CN104561387A - Real-time fluorescent quantitative PCR detection method for apple stem pitting virus - Google Patents
Real-time fluorescent quantitative PCR detection method for apple stem pitting virus Download PDFInfo
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Abstract
The invention provides a real-time fluorescent quantitative PCR detection method for an apple stem pitting virus, and belongs to the field of virus molecule detection. The method comprises the following steps: with cDNA of an infected material as a template, and ASPV-cp-F2 and ASPV-cp-R2 as primers, carrying out PCR amplification, so as to obtain positive recombinant plasmid standards; with copy number concentration of the positive recombinant plasmid standards and positive recombinant plasmid standards with the concentration as templates, building a standard curve by a Ct value of real-time fluorescent quantitative PCR employing specific primers and probes; carrying out real-time fluorescent quantitative PCR on a tested material according to the same condition; and achieving quantitative detection of the apple stem pitting virus of the tested material by comparing the Ct value with the standard curve. According to the method, quantitative determination of the apple stem pitting virus is achieved; the detection result can be directly read out through computer software; and the problems of false positivity of the detection result and environmental pollution are overcome.
Description
Technical field
The present invention relates to viruses molecule detection field, in particular to a kind of apple stem pitting virus real-time fluorescence quantitative PCR detection method.
Background technology
Apple stem pitting virus is one of latent virus that apple and pear tree generally occur, and this virus can cause the multiple diseases such as Apple stem acne is sick, pear vein yellow virus is sick, pears stone acne is sick, and can cause apple and pears production declining, product qualitative change is bad, has a strong impact on the production of fruit trees of China.The method mainly conventional RT-PCR technology of current detection ASPV.
The conventional RT-PCR detection technique scheme of ASPV comprises the following steps:
1, design conventional RT-PCR and detect primer;
2, the RNA of fruit tree test sample is extracted and reverse transcription becomes cDNA;
3, take cDNA as masterplate, utilize specific amplification primer to carry out standard PCR amplification;
4, amplified production is carried out 1.5% agarose gel electrophoresis;
5, carry out observation by gel imaging system to electrophoresis result to take pictures, contain ASPV if observe in the provable test sample of specific amplified band, instead then do not have.
But there is shortcoming in the conventional RT-PCR detection technique of ASPV:
(1), operation steps is comparatively loaded down with trivial details, wastes time and energy;
(2), need to carry out aftertreatment to pcr amplification product, only have and amplified production just can be obtained detected result through agarose gel electrophoresis, but need in sepharose making processes to add carcinogenic substance EB, easy pollution laboratory environment, also can constitute a threat to the health of experimenter, in last handling process easily there is crossed contamination in different sample room simultaneously, causes the false positive phenomenon of detected result;
(3), conventional RT-PCR technology can only carry out qualitative detection to virus, cannot detection by quantitative
In view of this, special proposition the present invention.
Summary of the invention
The object of the present invention is to provide a kind of apple stem pitting virus real-time fluorescence quantitative PCR detection method, this detection method have can realize for examination material apple stem pitting virus detection by quantitative, there is not false positive and environmental pollution, detection time short and highly sensitive technique effect.
In order to realize above-mentioned purpose of the present invention, spy by the following technical solutions:
The invention provides a kind of apple stem pitting virus real-time fluorescence quantitative PCR detection method, comprise the following steps:
1), with the cDNA of material of contaminating for template, with ASPV-cp-F2 and ASPV-cp-R2 for primer carries out pcr amplification, the amplified production that obtains is cut glue and is reclaimed and be transformed into competent cell after connecting with carrier after agarose gel electrophoresis, carry out plasmid extraction and qualification after picking positive colony Zengjing Granule, obtain positive recombinant plasmid standard substance;
2), with the copy number concentration of the described positive recombinant plasmid standard substance through gradient dilution and with the positive recombinant plasmid standard substance of this concentration be template, with ASPV-Probe3-F and ASPV-Probe3-R be primer, the Ct value of real-time fluorescence quantitative PCR of carrying out for probe with ASPV-Probe3 builds typical curve;
3), be template for the cDNA of examination material, according to step 2) identical amplification condition carries out real-time fluorescence quantitative PCR, the Ct value obtained and described typical curve are compared, and realize the detection by quantitative that confession tries material apple stem pitting virus;
Wherein, described ASPV-cp-F
2, ASPV-cp-R
2, ASPV-Probe3-F, ASPV-Probe3-R and ASPV-Probe3 nucleotide sequence respectively as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5.
