CN107475455A - NDV real-time fluorescence quantitative PCR detection method and its kit - Google Patents
NDV real-time fluorescence quantitative PCR detection method and its kit Download PDFInfo
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Abstract
The present invention relates to field of gene detection, in particular to NDV real-time fluorescence quantitative PCR detection method and its kit.For detecting the nucleotide sequence of NDV, the nucleotide sequence includes primer pair and probe, the primer pair such as SEQ ID NO：1 and SEQ ID NO：Shown in 2, the nucleotide sequence such as SEQ ID NO of the probe：Shown in 3.Nucleotide sequence provided by the present invention for detecting NDV, high specificity, stability is good, and good basis is provided for quantitative detection NDV.NDV real-time fluorescence quantitative PCR detection method provided by the invention, solve pollution, save manpower, and using the fluorescence quantitative PCR detection for exempting from nucleic acid extraction progress NDV, save testing cost, improve detection efficiency.
Description
Technical field
The present invention relates to field of gene detection, in particular to NDV real-time fluorescence quantitative PCR detection method
And its kit.
Background technology
Ewcastle disease (Newcastle disease) also known as philippine fowl disease, pseudo- checken pest, are by NDV (Newcastle
Disease virus, NDV) caused by birds are acute, hot, septic and highly contagious disease, it is tired with high fever, breathing
Hardly possible, diarrhea, neurological disorders, mucous membrane and serous coat bleeding are characterized.The World Health Organization (OIE), which is classified as, must circulate a notice of disease.
China's revision in 2008《First, two, the three bright record of class animal epidemic disease》It is classified as a kind of animal epidemic.
The technology of detection ewcastle disease has enzyme linked immunological (ELISA) and PCR (PCR), compares main flow at present
It is PCR detection techniques.PCR is divided into two kinds of regular-PCR and quantitative fluorescent PCR again, and compared with regular-PCR, quantitative fluorescent PCR has spy
The features such as opposite sex is stronger, effectively solves PCR pollution problems, and automaticity is high, and without electrophoresis process, avoid organic molten
The harm that agent is brought to experimenter.But need to extract the nucleic acid in sample before in general fluorogenic quantitative detection, this is with regard to invisible
In add testing cost, reduce detection efficiency.
In view of this, it is special to propose the present invention.
The content of the invention
The first object of the present invention is to provide the nucleotide sequence for detecting NDV, high specificity, stability
It is good, provide good basis for quantitative detection NDV.
The second object of the present invention is to provide a kind of NDV real-time fluorescence quantitative PCR detection method, solved
Pollution, manpower is saved, and uses and exempt from the fluorescence quantitative PCR detection that nucleic acid extraction carries out NDV, save testing cost,
Improve detection efficiency.
The third object of the present invention is the detection kit for providing NDV quantitative fluorescent PCR, for entering for detection
Row provides facility.
In order to realize the above-mentioned purpose of the present invention, spy uses following technical scheme：
For detecting the nucleotide sequence of NDV, the nucleotide sequence includes primer pair and probe, the primer pair
Such as SEQ ID NO：1 and SEQ ID NO：Shown in 2, the nucleotide sequence such as SEQ ID NO of the probe：Shown in 3.
Nucleotide sequence provided by the present invention for detecting NDV, high specificity, stability is good, for quantitative detection
NDV provides good basis.
Further, 5 ' end fluorescent reporter groups of the probe are FAM, and 3 ' end fluorescent quenching groups are BHQ1.
Present invention also offers NDV real-time fluorescence quantitative PCR detection method, more than viral nucleic acid to be detected
The SEQ ID NO stated：1 and SEQ ID NO：2 are used as primer, and with SEQID NO：3 as probe progress real time fluorescent quantitative
PCR is detected.
Specifically, SEQ ID NO：1 is upstream primer sequence, SEQ ID NO：2 be downstream primer sequence, SEQ ID NO：
3 be the sequence of probe, and the Y in its middle probe refers to annex base, Y=C+T.
The sequence being related to is as follows：
Sense primer：AGTGATGTGCTCGGACCTTC (5 ' -3 ' end)
Anti-sense primer：CCTGAGGAGAGGCATTTGCTA (5 ' -3 ' end)
Probe：TTCTCTAGCAGTGGRACAGCCTGY (5 ' -3 ' end)
NDV real-time fluorescence quantitative PCR detection method provided by the invention, solves pollution, automaticity
Height, save manpower.
