CN102634605A - 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
- Publication number
- CN102634605A CN102634605A CN2012100868231A CN201210086823A CN102634605A CN 102634605 A CN102634605 A CN 102634605A CN 2012100868231 A CN2012100868231 A CN 2012100868231A CN 201210086823 A CN201210086823 A CN 201210086823A CN 102634605 A CN102634605 A CN 102634605A
- Authority
- CN
- China
- Prior art keywords
- egg drop
- drop syndrome
- primer
- seq
- pcr
- 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.)
- Granted
Links
Images
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 the target sequence, fluorescence quantification PCR primer and the probe that are used to detect chicken egg drop syndrome virus, the invention still further relates to and utilize this target sequence to carry out method and test kit that chicken egg drop syndrome virus detects.
Background technology
The chicken egg drop syndrome (Egg drop syndrome, EDS) be called again chicken egg drop syndrome-76 or egg drop syndrome-76 (Egg drop syndrome-76, EDS-76).Chicken egg drop syndrome virus (EDSV) belongs to Adenoviridae Aviadenovirus III crowd (Avian adenovirus Group III).This sick characteristic is that chicken is inapparent infection before sexual maturity, and chicken opens and just shows clinical symptom postpartum, causes that mainly egg drop reduction and chorion form not congruent clinical symptom, cause the massive losses of egg productivity.The chicken egg drop syndrome is confined to Europe at first, and national generations such as Australia, Belgium, France, Britain should disease subsequently.Li Gang equals to be separated to EDSV in 1991, thereby has confirmed the existence of EDS in China.
The method of the EDS of laboratory detection at present is mainly etiology method, serological method, viral nucleic acid detection method; Method wherein commonly used is hemagglutination test, and hemagglutination test object commonly used often is the duck embryo allantoic liquid, and serology detects has high specificity; Characteristics such as sensitivity height; But preliminary preparation is complicated, and length consuming time, process of the test are loaded down with trivial details, and needs 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 higher than conventional PCR far away.Raue etc. are that the PCR on basis combines restricted enzyme cutting analysis in order to six adjacent bodies, successfully detect and differential diagnosis EDSV and 12 kinds of aviadenovirus.Wang Aihua etc. use colloid gold particle to be affinity tag, prepared the EDSV colloidal gold strip and this virus is detected.Utilization such as Dong Chunna isothermal ring mediation amplification technique detects EDSV, and its sensitivity reaches 60 copies, can accurately carry out the detection of chicken egg drop syndrome virus.But traditional P CR technology also exists deficiency in application, the one, can not be accurately quantitative, and the 2nd, easy crossed contamination produces false positive.
Real-time fluorescence quantitative PCR (real-time PCR) then can combine the advantage of conventional P CR and overcome deficiency, realizes the qualitative and quantitative relationship between detected result and the test sample, thereby can carry out accurately quantitatively sample as required.In addition; This technology not only realized to dna profiling quantitatively; And have highly sensitive, specificity and safety is stronger, can realize characteristics such as multiple reaction, level of automation height, nonstaining property, tool real-time and accuracy, be widely used in fields such as molecular biology research and medical research at present.The TaqMan fluorescence quantifying PCR method is expected in the diagnosis of chicken egg drop syndrome and prevention a kind of new method is provided, and also in the intravital Study on Pathogenicity of natural reservoir (of bird flu viruses) a kind of new technique means is provided for this virus simultaneously.
Summary of the invention
The object of the present invention is to provide the fluorescence quantifying PCR method that is used to detect chicken egg drop syndrome virus (EDSV), to improve detectivity to chicken egg drop syndrome virus.
The present invention also aims to provide a kind of test kit that detects chicken egg drop syndrome virus.
The invention provides the genetic marker that is used to detect chicken egg drop syndrome virus, its nucleotide sequence is 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 chicken egg drop syndrome virus.
Further, the present invention also is provided for specificity and expands the primer levy above-mentioned target sequence, and the fluorescent probe that is used with said primer.Can use such as Primer Express Version 3 softwares such as grade and come designing probe and primer.Usually primer length is a 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; Usually the Taqman probe length is a 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 applicable to that not only the external EDSV strain amplification of having reported also is suitable for the detection of domestic EDSV strain.
