ L, then doubling dilution to 1.0 * 10～1.0 * 10
8totally 8 extent of dilution are as standard form for copy/μ L, and-20 ℃ save backup.Using these standard substance as quantitative fluorescent PCR reaction, this plasmid called after pEASY-T1-edsv-h.
The design of embodiment 2EDSV TaqMan fluorescent quantitative PCR detection method the primer and probe is with synthetic
According to EDSV AV-127 pnca gene (Accession No.Y09598.1) sequence of GenBank login, by sequence alignment, select the hexon coding region gene of high conservative as amplification region, be identified for detecting the genetic marker of EDSV, its nucleotide sequence is as shown in SEQ ID NO.1, further by sequence alignment, design taqman probe and matching used design of primers thereof, its nucleotide sequence is respectively as shown in SEQ ID NO.4, SEQ ID NO.2, SEQ ID NO.3.
Described primer sequence is:
Taq-EHFP：5’-GACCGGTCACAAAAACTTCATCT-3’，
Taq-EHRP：5’-CACAATCCTGTTATCACCCACATTT-3’。
Probe sequence is:
5’FAM-CGCATCGTCCCGATCCAAACAGA-BHQ3’。
Although primer and probe and target sequence complete complementary are preferred, one skilled in the art will appreciate that in the situation that primer and template exist certain not complementary (especially 5 ' of primer end), also can specificly amplify required fragment.The test kit that contains these primers and the method for using these primers all within the scope of the present invention, as long as the amplified production that this primer amplification goes out contains target sequence of the present invention or its fragment.
The foundation of embodiment 3 egg drop syndrome virus (PRRSV) TaqMan fluorescence quantitative PCR detection methods
1,20 μ l reaction systems of real-time fluorescence quantitative PCR are: Premix Ex Taq 2 * buffer12.5 μ L, each 0.4 μ L of 10 μ mol/L upstream primer Taq-EHFP and 10 μ mol/L downstream primer Taq-EHRP, 10 μ M probe 0.8 μ L, ROX dyestuff 0.4 μ L, template (plasmid DNA) 2 μ L, mend ddH
2o to 20 μ L.
The response procedures of real-time fluorescence quantitative PCR is: 95 ℃ of denaturation 30s, 1 circulation; 95 ℃ of sex change 5s, 60 ℃ of annealing 30s, 40 circulations.
2, the making of system standard curve
The standard plasmid template that embodiment 1 is made is carried out quantitative fluorescent PCR according to above-mentioned condition and system, by the optimization to condition, accurate proportioning template concentrations, select most suitable annealing temperature, shorten the application of sample time, draw fluorescent quantitation amplification kinetic curve (Fig. 1) and the typical curve (Fig. 2) of EDSV.The concentration range of EDSV fluorescent quantitation plasmid control template is 1.0 * 10
2～1.0 * 10
8between copy/μ L, there are good linear relationship, coefficient R
2=0.992, amplification efficiency is 103.1%.
3, the judgement of result
When the present invention detects sample at every turn, must set up NTC contrast (without template contrast) and POS contrast (positive control), for result, interpretation plays a decisive role in two kinds of contrasts:
Effectively increase: NTC (-) AND POS (+)
Invalid amplification: NTC (+) AND POS (+) prompting system is polluted
Invalid amplification: NTC (-) AND POS (-) prompting system mistake or reagent lost efficacy.
Only have the sample detection result in the effective amplification situation of contrast just credible, otherwise test need repetition.
When in detection, two kinds of contrasts are effectively amplification, sample results judging criterion is as follows:
Ct value is less than or equal to 38 the positive result of sample;
The negative result of sample that Ct value is greater than 40;
The sample of Ct value between 38-40 needs repetition, and revision test, as Ct value is still judged to be positive amplification lower than 40, surpasses 40 and is judged to be negative amplification.
Susceptibility, specificity, repeatability and the estimation of stability of embodiment 4 EDSV real-time fluorescence quantitative PCR detection methods
1, sensitivity test
Choose 10 times of doubling dilutions, concentration is respectively 1 * 10
1～1 * 10
8the standard form of copies/ μ L carries out quantitative fluorescent PCR and conventional PCR simultaneously, observes both minimum recall rates, relatively its susceptibility.Conventional PCR reaction system is 20 μ L:10 * PCR buffer2.5 μ L, dNTPs (2.5mmol/L) 2 μ L, each 1 μ L of upstream and downstream primer (10 μ mol/L), upstream and downstream primer sequence is respectively as shown in SEQ ID No.6, SEQ ID No.7, template 2 μ L, EasyTaq enzyme 0.25 μ L, adds ddH
2o supplies 20 μ L.