This detection method provided by the invention, it adopts the cDNA of contamination material to be template, product is gone out by specific primer amplification, after this product is connected by carrier and transforms, obtain the fragment of massive duplication, picking positive colony Zengjing Granule also carries out plasmid extraction and qualification (enzyme cuts qualification) and then obtains positive recombinant plasmid standard substance.Copy number concentration is converted into according to this positive recombinant plasmid standard substance molecular weight, and gradient dilution; Then with the positive recombinant plasmid standard substance of this concentration be template, with ASPV-Probe3-F and ASPV-Probe3-R be primer, the Ct value of real-time fluorescence quantitative PCR of carrying out for probe with ASPV-Probe3 builds typical curve (Ct value-copy number concentration).In above-mentioned reaction, ASPV-Probe3-F, ASPV-Probe3-R and ASPV-Probe3 are specificity amplification primer and the probe of specific setting, and hold mark fluorescent group at 5 ' of ASPV-Probe3,3 ' end mark quenching group.
To be template carry out cDNA according to the condition identical with building above-mentioned typical curve to cDNA for examination material is template, by for the Ct value of examination material reaction and typical curve, directly obtains the content supplying the apple stem pitting virus tried in material.
The method achieve the quantitative assay for the apple stem pitting virus in examination material, and detected result can directly be read by computer software, do not need the operation such as gel electrophoresis and image checking, and then avoid the false-positive body harm that it also avoid environmental pollution and EB simultaneously and operator are caused of detected result.In addition, the whole testing process of this detection method only needs 1 hours, has and saves time and highly sensitive advantage.
Optionally, in step 1) in: the preparation method of the cDNA of described contamination material comprises:
Extract the total serum IgE infecting and have apple stem pitting virus material, and carry out reverse transcription with this RNA for template, obtain the cDNA of contamination material.
By extracting the total serum IgE infecting and have apple stem pitting virus material, then carry out the cDNA that reverse transcription obtains contamination material, the method is easy and simple to handle, is easy to realize.
Optionally, in step 1) in, the concentration of sepharose used in described agarose gel electrophoresis is 1-2%.
Optionally, in step 1) in: described carrier is pEASY-T
3, described competent cell is E.coli DH5 α.
Amplified production reclaims fragment by cutting glue after agarose gel electrophoresis, and in order to obtain the target fragment of massive duplication, this recovery fragment utilizes pEASY-T
3be transformed into after connection in E.coliDH5 α competent cell, carry out Zengjing Granule by picking positive colony, then after extracting with plasmid Mini Kit, order-checking and enzyme cut qualification.Through identifying that correct positive recombinant plasmid is as positive recombinant plasmid standard substance.
Optionally, in step 2) in:
The OD of described positive recombinant plasmid standard substance
260/ OD
280ratio is between 1.8-2.0.
OD
260/ OD
280the positive recombinant plasmid standard substance of ratio between 1.8-2.0, its purity is high, and the typical curve therefore made can react the relation of Ct value and initial template copy number concentration more accurately.
Optionally, in step 2) in:
The copy number concentration range of described positive recombinant plasmid standard substance is: 1.0 × 10
8copy/μ L ~ 1.0 × 10
1copy/μ L, and the multiple of described gradient dilution is 10 times.
Copy number concentration and its of the positive recombinant plasmid standard substance of 10 times of dilution gradients carry out the loose point coordinate of the Ct value of quantitative fluorescent PCR reaction evenly, more easily obtain the typical curve of high relation conefficient; And 1.0 × 10
8copy/μ L ~ 1.0 × 10
1copy/μ L sensing range is large, improves the popularity for examination material Viral diagnosis.
Optionally, in step 2) in:
Described typical curve with the logarithmic value of the copy number concentration of described positive recombinant plasmid standard substance for X-coordinate, with described Ct value for ordinate zou.