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 real time fluorescence quantifying PCR method detects chicken egg drop syndrome virus that is used for, it contains the EDSV specific fragment, and its nucleotide sequence is shown in SEQ ID NO.5, and the Auele Specific Primer of this sequence that is used to increase 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 zone of high conservative on the EDSV hexon gene order.After the specific fragment that amplifies was connected to carrier, sequencing result fitted like a glove with the expection fragment.Said carrier is preferably the pEASY-T1 carrier.
The invention provides a kind of fluorescence quantifying PCR method that detects chicken egg drop syndrome virus; Be to be standard form with above-mentioned standard plasmid; With the sample total DNA is template; (SEQ ID NO.1) is target sequence with above-mentioned genetic marker, utilizes primer and probe to carry out real-time fluorescence quantitative PCR, according to the amplification curve result of determination; Said 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 chicken 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 probes, 0.8 μ L, ROX dyestuff 0.4 μ L; Template (DNA) 2 μ L mend ddH
2O to 20 μ L; The response procedures of said real-time fluorescence quantitative PCR is: 95 ℃ of preparatory sex change 30s, 1 circulation; 95 ℃ of sex change 3s～5s, 58 ℃～60 ℃ annealing 30s～35s, 40 circulations.
The preferred reaction program of said real-time fluorescence quantitative PCR is: 95 ℃ of preparatory sex change 30s, 1 circulation; 95 ℃ of sex change 5s, 60 ℃ of annealing 30s, 40 circulations.
Must set up NTC contrast (no template contrast) and POS contrast (positive control) when the present invention detects sample at every turn, two kinds of contrasts play a decisive role for interpretation as a result:
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.
Have only the sample detection result under the effective amplification situation of contrast just credible, person's test does not need repetition.
When two kinds of contrasts were effectively amplification in detection, the sample results judgement criteria was following:
The Ct value is smaller or equal to the positive result of 38 sample;
The Ct value is greater than the negative result of 40 sample;
The sample of Ct value between 38-40 needs repetition, and revision test such as Ct value still are lower than 40 and are judged to be positive amplification, surpass 40 and are judged to be negative amplification.
The invention provides a kind of test kit that chicken egg drop syndrome virus detects that is used for, it comprises above-mentioned Taq Auele Specific Primer and the Taqman probe that cooperates 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 the fluorescent quantitation reaction solution, negative template and positive template, and said negative template is the stoning sour water, the total DNA of said positive template EDSV, positive template is preferably the standard plasmid that makes for the present invention.
The Taqman probe that the present invention also provides above-mentioned Taq Auele Specific Primer and cooperated primer to use detects the application in chicken egg drop syndrome virus test kit or the detection reagent in preparation.
Employed probe of quantitative fluorescent PCR of the present invention and supporting primer specificity are strong and highly stable, can collect good signal in the reaction process, and do not have the situation of cross reaction with other viruses, have brought into play well " double insurance " effect.In the replica test, display density is 1 * 10 as a result in group and between group
8Copies/ μ L and 1 * 10
1The template CV value of copies/ μ L is higher relatively, but still is lower than 2.5%, and the CV value of other concentration template all reaches 10 copy/μ L less than 1.2% this method minimum detectable activity, 1.0 * 10
2～1.0 * 10
8Good linear relationship is arranged, R between copy/μ L sensing range
2=0.992.The template concentrations of all samples of the inventive method detection all in the dynamics range of establishment method, needn't be done sample is done any dilution and concentrated.The detected result that EDSV artificial infection chicken crowd tests separating sample shows that 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 sample detectivity.The foundation of the inventive method can solve the problem of chicken egg drop syndrome early diagnosis, and can reflect the amplification situation of viral DNA in real time, dynamically reflects the distribution situation of virus in body tissue.
Description of drawings
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 specify content of the present invention, but should not be construed as limitation of the present invention.Under the situation that does not deviate from the present invention's spirit and essence, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.
If do not specialize the conventional means that used technique means is well known to those skilled in the art among the embodiment.
The structure of embodiment 1 EDSV standard plasmid
1 experiment material
1.1 bacterial strain, seed culture of viruses and carrier
Chicken egg drop syndrome virus (EDSV) NE4 strain, CAV (CIAV) CUX-1 strain, duck plague virus (DEV) CV strain, Mareks disease virus (MDV) 814 strains are preserved for this laboratory; DH5 α competent cell is by this prepared in laboratory and preservation; PEASY-T1 clone test kit is available from the Beijing Quanshijin Biotechnology Co., Ltd.
