CN103820561B - A kind of Citrus Huanglongbing pathogen Asia kind Chao Shi pcr amplification detection system and application - Google Patents

A kind of Citrus Huanglongbing pathogen Asia kind Chao Shi pcr amplification detection system and application Download PDF

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CN103820561B
CN103820561B CN201410085126.3A CN201410085126A CN103820561B CN 103820561 B CN103820561 B CN 103820561B CN 201410085126 A CN201410085126 A CN 201410085126A CN 103820561 B CN103820561 B CN 103820561B
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primer
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round pcr
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黄宏明
廖惠红
邢永秀
王茜
陈香玲
陈东奎
赵小龙
徐宁
方仁
李鸿莉
刘要鑫
李果果
欧智涛
张兰
黄其椿
赵洪涛
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Guangxi University
Horticultural Research Institute of Guangxi Academy of Agricultural Sciences
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Abstract

The present invention relates to a kind of Citrus Huanglongbing pathogen Asia kind PCR to detect, particularly relate to a kind of Citrus Huanglongbing pathogen Asia kind Chao Shi PCR detection system and application, belong to molecular biological arts.A kind of Citrus Huanglongbing pathogen Asia provided by the invention kind Chao Shi pcr amplification detection system, comprise first round PCR amplification system and second and take turns PCR amplification system, described first round PCR amplification system and second is taken turns PCR amplification system final volume and is 10 ~ 20 μ L, wherein include DNA profiling 1.0 μ L, 2 × Taq? PCR? Master? Mix5 μ L, primer 0.5 μ L, surplus is sterilizing distilled water; Wherein the second DNA profiling of taking turns PCR amplification system is obtained after diluting 10 ~ 20 times of volumes by first round pcr amplification product.Take turns that pcr amplification reaction system is little the first round of the present invention and second, first round PCR primer extension rate is little, and detect required reagent also few, corresponding expense is few.