Optionally, in step 2) in, in the process of described real-time fluorescence quantitative PCR, reaction system is:
TransStart Probe qPCR Super Mix 10 μ L, ASPV-Probe3-F and ASPV-Probe3-R 4-5pmol, ASPV-Probe34-5pmol, template 1.0 μ L, distilled water polishing to 20 μ L.
Optionally, in step 2) in, in the process of described real-time fluorescence quantitative PCR, amplification program is:
94 DEG C of denaturation 30-35s; 94 DEG C of sex change 4-7s, 55 DEG C of annealing 12-15s, 72 DEG C extend 15-20s, 35-40 circulation altogether.
Under this amplification program, its specific amplification is good.
Optionally, described real-time fluorescence quantitative PCR adopts Bio-Rad iQ
tM5 real-time fluorescence quantitative PCR instrument carry out.
Compared with prior art, beneficial effect of the present invention is:
(1), by typical curve realize the detection by quantitative for ASPV virus in examination material, and Standard PCR technology can only realize the qualitative detection of ASPV.
(2), detected result directly can read by computer software, do not need to carry out post-reaction treatment, so avoid the pollution of the false-positive generation of detected result and environment.
(3), this real-time fluorescence quantitative PCR testing process only needs 1 hours, compared with Standard PCR technology, enormously simplify detecting step, shortens the time of detection.
(4), by contrasting with Standard PCR technology, its sensitivity is 100 times of conventional RT-PCR technology.
Accompanying drawing explanation
In order to be illustrated more clearly in the embodiment of the present invention or technical scheme of the prior art, be briefly described to the accompanying drawing used required in embodiment or description of the prior art below.
The specific detection of the recombinant plasmid ASPV-cp real-time fluorescence quantitative PCR that Fig. 1 provides for the embodiment of the present invention 2;
Fig. 2 is the sensitivity test result figure that in prior art, Standard PCR detects ASPV;
The sensitivity test result figure of the detection method detection ASPV that Fig. 3 provides for the embodiment of the present invention 2.
Embodiment
Below in conjunction with embodiment, embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example only for illustration of the present invention, and should not be considered as limiting the scope of the invention.Unreceipted actual conditions person in embodiment, the condition of conveniently conditioned disjunction manufacturers suggestion is carried out.Agents useful for same or the unreceipted production firm person of instrument, be and can buy by commercially available the conventional products obtained.
It is to be noted, in all embodiments of the present invention and application examples, the Leaves of Apple Trees In The Field infecting ASPV picks up from Wen Ren village, Baoding, Hebei province in June, 2012, field test sample picks up from Hebei Science & Technology Normal College's gardening experiment centre in July, 2014, and apple tissue cultural seedlings of free is provided by Agricultural University Of Hebei's Biotechnology Experiment room.
In addition, for used key instrument and equipment source as follows:
RNAiso Plus, RNAiso-mate, EASY Dilution are purchased from the precious biotechnology company limited in Dalian;
2 × ES TaqMasterMix is purchased from Beijing CoWin Bioscience Co., Ltd.;
TransStart Probe qPCR Super Mix, TransScript One-Step gDNARemoval and cDNA Synthesis SuperMix and pEASY-T
3cloning Kit is purchased from Beijing Quanshijin Biotechnology Co., Ltd;
DNA gel recovery test kit and plasmid Mini Kit are purchased from Sangon Biotech (Shanghai) Co., Ltd.;
Bio-Rad S1000PCR instrument, Bio-Rad iQ
tM5 real-time fluorescence quantitative PCR instrument are U.S. Bio-Rad Products;
The micro-ultraviolet spectrometry degree of NanoDrop 2000 counts U.S. Thermo Scientific Products.
Embodiment 1
S11: with the cDNA of material of contaminating for template, with ASPV-cp-F
2and ASPV-cp-R
2for primer carries out pcr amplification, the amplified production that obtains is cut glue and is reclaimed and be transformed into competent cell after connecting with carrier after agarose gel electrophoresis, carries out plasmid extraction and qualification, obtain positive recombinant plasmid standard substance after picking positive colony Zengjing Granule;
S12: with the copy number concentration of the described positive recombinant plasmid standard substance through gradient dilution and with the positive recombinant plasmid standard substance of this concentration be template, with ASPV-Probe3-F and ASPV-Probe3-R be primer, the Ct value of real-time fluorescence quantitative PCR of carrying out for probe with ASPV-Probe3 builds typical curve;
S13: the cDNA for examination material is template, according to step 2) identical amplification condition carries out real-time fluorescence quantitative PCR, the Ct value obtained and described typical curve are compared, and realize the detection by quantitative that confession tries material apple stem pitting virus.