1.2 key instrument and reagent
ABI7900HT type quantitative real time PCR Instrument is available from u.s.a. applied biosystem company (ABI); Quantitative fluorescent PCR 2 * Ex premix Taq test kit, dna fragmentation reclaim test kit and all purchase the precious biotechnology ltd (TaKaRa) in Dalian; The little extraction reagent kit of plasmid is available from TIANGEN Biotech (Beijing) Co., Ltd..
2 methods and result
2.1 primer and probe design
EDSV AV-127 pnca gene (Accession No.Y09598.1) sequence according to the GenBank login; The hexon coding region gene of selecting high conservative through sequence alignment is as amplification region; 1 pair of special primer of Using P rimer 5.0 designs, and give birth to worker company by Shanghai and synthesize EHF:5 '-AAACGGTGTTACCACTGAC-3 '; EHR:5 '-AGCTGCTACCCATATCCA-3 '; Its pcr amplification product is 140bp, and nucleotide sequence is shown in SEQ ID No.5, and above-mentioned primer is used to make up standard plasmid.
2.2 the propagation and the evaluation of virus
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 behind the 120h; With this method, even passed for 3 generations, the fresh allantoic fluid that obtains is used for hemagglutination test; Choose hemagglutinative titer and be stored in-70 ℃ at the allantoic fluid more than 214, subsequent use.
2.3 the extraction of viral DNA
With reference to [Zhou Jinping, Li Gang, Zheng Mingqiu such as Li Gang [16]; Analyze [J] Deng. Ji Yuan and duck source egg drop syndrome viral DNA restriction enzyme mapping. Agricultural University Of Nanjing's journal, 1998,22 (2): 71～75] extract the method for allantoic fluid viral DNA; The virus of purifying is digested with SDS (10%) (final concentration is 1%)-Proteinase K (20mg/mL), saturated phenol/chloroform/primary isoamyl alcohol (25: 24: 1) extracting, deposition is used absolute ethanol washing; And, be stored in-20 ℃ of preservations with TE damping fluid dissolving DNA, subsequent use.
2.4 the foundation of standard form
DNA with EDSV is a template, carries out pcr amplification with Auele Specific Primer EHF and EHR.The 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 respectively are 1 μ L, EasyTaq archaeal dna polymerase (5U/ μ L) 0.25 μ L, dna profiling 2 μ L.PCR reaction conditions: preparatory 94 ℃ of 3min of sex change; 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 30s, totally 30 circulations; 72 ℃ of 7min.After the PCR product is identified with the 1.5.% agarose gel electrophoresis, use primer EHF and EHR to carry out the specific fragment that PCR reaction amplifies and be 140bp, conform to its intended purposes clip size.Reclaiming test kit with glue reclaims and the purified genes fragment.
The gene fragment that purifying is good is connected on the pEASY-T1 carrier; Be converted among the E.coli DH5 α; After PCR identifies; The order-checking of picking positive colony is identified through order-checking, and the target gene sequences of insertion reaches 100% with the consistence of the hexon gene conserved sequence of the AV-127 strain of announcing EDSV.Extract test kit with DNA and extract plasmid, measuring the pEASY-T1-edsv-h plasmid concentration through the uv-spectrophotometric appearance 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 * one 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, doubling dilution to 1.0 * 10～1.0 * 10 then
8Totally 8 extent of dilution are as standard form for copy/μ L, and-20 ℃ of preservations are subsequent use.With these standard substance, this plasmid called after pEASY-T1-edsv-h as the quantitative fluorescent PCR reaction.
The design of embodiment 2EDSV TaqMan fluorescent quantitative PCR detection method the primer and probe is with synthetic
EDSV AV-127 pnca gene (Accession No.Y09598.1) sequence according to the GenBank login; The hexon coding region gene of selecting high conservative through sequence alignment is as amplification region; Confirm to be used to detect the genetic marker of EDSV; Its nucleotide sequence is shown in SEQ ID NO.1, and further through sequence alignment design taqman probe and matching used design of primers thereof, its nucleotide sequence is respectively shown in SEQ ID NO.4, SEQ ID NO.2, SEQ ID NO.3.
Said primer sequence is:
Taq-EHFP：5’-GACCGGTCACAAAAACTTCATCT-3’，
Taq-EHRP：5’-CACAATCCTGTTATCACCCACATTT-3’。
Probe sequence is:
5’FAM-CGCATCGTCCCGATCCAAACAGA-BHQ3’。
Though the complete complementation of primer and probe and target sequence is preferred, one skilled in the art will appreciate that at primer and template to exist under the situation of 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 are all within 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 chicken 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 probes, 0.8 μ L; ROX dyestuff 0.4 μ L, template (DNA) 2 μ L mend ddH
2O to 20 μ L.