Further, the viral nucleic acid to be detected is extracted using following steps：
Thing to be detected is taken, lysate is added, vibration, then adds neutralizer, mix, centrifuge, obtained supernatant is
The viral nucleic acid to be detected；
The lysate is mixed by following component：The EDTA solution 0.3 that molar concentration is 0.05M and pH is 8 ±
0.01ml, Tris-HCl 0.75 ± 0.01ml, Triton 0.1 ± 0.01ml of X-100 that molar concentration is 0.1M and pH is 8,
0.2 ± 0.01ml of NP-40, CHAPS0.4 ± 0.01g, molar concentration is 100mM DTT 0.25 ± 0.01ml, SDS 0.46
± 0.01g, pH are 7.4 3.54 ± 0.05ml of PBS, and molar concentration is 0.15M 4.1 ± 0.05ml of NaCl；
The neutralizer is mixed by following component：1 ± 0.01ml of glycerine, molar concentration is 0.1M and pH is 8
Tris-HCl 0.75 ± 0.01ml, Triton 0.1 ± 0.01ml of X-100, the PMSF 0.4 that molar concentration is 5mM ±
0.01ml, molar concentration are 100mM DTT 0.2 ± 0.01ml of 0.2 ± 0.01ml, pharmalyte, and molar concentration is
0.15M 7.35 ± 0.05ml of NaCl.
The extracting method general step of existing nucleic acid is more, and the extracting method provided by the invention only adds lysate
With two steps of neutralizer, directly centrifugation can complete the extraction of genome, and method is easy, belongs to the technique for exempting from nucleic acid extraction, and
And after testing, extraction effect is stable, is well positioned to meet the demand of real-time fluorescence quantitative PCR detection method.
Further, the adding proportion of the lysate and the neutralizer is 1:1±0.1.
Experiment proves that the lysate and extract solution of above composition, are added in the ratio, extract obtained genome effect
It is stable, it is well positioned to meet the demand of real-time fluorescence quantitative PCR detection method.
Further, the vibration uses vortex oscillation, and time 50-80s, the rotating speed of the centrifugation is 8000-
12000rpm centrifuges 1-3min.Pass through vibration so that extract is fully broken, then by centrifugation, goes the removal of impurity, obtains more
Pure viral nucleic acid to be detected.
Further, the reaction condition of the real-time fluorescence quantitative PCR detection：
The first step：45 DEG C of holding 15min；
Second step：95 ± 1 DEG C of holding 2-5min；
3rd step：95 ± 1 DEG C of holding 10-15s, 60 DEG C of holding 1min, 40 ± 2 circulate, wherein 60 DEG C set collection glimmering
Optical signal.
When carrying out real-time fluorescence quantitative PCR detection, also comprising negative control and positive control, and according to collecting
CT values judge whether thing to be detected is positive.
Positive control：CT values ＜ 30 simultaneously has obvious amplification curve.
Negative control：Without CT values or C T values>37, without obvious amplification curve.
Requirements above need to meet that otherwise this experiment is invalid, it is necessary to re-start simultaneously.
Thing to be detected is that the positive of sample is judged in the following manner：
Sample CT values<30 and there is obvious amplification curve, be then judged to the positive；
Sample CT values>37 have no amplification curve, then are judged to feminine gender；
Sample CT values be then judged between 30 and 37 it is suspicious, need to repeat detect.
It is determined as the positive still for 30-37 and if having obvious amplification curve if second of detection CT value, is otherwise feminine gender.
Further, the thing to be detected include birds pharyngolaryngeal cavity swab collection thing, cloacal swab collection thing, blood,
Serum, blood plasma, tissue.
Usually, the thing to be detected of liquid type takes 10-50 μ l, adds 4-6 times of lysate, addition and lysate after vibration
Isometric neutralizer, mix, centrifugation, take 50-120 μ l supernatant to carry out fluorescence quantitative PCR detection；Correspondingly, it is right
For solid tissue, take 1-5mg tissue to be ground, then add 1ml PBS solutions and mix, centrifuging and taking 10-50 μ l, add
Enter 4-6 times of lysate, addition and the isometric neutralizer of lysate, are mixed after vibration, centrifugation, take 50-120 μ l supernatant
Carry out fluorescence quantitative PCR detection.