Wherein, described ASPV-cp-F
2, ASPV-cp-R
2, ASPV-Probe3-F, ASPV-Probe3-R and ASPV-Probe3 nucleotide sequence respectively as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5.
Table 1 primer and probe sequence
Embodiment 2
S21: obtain primer and probe
According to the nucleotide sequence (accession number: NC003462, AF491930, JF946775, HM125159) of the apple stem pitting virus logged in GenBank (ASPV) coat protein gene (cp), Primer Primer 5.0 is utilized to design the primer ASPV-cp-F of amplification cp gene complete sequence
2/ R
2, for the clone of positive plasmid standard substance sequence.
ClustalX (1.81) is utilized to carry out multiple ratio pair to cloning the apple stem pitting virus cp gene order delivered in the apple stem pitting virus cp gene order and GenBank that obtain in this invention, select metastable conservative region as detection target sequence, utilize PrimerPrimer 5.0 and Primer Express v3.0 software design specificity amplification primer (ASPV-Probe3-F and ASPV-Probe3-R) and TaqMan probe (please refer to table 1), mark fluorescent group held by probe 5 ', 3 ' end mark quenching group.
S22: the extraction of total serum IgE and the synthesis of cDNA
Test kit is utilized to extract contamination material and the total serum IgE for examination material, and with this total serum IgE for template utilizes Reverse Transcription box to synthesize cDNA respectively, concrete, take 0.1g Apple Leaves, RNAiso Plus and RNAiso-mate is utilized to extract total serum IgE according to test kit specification sheets, obtain after cDNA for primer utilizes TransScript One-Step gDNARemoval and cDNA Synthesis SuperMix test kit to carry out reverse transcription with Oligo (dT) 18 again, carry out standard PCR amplification.
S23: the structure of positive recombinant plasmid standard substance
Utilize primer ASPV-cp-F
2/ R
2the pcr amplification of ASPV-cp gene is carried out to the catch an illness cDNA of Apple Leaves of field.Amplified production, after 1.5% agarose gel electrophoresis, adopts DNA gel to reclaim test kit and cuts glue recovery to the specific band meeting target fragment size (1444bp), be connected to pEASY-T after purifying
3on carrier, and be converted into E.coli DH5 α competent cell, picking positive colony carries out Zengjing Granule, and after extracting with plasmid Mini Kit, order-checking and enzyme cut qualification.Through identifying that correct positive recombinant plasmid is as positive recombinant plasmid standard substance.
S24: production standard curve
The OD extracting plasmid is measured with NanoDrop 2000
260and OD
280value and concentration thereof, OD
260/ OD
280ratio is used for production standard curve at the plasmid of 1.8-2.0.Its mass concentration is scaled copy number concentration by the molecular weight according to plasmid: copies/ μ L=(ng/ μ L × 10
-9) × (6.02 × 10
23)/(DNA length × 660); With Easy Dilution, it is diluted to 1.0 × 10 successively again
8copy/μ L ~ 1.0 × 10
1copy/μ L, totally 8 concentration gradients, and set up 20 μ L amplification systems as template reference reagent box specification sheets:
TransStart Probe qPCR Super Mix 10μL，
ASPV-Probe3-F, ASPV-Probe3-R (10pmol/ μ L) each 0.4 μ L,
ASPV-Probe3(10pmol/μL)0.4μL，
Template (8 gradients) 1.0 Μ l,
Distilled water 7.8 μ L.
At Bio-Rad iQ
tM5 real-time fluorescence quantitative PCR instrument increase by following reaction parameter: 94 DEG C of denaturation 30s; 94 DEG C of sex change 5s, 55 DEG C of annealing 15s, 72 DEG C extend 15s, totally 40 circulations.After reaction terminates, analysis software is utilized automatically to generate typical curve.