The standard plasmid of 10 times of dilutions of take carries out quantitative fluorescent PCR test with reference to the method for embodiment 3 as template, draw amplification kinetic curve (Fig. 1), show that the low energy of method that this experiment sets up detects the DNA that starting point concentration is 1.0 * 10copies/ μ L.With identical template, carry out conventional PCR reaction, with 1.5% agarose gel electrophoresis, show (Fig. 3), starting point concentration can be detected is 1.0 * 10
4the DNA of copies/ μ L.It is high 1000 times that the fluorescence quantifying PCR method that presentation of results the present invention sets up detects EDSV susceptibility than conventional PCR method.
2, specific test
Extract the DNA of Chicken Anemia Virus (CAV) (CAIV), duck plague virus (DEV), Mareks disease virus (MDV), get the template of equivalent and carry out quantitative fluorescent PCR test by the method that embodiment 3 sets up, simultaneously with ddH
2o makes negative control, and the EDSV standard plasmid making with embodiment 1 is made positive control, identifies the specificity of the inventive method.
Select 1.0 * 10
6copies/ μ L and 1.0 * 10
2the standard plasmid of copies/ μ L, the DNA of CIAV, DEV, MDV carries out quantitative fluorescent PCR reaction simultaneously.The results are shown in Figure 4, display standard plasmid has good amplification curve and specificity, and CIAV, DEV, tri-negative controls of MDV and blank only have slight fluorescent signal to ignore, positive control has good amplification curve, thereby shows that the method for the fluorescence quantitative RT-RCR detection EDSV that this experiment is set up has good specificity.
3, repeatability and stability test
Between group, replica test need carry out 3 times with identical sample independently fluorescence quantitative PCR detection under same reaction conditions, each sample arranges 3 repetitions, in group, replica test needs each sample to do 3 repetitions, under same reaction conditions, carry out fluorescence quantitative PCR detection, calculate respectively the variation coefficient of CT value, the variation coefficient is standard deviation/mean number, heavier renaturation and stability.
By l * 10
1～1 * 10
8copies/ μ L standard plasmid is used for organizing replica test between interior and group, result (table 1) shows that the replica test variation coefficient (CV) of all samples is all less than 2.5%, illustrates that the fluorescence quantifying PCR method of the detection EDSV that this experiment is set up has good repeatability and stability.
Between table 1 fluorescence quantitative PCR detection sample sets and group in replica test result
10 of every laying hen intramuscular injection of experimental group 200 μ L sterilizing PBS dilutions
6tCID
50virus allantoic fluid.The normal duck embryo allantoic liquid of every laying hen intramuscular injection of control group 200 μ L sterilizing PBS dilutions.Open and collect chicken group postpartum and lay eggs and movement, and as template, carry out the detection of quantitative fluorescent PCR with 32 separated samples, set feminine gender and positive control simultaneously.
Infect the detection of EDSV chicken group clinical isolates
From EDSV attacks malicious chicken group the institute of 15 days～12 weeks lay eggs and ight soil in separated 32 duplicate samples, carry out respectively the detection of quantitative fluorescent PCR and conventional PCR, result (table 2) shows that the positive rate of fluorescence quantifying PCR method reaches 90.6%, and the positive rate of conventional PCR method is 56.3%, by two kinds of methods, negative control group chicken group isolate is detected, EDSV all do not detected, the method that the fluorescence quantitative PCR detection EDSV that this experiment is set up is described has very high susceptibility, far above regular-PCR method.
Table 2 TaqMan real time fluorescent quantitative and regular-PCR detect the comparison of EDSV sample result
The template concentrations of all samples of this experiment detection all, in the dynamics range of establishment method, needn't be done any dilution and concentrated to sample.The method of the TaqMan fluorescence quantitative PCR detection egg drop syndrome virus that this research is set up has good specificity, susceptibility, repeatability, stability, process is simple, reaction fast, for diagnosis and the prevention of egg drop syndrome provides a kind of new method, simultaneously also for the Study on Pathogenicity of this virus in natural reservoir (of bird flu viruses) body provides a kind of new technique means.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements, all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (8)
1. for detection of a genetic marker for egg drop syndrome virus, it has the sequence shown in SEQ ID No.1, and classifies as for the nucleotides sequence of the Auele Specific Primer of this genetic marker that increases:
Taq-EHFP：5’-GACCGGTCACAAAAACTTCATCT-3’，
Taq-EHRP：5’-CACAATCCTGTTATCACCCACATTT-3’；
With the fluorescent probe that this Auele Specific Primer is used in conjunction with, its nucleotides sequence is classified as:
5’FAM-CGCATCGTCCCGATCCAAACAGA-BHQ3’。
2. for the Auele Specific Primer of genetic marker described in the claim 1 that increases, its nucleotides sequence is classified as:
Taq-EHFP：5’-GACCGGTCACAAAAACTTCATCT-3’，
Taq-EHRP：5’-CACAATCCTGTTATCACCCACATTT-3’。
3. with the fluorescent probe that is used in conjunction with of primer described in claim 2, its nucleotides sequence is classified as:
5’FAM-CGCATCGTCCCGATCCAAACAGA-BHQ3’。
4. for real time fluorescence quantifying PCR method, detect a standard plasmid for egg drop syndrome virus, it contains EDSV specific fragment, and its nucleotide sequence, as shown in SEQ ID NO.5, for the Auele Specific Primer of this sequence that increases is:
EHF：5’-AAACGGTGTTACCACTGAC-3’，
EHR：5’-AGCTGCTACCCATATCCA-3’；
When described standard plasmid is viral for detection of egg drop syndrome, take sample total DNA as template, take genetic marker claimed in claim 1 as target sequence, utilize probe described in primer described in claim 2 and claim 3 to carry out real-time fluorescence quantitative PCR, according to amplification curve result of determination.