The response procedures of real-time fluorescence quantitative PCR is: 95 ℃ of preparatory sex change 30s, 1 circulation; 95 ℃ of sex change 5s, 60 ℃ of annealing 30s, 40 circulations.
2, the making of system typical curve
The standard plasmid template that embodiment 1 makes is carried out quantitative fluorescent PCR according to above-mentioned condition and system; Through optimization to condition; The precise proportioning template concentrations; Select only 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 the copy/μ L good linear relationship is arranged, coefficient R
2=0.992, amplification efficiency is 103.1%.
3, result's judgement
Must set up NTC contrast (no template contrast) and POS contrast (positive control) when the present invention detects sample at every turn, two kinds of contrasts play a decisive role for interpretation as a result:
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.
Have only the sample detection result under the effective amplification situation of contrast just credible, otherwise test need repetition.
When two kinds of contrasts were effectively amplification in detection, the sample results judgement criteria was following:
The Ct value is smaller or equal to the positive result of 38 sample;
The Ct value is greater than the negative result of 40 sample;
The sample of Ct value between 38-40 needs repetition, and revision test such as Ct value still are lower than 40 and are judged to be positive amplification, surpass 40 and are 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); The upstream and downstream primer sequence is respectively 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.
With the standard plasmid of 10 times of dilutions is that template is carried out the quantitative fluorescent PCR test with reference to the method for embodiment 3, draws amplification kinetic curve (Fig. 1), shows that the low energy of method that this experiment sets up detects the DNA that starting point concentration is 1.0 * 10copies/ μ L.Carry out conventional PCR reaction with identical template, show (Fig. 3) with 1.5% agarose gel electrophoresis, can detect starting point concentration 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 the EDSV susceptibility than conventional PCR method.
2, specificity test
Extract the DNA of CAV (CAIV), duck plague virus (DEV), Mareks disease virus (MDV), get the template of equivalent and carry out the quantitative fluorescent PCR test, simultaneously with ddH with the method for embodiment 3 foundation
2O makes negative control, makes positive control with the EDSV standard plasmid that embodiment 1 makes, and 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 carry out the quantitative fluorescent PCR reaction simultaneously.The result sees Fig. 4; The display standard plasmid has good amplification curve and specificity; And CIAV, DEV, three negative controls of MDV and blank only have slight fluorescent signal to ignore; Positive control has good amplification curve, thereby the method that shows the fluorescence quantitative RT-RCR detection EDSV that this experiment is set up has good specificity.
3, repeatability and stability test
Replica test needs under same reaction conditions, to carry out independently fluorescence quantitative PCR detection 3 times with identical sample between group; Each sample is provided with 3 repetitions; Replica test then needs each sample to do 3 repetitions in the group, under same reaction conditions, carries out fluorescence quantitative PCR detection, calculates the variation coefficient of CT value respectively; The variation coefficient is standard deviation/mean number, heavier renaturation and stability.
With l * 10
1～1 * 10
8Copies/ μ L standard plasmid is used in the group and replica test between group; Result's (table 1) shows the replica test variation coefficient (CV) of all samples all less than 2.5%, explains that the fluorescence quantifying PCR method of the detection EDSV that this experiment is set up has good repeatability and stable.
Reach replica test result in the group between table 1 fluorescence quantitative PCR detection sample sets
10 of every laying hen intramuscular injection of experimental group 200 μ L sterilization PBS dilution
6TCID
50The virus allantoic fluid.The normal duck embryo allantoic liquid of every laying hen intramuscular injection of control group 200 μ L sterilization PBS dilution.Open and collect the chicken crowd postpartum and lay eggs and movement, and carry out the detection of quantitative fluorescent PCR as template, set feminine gender and positive control simultaneously with isolating 32 samples.
Infect the detection of EDSV chicken crowd clinical isolates
After EDSV attacks malicious chicken crowd 15 days～12 the week the institute lay eggs and ight soil separated 32 duplicate samples; Carry out the detection of quantitative fluorescent PCR and conventional PCR respectively; Result's (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%, with two kinds of methods negative control group chicken crowd isolate is detected, and does not all detect EDSV; The method that the fluorescence quantitative PCR detection EDSV that this experiment is set up is described has very high susceptibility, far above the regular-PCR method.