Copy number concentration and the Ct value of positive recombinant plasmid standard substance all present good linear relationship, relation conefficient (R
2)reach 0.999, amplification efficiency (E) is 99.3%, and straight-line equation is y=-3.34log (x)+44.117, and wherein, the logarithmic value of the copy number concentration of positive recombinant plasmid standard substance is x-axis, and the Ct value of reaction is y-axis.
S25: for the detection of ASPV in examination material
To supply the cDNA of examination material for template in step S22, real-time fluorescence quantitative PCR is carried out according to the amplification condition identical with step S24, the Ct value obtained and described typical curve are compared, and directly read the detected result for the ASPV in examination material by computer software.
In addition, in order to verify the Detection results of this real-time fluorescence quantitative PCR detection method provided by the invention, spy carries out following experimental analysis:
Specificity analyses
With embodiment 2 prepare recombinant plasmid ASPV-cp (positive recombinant plasmid standard substance) be positive template, with recombinant plasmid ACLSV-cp, ASGV-cp for negative template, take distilled water as blank, carry out real-time fluorescence quantitative PCR detection, result is (wherein, 1ASPV-cp recombinant plasmid as shown in Figure 1; 2ACLSV-cp recombinant plasmid; 3ASGV-cp recombinant plasmid; 4 distilled waters), found that, except the fluorescent signal detected in ASPV-cp plasmid reaction tubes in obvious ascendant trend, all the other detected results are feminine gender, show the detection ASPV that the method can be special.
Sensitivity analysis
After the ASPV-cp positive plasmid (positive recombinant plasmid standard substance) provided the embodiment of the present invention 2 carries out 10 times of doubling dilutions, carry out Standard PCR respectively and real-time fluorescence quantitative PCR detects.
Please refer to Fig. 2 (wherein, M:DNA ladder marker; 1:1.0 × 10
8copies/ μ L; 2:1.0 × 107copies/ μ L; 3:1.0 × 10
6copies/ μ L; 4:1.0 × 10
5copies/ μ L; 5:1.0 × 10
4copies/ μ L; 6:1.0 × 10
3copies/ μ L; 7:1.0 × 10
2copies/ μ L; 8:1.0 × 10
1copies/ μ L; 9:No template control), result shows, and the Monitoring lower-cut of Standard PCR is 1.0 × 10
4copy/μ L.Please refer to Fig. 3 (M:DNA ladder marker; 1:1.0 × 10
8copies/ μ L; 2:1.0 × 107copies/ μ L; 3:1.0 × 10
6copies/ μ L; 4:1.0 × 10
5copies/ μ L; 5:1.0 × 10
4copies/ μ L; 6:1.0 × 10
3copies/ μ L; 7:1.0 × 10
2copies/ μ L; 8:1.0 × 10
1copies/ μ L; N:No template control, X-coordinate is cycle number, and ordinate zou is fluorescence signal intensity), find that the Monitoring lower-cut of real-time fluorescence quantitative PCR is 1.0 × 10
2copy/μ L, shows that the sensitivity of real-time fluorescence quantitative PCR is 100 times of Standard PCR.
Repeatability is analyzed
Choose the positive recombinant plasmid standard substance 1.0 × 10 that the embodiment of the present invention 2 provides
7, 1.0 × 10
5, 1.0 × 10
3the dilution ASPV-cp plasmid of copies/ μ L 3 is that template is carried out in group and real-time fluorescence quantitative PCR reaction between group, result please refer to table 2, the variation coefficient of the Ct value find in each concentration plasmid group, repeating between group is all less than 1%, shows that the method has good repeatability.
The repeatability analysis of table 2 real-time fluorescence quantitative PCR
Application examples
Utilize the method for the embodiment of the present invention 2 to detect 15 parts of Leaves of Apple Trees In The Fields, knot please refer to table 3, and the recall rate of test sample ASPV is 100%, Ct value is 24.15 ~ 30.79, and viral level is 9.51 × 10
5~ 9.78 × 10
3copies/ μ L
,tissue cultural seedlings of free and blank detected result are feminine gender, and the Ct value of positive control is 22.15, and viral level is 3.78 × 10
6copies/ μ L, shows that the method can be used for the detection of ASPV in apple sample.