5. contain described in claim 2 test kit of probe described in primer and claim 3.
6. test kit as claimed in claim 5, it is characterized in that, take standard plasmid claimed in claim 4 as standard form, take sample total DNA as template, take genetic marker claimed in claim 1 as target sequence, utilize probe described in primer described in claim 2 and claim 3 to carry out real-time fluorescence quantitative PCR, according to amplification curve result of determination.
7. test kit as claimed in claim 5, it is characterized in that, 20 μ l reaction systems of real-time fluorescence quantitative PCR are: Premix Ex Taq2 * buffer 12.5 μ L, each 0.4 μ L of 10 μ mol/L upstream primer EHFP and 10 μ mol/L downstream primer EHRP, 10 μ M probe 0.8 μ L, ROX dyestuff 0.4 μ L, sample total DNA 2 μ L, mend ddH
2o to 20 μ L; The response procedures of described real-time fluorescence quantitative PCR is: 95 ℃ of denaturation 30s, 1 circulation; 95 ℃ of sex change 5s, 60 ℃ of annealing 30s, 40 circulations.
8. described in claim 2, described in primer and claim 3, probe detects the application in egg drop syndrome virus test kit or detection reagent in preparation.
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| CN105671203A (en) * | 2016-03-09 | 2016-06-15 | 福建省农业科学院畜牧兽医研究所 | Egg drop important virus loop-mediated multiplex PCR (polymerase chain reaction) quick detection primers |
| CN117144065A (en) * | 2023-10-13 | 2023-12-01 | 河南农业大学 | Primer probe set, kit and detection method for fluorescence RAA detection of avian egg drop syndrome virus |
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| CN102002533A (en) * | 2010-05-25 | 2011-04-06 | 山东省农业科学院畜牧兽医研究所 | Kit and method for simultaneously detecting four common epidemic diseases causing egg drop |
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2012
- 2012-03-28 CN CN201210086823.1A patent/CN102634605B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102002533A (en) * | 2010-05-25 | 2011-04-06 | 山东省农业科学院畜牧兽医研究所 | Kit and method for simultaneously detecting four common epidemic diseases causing egg drop |
Non-Patent Citations (10)
| Title |
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| Development of a real-time PCR assay for the detection of egg drop syndrome 1976 virus;Martina Regina Schybli;《University of Zurich, Vetsuisse Faculty》;20101231;摘要、材料与方法部分和表4引物与探针 * |
| Establishing a TaqMan-Based Real-Time PCR Assay for the rapid detection and quantification of the newly emerged duck tembusu virus;Yan et al;《Virology Journal》;20111031;第8卷;全文 * |
| Hexon based PCRs combined with restriction enzyme analysis for rapid detection and differentiation of fowl adenoviruses and egg drop syndrome virus;Raue and Hess;《Journal of Virological Methods》;19980831;第73卷(第2期);摘要及第217页 * |
| Martina Regina Schybli.Development of a real-time PCR assay for the detection of egg drop syndrome 1976 virus.《University of Zurich, Vetsuisse Faculty》.2010,摘要、材料与方法部分和表4引物与探针. |
| Raue and Hess.Hexon based PCRs combined with restriction enzyme analysis for rapid detection and differentiation of fowl adenoviruses and egg drop syndrome virus.《Journal of Virological Methods》.1998,第73卷(第2期),摘要及第217页. |
| Yan et al.Establishing a TaqMan-Based Real-Time PCR Assay for the rapid detection and quantification of the newly emerged duck tembusu virus.《Virology Journal》.2011,第8卷全文. |
| 周云雷等.鸡毒支原体实时荧光定量PCR 检测方法的建立.《中国农业科学》.2011,第44卷(第11期),全文. |
| 肠炎沙门菌种特异性PCR及对感染鸭快速检测;邓树轩等;《中国兽医学报》;20090227;第29卷(第2期);全文 * |
| 邓树轩等.肠炎沙门菌种特异性PCR及对感染鸭快速检测.《中国兽医学报》.2009,第29卷(第2期),全文. |
| 鸡毒支原体实时荧光定量PCR 检测方法的建立;周云雷等;《中国农业科学》;20111230;第44卷(第11期);全文 * |
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