Table 2 TaqMan real time fluorescent quantitative and regular-PCR detect the EDSV sample result relatively
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 chicken egg drop syndrome virus that this research is set up has good specificity, susceptibility, repeatability, stability; Process is simple; Reaction fast; For the diagnosis and the prevention of chicken egg drop syndrome provides a kind of new method, also in the intravital Study on Pathogenicity of natural reservoir (of bird flu viruses) a kind of new technique means is provided simultaneously for this virus.
Though, the present invention has been done detailed description in the preceding text with general explanation and specific embodiments, on basis of the present invention, can to some modifications of do or improvement, this will be apparent to those skilled in the art.Therefore, these modifications or the improvement on the basis of not departing from spirit of the present invention, made all belong to the scope that requirement of the present invention is protected.
Claims (10)
1. one kind is used to detect the viral genetic marker of chicken egg drop syndrome, and it has the sequence shown in the SEQ ID No.1 or its specific fragment.
2. the Auele Specific Primer of the said genetic marker of claim 1 is used to increase.
3. primer according to claim 2, its nucleotides sequence is classified as:
Taq-EHFP：5’-GACCGGTCACAAAAACTTCATCT-3’，
Taq-EHRP：5’-CACAATCCTGTTATCACCCACATTT-3’。
4. the fluorescent probe that is used with claim 2 or 3 said primers.
5. fluorescent probe according to claim 4, its nucleotides sequence is classified as:
5’FAM-CGCATCGTCCCGATCCAAACAGA-BHQ3’。
6. one kind is used for the standard plasmid that real time fluorescence quantifying PCR method detects chicken egg drop syndrome virus, and it contains the EDSV specific fragment, and its nucleotide sequence is shown in SEQ ID NO.5, and the Auele Specific Primer of this sequence that is used to increase is:
EHF：5’-AAACGGTGTTACCACTGAC-3’，
EHR：5’-AGCTGCTACCCATATCCA-3’。
7. the detection method of chicken egg drop syndrome virus; It is characterized in that; With the described standard plasmid of claim 6 is standard form, is template with the sample total DNA, is target sequence with the described genetic marker of claim 1; Utilize claims 2 or 3 described primers and claim 4 or 5 said probes to carry out real-time fluorescence quantitative PCR, according to the amplification curve result of determination.
8. the detection method of chicken egg drop syndrome virus as claimed in claim 7; 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 probes, 0.8 μ L; ROX dyestuff 0.4 μ L, template (DNA) 2 μ L mend ddH
2O to 20 μ L; The response procedures of said real-time fluorescence quantitative PCR is: 95 ℃ of preparatory sex change 30s, 1 circulation; 95 ℃ of sex change 5s, 60 ℃ of annealing 30s, 40 circulations.
9. the test kit that contains claim 2 or 3 said primers and/or claim 4 or 5 said probes.
10. claim 2 or 3 said primers and/or claim 4 or 5 said probes detect the application in chicken egg drop syndrome virus test kit or the detection reagent in preparation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210086823.1A CN102634605B (en) | 2012-03-28 | 2012-03-28 | Method for detecting egg drop syndrome viruses and kit for method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210086823.1A CN102634605B (en) | 2012-03-28 | 2012-03-28 | Method for detecting egg drop syndrome viruses and kit for method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102634605A true CN102634605A (en) | 2012-08-15 |
| CN102634605B CN102634605B (en) | 2014-04-23 |
Family
ID=46619179
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210086823.1A Expired - Fee Related CN102634605B (en) | 2012-03-28 | 2012-03-28 | Method for detecting egg drop syndrome viruses and kit for method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102634605B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103320538A (en) * | 2013-07-11 | 2013-09-25 | 广西壮族自治区兽医研究所 | Duplex RT-PCR kit of duck Tembusu virus and egg drop syndrome virus |
| CN103320540A (en) * | 2013-07-11 | 2013-09-25 | 广西壮族自治区兽医研究所 | Triplex RT-PCR kit of duck Tembusu virus, egg drop syndrome virus and newcastle disease virus |
| CN104498631A (en) * | 2014-12-26 | 2015-04-08 | 福建省农业科学院畜牧兽医研究所 | Multiplex PCR detection primer and method for important viruses causing egg-laying abnormality |
| 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 |
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 |
-
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 (5)
| Title |
|---|