Quantitative fluorescent PCR reaction system can use 10-50 μ l systems, it is preferred to use 20 μ l systems, be specially：
Negative control, positive control or the μ l of sample 2, the μ l of reaction solution 10, the μ l of enzyme 0.5, the μ l of primed probe 1.8, seedless sour water
5.7 μ l, cumulative volume are 20 μ l.
During fluorescence quantitative PCR detection, in the positive control of addition, viral nucleic acid copies in 10-20 ten thousand；Sample then according to
Step is extracted, and adds 2 μ l.
The volume of quantitative fluorescent PCR reaction system is expanded or shunk, and each composition is expanded or shunk accordingly.
PCR amplification instrument used in the present invention is ABI 7500, expand reaction system liquid used and reverse transcriptase purchase in
Promega brands.
Present invention also offers the detection kit of NDV quantitative fluorescent PCR, including above-mentioned primer pair and spy
Pin.
Further, in addition to negative control, positive control, reaction amplification liquid, reverse transcriptase, any in seedless sour water
Kind is a variety of.
The detection kit of NDV quantitative fluorescent PCR provided by the invention, is easy to the detection of NDV to enter
OK, good condition is provided for the detection of NDV.
Further, the kit includes box body, is provided with the box body with porose pad；
Different reagents is placed in the container for posting different colours label or different colours, and the container inserts the hole
In, the number in the hole is not less than the number of container.
Kit provided by the invention, by the color of container such as bottle or the difference of label, preferably distinguish what is be put into
Whether constituent is correct, reduces error probability.
Preferably, the pad is preferably highly dense property foaming mould.Highly dense property foaming mould, there is fixed, buffering, support
Effect.
Compared with prior art, beneficial effects of the present invention are：
(1) provided by the present invention for the nucleotide sequence of detection NDV, high specificity, stability is good, is quantitative
Detect NDV and good basis is provided.
(2) NDV real-time fluorescence quantitative PCR detection method provided by the invention, solve pollution, save manpower,
And using the fluorescence quantitative PCR detection for exempting from nucleic acid extraction progress NDV, testing cost is saved, improves detection efficiency.
(3) present invention also offers the lysate for exempting from nucleic acid extraction use and the specific composition of neutralizer so that extraction effect
Fruit is stable, is well positioned to meet the demand of real-time fluorescence quantitative PCR detection method.
(4) present invention also offers the system and reaction condition of real-time fluorescence quantitative PCR detection, and then sentenced by CT values
Whether the thing to be detected that breaks is the positive, as a result accurately and reliably.
(5) present invention also offers the detection kit of NDV quantitative fluorescent PCR, the color of container such as bottle is passed through
Or the difference of label, whether correct, reduce error probability if preferably distinguishing the constituent being put into, while is the progress of detection
Facility is provided.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
There is the required accompanying drawing used in technology description to be briefly described.
Fig. 1 is the amplification curve diagram that the nucleic acid that the normal method for extracting nucleic acid in the embodiment of the present invention 1 obtains is carried out；
Fig. 2 provides to crack for the present invention in the embodiment of the present invention 1 exempts from the amplification song that the nucleic acid that extracting method obtains is carried out
Line chart；
Fig. 3 is the amplification curve diagram that the nucleic acid that the normal method for extracting nucleic acid in the embodiment of the present invention 2 obtains is carried out；
Fig. 4 provides to crack for the present invention in the embodiment of the present invention 2 exempts from the amplification song that the nucleic acid that extracting method obtains is carried out
Line chart；
Fig. 5 is the structural representation for the kit that the embodiment of the present invention 4 provides.
In figure：
1- box bodys；2- holes；3- is padded.
Embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the present invention.It is unreceipted specific in embodiment
Condition person, the condition suggested according to normal condition or manufacturer are carried out.Agents useful for same or the unreceipted production firm person of instrument, it is
The conventional products that can be obtained by commercially available purchase.