The detection method of table 3 embodiment 2 is to the detected result of 15 parts of Leaves of Apple Trees In The Fields
| Sample | Detected result | Ct value | ASPV concentration (copies/ μ L) |
| 1 | + | 24.15 | 9.51×10 5 |
| 2 | + | 26.22 | 2.28×10 5 |
| 3 | + | 25.78 | 3.09×10 5 |
| 4 | + | 30.79 | 9.78×10 3 |
| 5 | + | 25.54 | 3.65×10 5 |
| 6 | + | 26.37 | 2.06×10 5 |
| 7 | + | 28.87 | 3.67×10 4 |
| 8 | + | 26.39 | 2.03×10 5 |
| 9 | + | 29.03 | 3.29×10 4 |
| 10 | + | 26.35 | 2.09×10 5 |
| 11 | + | 28.02 | 6.60×10 4 |
| 12 | + | 27.48 | 9.57×10 4 |
| 13 | + | 29.63 | 2.17×10 4 |
| 14 | + | 26.85 | 1.48×10 5 |
| 15 | + | 28.31 | 5.40×10 4 |
| Positive control | + | 22.15 | 3.78×10 6 |
| The virus-free plantlets | - | 0 | 0 |
| CK | - | 0 | 0 |
Wherein, in table 3, "+" is that positive "-" is for negative.
In sum, the embodiment of the present invention is Auele Specific Primer and Taqman probe according to the conserved sequence design and synthesis of ASPV cp gene, and apply ASPV-cp recombinant plasmid and construct typical curve as standard substance, establish the Taqman probe for real-time fluorescence quantitative PCR detecting method for ASPV, achieve the detection by quantitative of ASPV first, this is also domestic first about the report of ASPV real-time fluorescence quantitative PCR detection method.
The whole testing process of application the method only needs about 1h, and result can directly draw from computer software, does not need to carry out PCR aftertreatment, effectively can prevent pollution and the false positive of testing process.The method can detect the ASPV-cp recombinant plasmid of 100 copies/μ L in 20 μ L reaction systems, and sensitivity is 100 times of Standard PCR; And to other latent virus recombinant plasmid (ASGV-cp, ACLSV-cp) without specific amplified, batch in repeat and batch between repeat the variation coefficient be all less than 1%, compare with detection method in the past, there is the advantages such as easy, quick, accurate, sensitive, stable.The application of the method achieves ASPV detection means by the leap of qualitative detection to detection by quantitative, by for studying the infecting of ASPV further, breed, to distribute and the rule such as timing variations provides reliable technique means, and establish solid basis for the multiple quantitative PCR detection system of research and development Apple virus.
Although illustrate and describe the present invention with specific embodiment, however it will be appreciated that can to make when not deviating from the spirit and scope of the present invention many other change and amendment.Therefore, this means to comprise all such changes and modifications belonged in the scope of the invention in the following claims.
Claims (10)
1. an apple stem pitting virus real-time fluorescence quantitative PCR detection method, is characterized in that, comprises the following steps:
1), with the cDNA of material of contaminating for template, with ASPV-cp-F
2and ASPV-cp-R
2for primer carries out pcr amplification, the amplified production that obtains is cut glue and is reclaimed and be transformed into competent cell after connecting with carrier after agarose gel electrophoresis, carries out plasmid extraction and qualification, obtain positive recombinant plasmid standard substance after picking positive colony Zengjing Granule;
2), with the copy number concentration of the described positive recombinant plasmid standard substance through gradient dilution and with the positive recombinant plasmid standard substance of this concentration be template, with ASPV-Probe3-F and ASPV-Probe3-R be primer, the Ct value of real-time fluorescence quantitative PCR of carrying out for probe with ASPV-Probe3 builds typical curve;
3), be template for the cDNA of examination material, according to step 2) identical amplification condition carries out real-time fluorescence quantitative PCR, the Ct value obtained and described typical curve are compared, and realize the detection by quantitative that confession tries material apple stem pitting virus;
Wherein, described ASPV-cp-F
2, ASPV-cp-R
2, ASPV-Probe3-F, ASPV-Probe3-R and ASPV-Probe3 nucleotide sequence respectively as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5.