| MARTINA REGINA SCHYBLI: "Development of a real-time PCR assay for the detection of egg drop syndrome 1976 virus", 《UNIVERSITY OF ZURICH, VETSUISSE FACULTY》 * |
| 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》 * |
| 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》 * |
| 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》 * |
| 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》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103320538A (en) * | 2013-07-11 | 2013-09-25 | 广西壮族自治区兽医研究所 | Duplex RT-PCR kit of duck Tembusu virus and egg drop syndrome virus |
| CN103320540A (en) * | 2013-07-11 | 2013-09-25 | 广西壮族自治区兽医研究所 | Triplex RT-PCR kit of duck Tembusu virus, egg drop syndrome virus and newcastle disease virus |
| CN103320540B (en) * | 2013-07-11 | 2016-08-10 | 广西壮族自治区兽医研究所 | Duck tembusu virus, egg-decreasing syndrome virus and the Triplex RT-PCR kit of Avian pneumo-encephalitis virus |
| CN103320538B (en) * | 2013-07-11 | 2016-09-07 | 广西壮族自治区兽医研究所 | Duck tembusu virus and the duplex RT-PCR kit of egg-decreasing syndrome virus |
| CN104498631A (en) * | 2014-12-26 | 2015-04-08 | 福建省农业科学院畜牧兽医研究所 | Multiplex PCR detection primer and method for important viruses causing egg-laying abnormality |
| 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 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102634605B (en) | 2014-04-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Herrera et al. | Strain-dependent variation in 18S ribosomal DNA copy numbers in Aspergillus fumigatus | |
| Mahmoudian et al. | Development of a SYBR Green quantitative polymerase chain reaction assay for rapid detection and quantification of infectious laryngotracheitis virus | |
| CN105039586A (en) | Primer and kit for detecting duck type-II adenovirus | |
| CN102634605B (en) | Method for detecting egg drop syndrome viruses and kit for method | |
| CN110872637A (en) | Reagent for identifying African swine fever gene deletion vaccine, detection method and application | |
| Zhang et al. | Development of a directly visualized recombinase polymerase amplification–sybr green i method for the rapid detection of African swine fever virus | |
| CN106350608A (en) | Detection kit and detection method for Cyprinid herpesvirus III | |
| CN103725796A (en) | BVDV (bovine viral diarrhea virus) internal control typing fluorescent PCR (polymerase chain reaction) detection kit and preparation thereof | |
| CN101831507A (en) | Real-time fluorescence quantitative PCR (polymerase chain reaction) detection method of grass carp reovirus SYBR Green I | |
| CN112410472A (en) | Primer probe combination and detection kit for detecting mycoplasma pneumoniae, chlamydia pneumoniae and adenovirus | |
| CN104673934A (en) | Koi herpesvirus RT-LAMP detection primer group, kit and detection method thereof | |
| CN105002298A (en) | Fluorescent quantitative PCR detection method of spring viraemia of carp virus | |
| CN1724686B (en) | Target sequence used for detecting mycoplasma pnoumoniae and reagent box | |
| CN103667271B (en) | The primer of detection by quantitative Candida albicans bacterium and probe and application thereof | |
| CN102154270B (en) | Cronobacter sakazakii O antigen specific nucleotides and use thereof | |
| CN106435007A (en) | Primer, probe, kit and method for detecting bacillus erysipelatos-suis by fluorescent quantitative PCR (Polymerase Chain Reaction) | |
| CN103397108A (en) | HCLV (hog cholera lapinized virus) fluorescent quantitation RT-PCR (reverse transcription-polymerase chain reaction) detection kit | |
| CN102676697A (en) | Primers and probe for detecting peste des petits ruminants virus and kit | |
| CN104745722A (en) | Primers, probe and kit used for detecting varicella zoster viruses (VZVs) | |
| CN102286642A (en) | Giant salamander iridovirus TaqMan real-time fluorescent quantitative PCR (polymerase chain reaction) kit and application thereof | |
| CN104745724A (en) | Primers, probe and kit used for detecting JC viruses (JCVs) | |
| CN103361444A (en) | Kit for testing Marek's disease virus of chicken by using MEQ gene and detection method thereof | |
| CN106399512A (en) | Detecting kit for aeromonas hydrophila and detecting method of detecting kit | |
| RU2639498C1 (en) | Set of oligonucleotide primers and fluorescence marked probes for identifying of blastomycesis dermatitis irritant | |
| Tomasek et al. | Comparison of three template preparation methods for routine detection of beak and feather disease virus and avian polyomavirus with single and nested polymerase chain reaction in clinical specimens |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20140423 |