Embodiment 1
1st, solution is configured：
Lysate is mixed by following component：EDTA 0.3 ± 0.01ml of solution that molar concentration is 0.05M and pH is 8,
Tris-HCl 0.75 ± 0.01ml, Triton the X-100 0.1 ± 0.01ml, NP-40 that molar concentration is 0.1M and pH is 8
0.4 ± 0.01g of 0.2 ± 0.01ml, CHAPS, the DTT 0.25 ± 0.01ml, SDS 0.46 that molar concentration is 100mM ±
0.01g, pH are 7.4 PBS3.54 ± 0.05ml, and molar concentration is 0.15M 4.1 ± 0.05ml of NaCl；
Neutralizer is mixed by following component：1 ± 0.01ml of glycerine, the Tris- that molar concentration is 0.1M and pH is 8
HCl 0.75 ± 0.01ml, Triton 0.1 ± 0.01ml of X-100, molar concentration are 5mM 0.4 ± 0.01ml of PMSF, are rubbed
DTT 0.2 ± 0.01ml of 0.2 ± 0.01ml, pharmalyte that your concentration is 100mM, molar concentration are 0.15M NaCl
7.35±0.05ml。
2nd, the viral nucleic acid of thing to be detected is obtained：
Group 1：Normal extraction nucleic acid
5mg tissue is taken to be ground with mill, the tissue ground；
Add 150 μ l SDS extract solution (constituents:1%SDS, 0.01mol/L EDTA, 0.705mol/L NaCl,
0.05mol/L Tris, 0.5% sorbierite, 1%PVP, 1% beta -mercaptoethanol), after whirlpool is abundant, 65 DEG C of water-bath 30min,
Every 10min or so jogs once；
Add isometric 150 μ l phenol:Chloroform:Isoamyl alcohol (25:24:1), mix to not stratified, 12000r/min centrifugations
10min, supernatant is taken, add 0.2 μ l RNase A (10mg/mL), 37 DEG C of water-bath 30min；
Extracting once rear centrifuging and taking supernatant is repeated, 0.7 times of volume isopropanol is added, slowly mixes to the agglomerating precipitations of DNA, room
Temperature stands 30min；
70% ethanol washing DNA is precipitated 2 times, and absolute ethyl alcohol is washed 1 time, and inversion is dried, and adds 200 μ l ddH2O is fully molten
DNA is solved, it is standby.
Group 2：Extracting method is exempted from cracking
Take 5mg tissue to be ground with mill, the tissue ground, then add 1ml PBS solutions and mix, from
The heart takes 20 μ l supernatants to add in 1.5ml centrifuge tubes, adds 100 μ l lysate, vortex vibration 1min；
100 μ l neutralizers are added, concussion mixes；
12000rpm centrifuges 2min, takes the μ l of supernatant 100 to obtain stoste in new centrifuge tube.
Stoste, 10 times of gradient dilutions are used respectively, until dilution 103For last concentration, various concentration are then contrasted
Tissue treatment liquid sample, the normal sample expanding effect for exempting from nucleic acid extraction extracted in nucleic acid and the present invention.
3rd, real-time fluorescence quantitative PCR detects
The reaction system (20 μ l) of 3.1 PCR kit for fluorescence quantitative：
The μ l of sample 2 to be detected, the μ l of reaction solution 10, the μ l of enzyme 0.5, primed probe 1.8 μ l, the seedless μ l of sour water 5.7.Upstream and downstream is drawn
The concentration of thing is 20/3pmol/L, and the concentration of probe is 10/3pmol/L.
Wherein, primed probe is following three kinds of sequences using molar concentration as 2:2：The mixture of 1 mixing, including：
Sense primer：AGTGATGTGCTCGGACCTTC (5 ' -3 ' end)；
Anti-sense primer：CCTGAGGAGAGGCATTTGCTA (5 ' -3 ' end)；
Probe：TTCTCTAGCAGTGGRACAGCCTGY (5 ' -3 ' end).
Negative control (seedless sour water) or positive control (Newcastle Disease Virus that 10-20 ten thousand is copied) and sample to be detected
Equally, the reaction template of addition is different unlike.
The reaction condition of 3.2 real-time fluorescence quantitative PCRs detection：
The first step：45 DEG C of holding 15min；
Second step：95 DEG C of holding 2-5min；
3rd step：95 DEG C of holding 15s, 60 DEG C of holding 1min, 40 circulate, wherein 60 DEG C set collection fluorescence signals.
PCR amplification instrument used in the present invention is ABI 7500, expand reaction system liquid used and reverse transcriptase purchase in
Promega brands.
Obtained amplification curve diagram is as depicted in figs. 1 and 2.