2. apple stem pitting virus real-time fluorescence quantitative PCR detection method according to claim 1, is characterized in that, in step 1) in: the preparation method of the cDNA of described contamination material comprises:
Extract the total serum IgE infecting and have apple stem pitting virus material, and carry out reverse transcription with this RNA for template, obtain the cDNA of contamination material.
3. apple stem pitting virus real-time fluorescence quantitative PCR detection method according to claim 1, is characterized in that, in step 1) in, the concentration of sepharose used in described agarose gel electrophoresis is 1-2%.
4. the apple stem pitting virus real-time fluorescence quantitative PCR detection method according to any one of claim 1-3, is characterized in that, in step 1) in:
Described carrier is pEASY-T
3, described competent cell is E.coli DH5 α.
5. apple stem pitting virus real-time fluorescence quantitative PCR detection method according to claim 4, is characterized in that, in step 2) in:
The OD of described positive recombinant plasmid standard substance
260/ OD
280ratio is between 1.8-2.0.
6. apple stem pitting virus real-time fluorescence quantitative PCR detection method according to claim 5, is characterized in that, in step 2) in:
The copy number concentration range of described positive recombinant plasmid standard substance is: 1.0 × 10
8copy/μ L ~ 1.0 × 10
1copy/μ L, and the multiple of described gradient dilution is 10 times.
7. apple stem pitting virus real-time fluorescence quantitative PCR detection method according to claim 6, is characterized in that, in step 2) in:
Described typical curve with the logarithmic value of the copy number concentration of described positive recombinant plasmid standard substance for X-coordinate, with described Ct value for ordinate zou.
8. apple stem pitting virus real-time fluorescence quantitative PCR detection method according to claim 7, is characterized in that, in step 2) in, in the process of described real-time fluorescence quantitative PCR, reaction system is:
TransStart Probe qPCR Super Mix 10 μ L, ASPV-Probe3-F and ASPV-Probe3-R 4-5pmol, ASPV-Probe34-5pmol, template 1.0 μ L, distilled water polishing to 20 μ L.
9. apple stem pitting virus real-time fluorescence quantitative PCR detection method according to claim 8, is characterized in that, in step 2) in, in the process of described real-time fluorescence quantitative PCR, amplification program is:
94 DEG C of denaturation 30-35s; 94 DEG C of sex change 4-7s, 55 DEG C of annealing 12-15s, 72 DEG C extend 15-20s, 35-40 circulation altogether.
10. apple stem pitting virus real-time fluorescence quantitative PCR detection method according to claim 8, is characterized in that, described real-time fluorescence quantitative PCR adopts Bio-Rad iQ
tM5 real-time fluorescence quantitative PCR instrument carry out.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106957915A (en) * | 2017-04-13 | 2017-07-18 | 河北农业大学 | Detection primer, kit and the quantitative detecting method of apple tree/Fungus of Pear Canker Disease bacterium |
| CN111172322A (en) * | 2020-01-08 | 2020-05-19 | 中国农业大学 | Real-time fluorescent quantitative PCR detection method for four pear virus pathogens |
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| CN104164517A (en) * | 2014-07-21 | 2014-11-26 | 中国农业大学 | Multiple RT-PCR detection method for apple latent viruses and viroid |
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| CN104164517A (en) * | 2014-07-21 | 2014-11-26 | 中国农业大学 | Multiple RT-PCR detection method for apple latent viruses and viroid |
Non-Patent Citations (1)
| Title |
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| 王鹏: "苹果和桃中3种潜隐性病毒的Real-Time RT-PCR检测", 《吉林农业大学硕士学位论文》, no. 2, 15 December 2013 (2013-12-15) * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106957915A (en) * | 2017-04-13 | 2017-07-18 | 河北农业大学 | Detection primer, kit and the quantitative detecting method of apple tree/Fungus of Pear Canker Disease bacterium |
| CN106957915B (en) * | 2017-04-13 | 2020-09-29 | 河北农业大学 | Primer and kit for detecting apple tree/pear tree rot pathogen and quantitative detection method |
| CN111172322A (en) * | 2020-01-08 | 2020-05-19 | 中国农业大学 | Real-time fluorescent quantitative PCR detection method for four pear virus pathogens |
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