Concentration of specimens corresponding to Fig. 1 and Fig. 2 is as shown in table 1 with CT values.
The concentration of specimens of table 1 and CT values
It can be regarded as from Fig. 1 and Fig. 2 and table 1, the detection of nucleic acids result extracted by the way of different using two kinds is without obvious
Difference, it was demonstrated that it is provided by the invention cracking it is hands-free take nucleic acid method it is feasible, traditional nucleic acid extraction step can be substituted.
Embodiment 2
1st, solution is configured：
Lysate is mixed by following component：EDTA 0.3 ± 0.01ml of solution that molar concentration is 0.05M and pH is 8,
Tris-HCl 0.75 ± 0.01ml, Triton the X-100 0.1 ± 0.01ml, NP-40 that molar concentration is 0.1M and pH is 8
0.4 ± 0.01g of 0.2 ± 0.01ml, CHAPS, the DTT 0.25 ± 0.01ml, SDS 0.46 that molar concentration is 100mM ±
0.01g, pH are 7.4 PBS3.54 ± 0.05ml, and molar concentration is 0.15M 4.1 ± 0.05ml of NaCl；
Neutralizer is mixed by following component：1 ± 0.01ml of glycerine, the Tris- that molar concentration is 0.1M and pH is 8
HCl 0.75 ± 0.01ml, Triton 0.1 ± 0.01ml of X-100, molar concentration are 5mM 0.4 ± 0.01ml of PMSF, are rubbed
DTT 0.2 ± 0.01ml of 0.2 ± 0.01ml, pharmalyte that your concentration is 100mM, molar concentration are 0.15M NaCl
7.35±0.05ml。
2nd, the viral nucleic acid of thing to be detected is obtained：
Group 1：Normal extraction nucleic acid
The μ l of blood sample 20 are taken, add 100 μ l SDS extract solution (constituents:1%SDS, 0.01mol/L EDTA,
0.705mol/L NaCl, 0.05mol/L Tris, 0.5% sorbierite, 1%PVP, 1% beta -mercaptoethanol), after whirlpool is abundant,
65 DEG C of water-bath 30min, interval 10min or so jogs are once；
Add isometric 100 μ l phenol:Chloroform:Isoamyl alcohol (25:24:1), mix to not stratified, 12000r/min centrifugations
10min, supernatant is taken, add 0.2 μ l RNase A (10mg/mL), 37 DEG C of water-bath 30min；
Extracting once rear centrifuging and taking supernatant is repeated, 0.7 times of volume isopropanol is added, slowly mixes to the agglomerating precipitations of DNA, room
Temperature stands 30min；
70% ethanol washing DNA is precipitated 2 times, and absolute ethyl alcohol is washed 1 time, and inversion is dried, and adds 200 μ l ddH2O is fully molten
DNA is solved, it is standby.
Group 2：Extracting method is exempted from cracking
20 μ l blood samples are taken, are added in 1.5ml centrifuge tubes, add 100 μ l lysate, vortex vibration 1min；
100 μ l neutralizers are added, concussion mixes；
12000rpm centrifuges 2min, takes the μ l of supernatant 100 to obtain stoste in new centrifuge tube.
Stoste, 10 times of gradient dilutions are used respectively, until dilution 104For last concentration, various concentration are then contrasted
Tissue treatment liquid sample, the normal sample expanding effect for exempting from nucleic acid extraction extracted in nucleic acid and the present invention.
3rd, real-time fluorescence quantitative PCR detects
The reaction system (20 μ l) of 3.1 PCR kit for fluorescence quantitative：
The μ l of sample 2 to be detected, the μ l of reaction solution 10, the μ l of enzyme 0.5, primed probe 1.8 μ l, the seedless μ l of sour water 5.7.
Negative control (seedless sour water) or positive control (Newcastle Disease Virus that 10-20 ten thousand is copied) and sample to be detected
Equally, the reaction template of addition is different unlike.
The reaction condition of 3.2 real-time fluorescence quantitative PCRs detection：
The first step：45 DEG C of holding 15min；
Second step：95 DEG C of holding 2min；
3rd step：95 DEG C of holding 15s, 60 DEG C of holding 1min, 40 circulate, wherein 60 DEG C set collection fluorescence signals.
PCR amplification instrument used in the present invention is ABI 7500, expand reaction system liquid used and reverse transcriptase purchase in
Promega brands.
Obtained amplification curve diagram is as shown in Figure 3 and Figure 4.
Concentration of specimens corresponding to Fig. 3 and Fig. 4 is as shown in table 2 with CT values.
The concentration of specimens of table 2 and CT values
It can be regarded as from Fig. 3 and Fig. 4 and table 2, the detection of nucleic acids result extracted by the way of different using two kinds is without obvious
Difference, it was demonstrated that it is provided by the invention cracking it is hands-free take nucleic acid method it is feasible, traditional nucleic acid extraction step can be substituted.
Embodiment 3
Altogether detect 100 birds, each birds take respectively pharyngolaryngeal cavity swab collection thing, cloacal swab collection thing, tissue,
Blood, serum, blood plasma are detected accordingly.
100 parts of birds pharyngolaryngeal cavity swab collection things, 100 parts of cloacal swab collection things and 100 parts of tissues, using embodiment
The hands-free method for taking nucleic acid of 1 cracking extracts genome, and obtained extract carries out quantitative fluorescent PCR (step is with embodiment 1),
Obtain following result：
The CT values of 30 parts of birds pharyngolaryngeal cavity swab collection things<30 and there is obvious amplification curve, be judged to the positive, 70 parts of birds
Pharyngolaryngeal cavity swab gathers the CT values of thing>37 have no amplification curve, then are judged to feminine gender；
The CT values of 30 parts of cloacal swab collection things<30 and there is obvious amplification curve, be judged to the positive, 70 parts of cloacas are wiped
The CT values of son collection thing>37 have no amplification curve, then are judged to feminine gender；
The CT values of 30 parts of tissues<30 and there is obvious amplification curve, be judged to the positive, the CT values of 70 parts of tissues>37 have no expansion
Increase curve, be then judged to feminine gender.
100 parts of blood, 100 parts of blood plasma and 100 parts of serum, extracted using the hands-free method for taking nucleic acid of cracking of embodiment 2
Genome, obtained extract carry out quantitative fluorescent PCR (step is with embodiment 2), obtain following result：
The CT values of 30 parts of blood<30 and there is obvious amplification curve, be judged to the positive, the CT values of 70 parts of blood>37 have no expansion
Increase curve, be then judged to feminine gender；
The CT values of 30 parts of serum<30 and there is obvious amplification curve, be judged to the positive, the CT values of 70 parts of serum>37 have no expansion
Increase curve, be then judged to feminine gender；
The CT values of 30 parts of blood plasma<30 and there is obvious amplification curve, be judged to the positive, the CT values of 70 parts of blood plasma>37 have no expansion
Increase curve, be then judged to feminine gender.
Through subsequent authentication, Detection accuracy 100%.
Embodiment 4
Present invention also offers the detection kit of NDV quantitative fluorescent PCR, as shown in figure 5, the kit
Including box body 1, it is provided with the box body 1 with porose pad 3；
Different reagents is placed in the container such as bottle for posting different colours label or different colours, the container insertion
In the hole 2, the number in the hole 2 is not less than the number of container.
Different reagents includes above-mentioned primer pair and probe, further, in addition to it is negative control, positive control, anti-
Any of liquid, reverse transcriptase, seedless sour water or a variety of should be expanded.
Specifically, hole 2 different in figure may be inserted into such as bottle of the container containing different reagents.But in order to preferably distinguish
Whether the constituent being put into is correct, reduces error probability, is put into the container cover of different component and pastes generally according to following sequence
There is the difference label of different colours, blue label (primer) bottle, brown label (probe) bottle, Huang are sequentially placed into from upper left side
Colour code label (enzyme) bottle, water white transparency label (seedless sour water) bottle, green-ticket (negative control) bottle, red-ticket (sun
Property control) bottle.Separately there are sample lysate and neutralizer to be placed in side gap.
Kit provided by the invention, by the color of bottle or the difference of label, preferably distinguish the composition that is put into
Divide whether correct, reduction error probability.
Preferably, the pad 3 is preferably highly dense property foaming mould.Highly dense property foaming mould, there is fixed, buffering, branch
The effect of support.
The detection kit of NDV quantitative fluorescent PCR provided by the invention, is easy to the detection of NDV to enter
OK, good condition is provided for the detection of NDV.
Although illustrate and describing the present invention with specific embodiment, but will be appreciated that without departing substantially from the present invention's
Many other change and modification can be made in the case of spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
<110>Beijing great You Tai Lays Bioisystech Co., Ltd
<120>NDV real-time fluorescence quantitative PCR detection method and its kit
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence
<400> 1
agtgatgtgc tcggaccttc 20
<210> 2
<211> 21
<212> DNA
<213>Artificial sequence
<400> 2
cctgaggaga ggcatttgct a 21
<210> 3
<211> 24
<212> DNA
<213>Artificial sequence
<400> 3
ttctctagca gtggracagc ctgy 24
Claims (10)
1. the nucleotide sequence for detecting NDV, it is characterised in that the nucleotide sequence includes primer pair and probe, institute
State primer pair such as SEQ ID NO：1 and SEQ ID NO：Shown in 2, the nucleotide sequence such as SEQ ID NO of the probe：Shown in 3.
2. nucleotide sequence according to claim 1, it is characterised in that 5 ' end fluorescent reporter groups of the probe are FAM,
3 ' end fluorescent quenching groups are BHQ1.
3. NDV real-time fluorescence quantitative PCR detection method, it is characterised in that viral nucleic acid to be detected is with claim
SEQ ID NO described in any one of 1-2：1 and SEQ ID NO：2 are used as primer, and with SEQ ID NO：3 are carried out in fact as probe
When fluorescence quantitative PCR detection.
4. detection method according to claim 3, it is characterised in that the viral nucleic acid to be detected uses following steps
Extraction：
Thing to be detected is taken, lysate is added, vibration, then adds neutralizer, mix, centrifugation, obtained supernatant is described
Viral nucleic acid to be detected；
The lysate is mixed by following component：EDTA 0.3 ± 0.01ml of solution that molar concentration is 0.05M and pH is 8,
Tris-HCl 0.75 ± 0.01ml, Triton the X-100 0.1 ± 0.01ml, NP-40 that molar concentration is 0.1M and pH is 8
0.4 ± 0.01g of 0.2 ± 0.01ml, CHAPS, the DTT 0.25 ± 0.01ml, SDS 0.46 that molar concentration is 100mM ±
0.01g, pH are 7.4 3.54 ± 0.05ml of PBS, and molar concentration is 0.15M 4.1 ± 0.05ml of NaCl；
The neutralizer is mixed by following component：1 ± 0.01ml of glycerine, the Tris- that molar concentration is 0.1M and pH is 8
HCl 0.75 ± 0.01ml, Triton 0.1 ± 0.01ml of X-100, molar concentration are 5mM 0.4 ± 0.01ml of PMSF, are rubbed
DTT 0.2 ± 0.01ml of 0.2 ± 0.01ml, pharmalyte that your concentration is 100mM, molar concentration are 0.15M NaCl
7.35±0.05ml。
5. detection method according to claim 4, it is characterised in that the adding proportion of the lysate and the neutralizer
For 1:1±0.1.
6. detection method according to claim 4, it is characterised in that the vibration uses vortex oscillation, time 50-
80s, the rotating speed of the centrifugation centrifuge 1-3min for 8000-12000rpm.
7. detection method according to claim 4, it is characterised in that the reaction bar of the real-time fluorescence quantitative PCR detection
Part：
The first step：45 DEG C of holding 15min；
Second step：95 ± 1 DEG C of holding 2-5min；
3rd step：95 ± 1 DEG C of holding 10-15s, 60 DEG C of holding 1min, 40 ± 2 circulate, wherein 60 DEG C set collection fluorescence letter
Number.
8. according to the detection method described in claim any one of 4-7, it is characterised in that the thing to be detected includes birds throat
Chamber swab collection thing, cloacal swab collection thing, blood, serum, blood plasma, tissue.
9. the detection kit of NDV quantitative fluorescent PCR, it is characterised in that including described in claim any one of 1-2
Primer pair and probe；
Further, in addition to negative control, positive control, reaction amplification liquid, reverse transcriptase, any of seedless sour water or
It is a variety of.
10. kit according to claim 7, it is characterised in that the kit includes box body, is set in the box body
Have with porose pad；
Different reagents is placed in the container for posting different colours label or different colours, and the container is inserted in the hole,
The number in the hole is not less than the number of container；
The pad is preferably highly dense property foaming mould.
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