Description

A kind of Citrus Huanglongbing pathogen Asia kind Chao Shi pcr amplification detection system and application
Technical field
The present invention relates to a kind of Citrus Huanglongbing pathogen Asia kind PCR to detect, particularly relate to a kind of Citrus Huanglongbing pathogen Asia kind Chao Shi PCR detection system and application, belong to molecular biological arts.
Background technology
At present, Chao Shi PCR(Nested-PCR) be relatively more conventional a kind of detection method of Citrus Huanglongbing pathogen, there is higher than Standard PCR sensitivity, that specificity is stronger advantage.Nest-type PRC reaction utilizes two cover PCR primer to carry out two-wheeled pcr amplification reaction, and in first round amplification, overcoat primer is in order to produce amplified production, and amplification products therefrom carries out second and takes turns amplification under the existence of embedded primer.
Although nest-type PRC reaction adds the complicacy of amplified reaction but reduces the possibility of multiple target site that increases, adds the specificity of detection, but all ingredients needed for the kind cause of disease PCR detection reaction system of Citrus Huanglongbing pathogen Asia is expensive, the expense that needs are born is comparatively large, therefore adopts little system as far as possible.When taking turns the template of PCR using first round PCR primer as second, wherein can be divided into again two kinds of approach:
(1) first round PCR primer through agarose gel electrophoresis, cut glue and put-20 DEG C and spend the night, the centrifugal 10min of 12000rpm, get supernatant liquor and do template and reclaim or directly film rubber tapping direct purification is carried out second again and take turns PCR.This kind of method cuts glue with a large amount of, and glue has shredded and added TE again, multigelation centrifuging and taking supernatant, and the rate of recovery is very low, loses a lot of DNA in process; Or need to prepare the equipment such as pillar and other reagent separately, complex steps, time-consuming.
(2) using the template of taking turns PCR after first round PCR primer dilution as second, at primer, ddH 2o, 2 × TaqPCRMasterMix(be not containing dyestuff) under the fixing prerequisite of consumption, the extension rate of first round PCR primer just becomes the deciding factor of whole Chao Shi PCR final effect.At present, dilute volume multiple and be often 50 ~ 10000 times.The most frequently used dilution volume multiple is 50 times, 100 times, 1000 times, 10000 times.For preventing taking turns RNA interference in PCR process second, to reach better effect, general RNase-freewater or 0.1%DEPC that use dilutes, and these two kinds of reagent prices are high.RNase-freewater price is 48 yuan/100ml(Beijing CoWin Bioscience Co., Ltd.), 0.1%DEPC working fluid price is 100 yuan/100ml(Beijing DingGuo ChangSheng Biology Technology Co., Ltd).Therefore, using Chao Shi round pcr to detect in the process of Citrus Huanglongbing pathogen, although highly diluted multiple easily realizes final detected result, extension rate is higher, the working fluid of required RNase-freewater or 0.1%DEPC is more, also just means more energy consumption.If first round PCR primer extension rate significantly can be reduced and finally can meet the requirement of Chao Shi round pcr, so not only save reagent dosage, the object of energy efficient can also be reached, because in the process making the reagent such as DEPC, could use after generally all using high temperature (120 DEG C) autoclaving process.
Summary of the invention
The object of this invention is to provide a kind of Citrus Huanglongbing pathogen Asia kind Chao Shi pcr amplification detection system and application.
Object of the present invention is achieved through the following technical solutions:
A kind of Citrus Huanglongbing pathogen Asia kind Chao Shi pcr amplification detection system, comprise first round PCR amplification system and second and take turns PCR amplification system, described first round PCR amplification system and second is taken turns PCR amplification system final volume and is 10 ~ 20 μ L, wherein include DNA profiling 1.0 μ L, 2 × TaqPCRMasterMix5 μ L, primer 0.5 μ L, surplus is sterilizing distilled water; It is obtained after wherein the second DNA profiling of taking turns PCR amplification system dilutes 10 ~ 20 times of volumes by first round pcr amplification product.
Preferably: the DNA profiling concentration of described first round PCR amplification system is 2.75ng/ μ l.
Preferably: 2 described × TaqPCRMasterMix containing dyestuff, is not dATP, dCTP, dGTP, dTTP of 0.4mmol/L comprising concentration; Concentration is the Mgcl of 4mmol/L 2; Total concn is Taq enzyme and the PfuDNA polysaccharase of 0.05U/ μ L.
Preferably: described primer comprises upstream primer and downstream primer, described upstream primer and downstream primer concentration are 5 μm of ol/L, and volume ratio is 1:1.
Above-mentioned Citrus Huanglongbing pathogen Asia kind Chao Shi pcr amplification detection system is utilized to carry out pcr amplification reaction, this amplified reaction comprises first round pcr amplification and second and takes turns pcr amplification, second DNA profiling of taking turns pcr amplification is obtained first round pcr amplification product dilution, first round pcr amplification reaction condition is 94 DEG C of denaturation 4min, 94 DEG C of sex change 30sec, 58 DEG C of annealing 45sec, 72 DEG C extend 45sec, 35 circulations, 72 DEG C finally extend 7min; Second to take turns pcr amplification reaction condition be 94 DEG C of denaturation 4min, and 94 DEG C of sex change 45sec, 55 DEG C of annealing 45sec, 72 DEG C extend 45sec, and 35 circulations, 72 DEG C finally extend 7min.
Beneficial effect of the present invention:
(1) first round and second takes turns pcr amplification reaction system is 10 ~ 20 μ L, and reaction system is less, and detect required amount of reagent also few, corresponding expense is few.
(2) will take turns the template of PCR after first round PCR primer dilution 10 ~ 20 times of volumes as second, extension rate is less, and detect required reagent also few, corresponding expense is few.
(3) the present invention uses amount of reagent few, can also alleviate experiment waste to the pollution of environment.
(4) the present invention uses amount of reagent few, make to detect on a large scale Citrus Huanglongbing pathogen Asia kind to detect application on a large scale and become possibility, integrated control for Citrus Huanglongbing pathogen provides solid technical support, and promote Citrus Industry and develop in a healthy way, the social benefit scale that it causes is immeasurable.
Accompanying drawing explanation
To be 7 granulated sugar tangerine leaf samples carry out repeating second for three times through first round pcr amplification reaction products therefrom through three kinds of concentration DNA profilings that 10 times, 20 times, 100 times volume dilution obtain to Fig. 1 takes turns pcr amplification reaction result;
M swimming lane representation DNA markerDM2000;
Swimming lane 1-7 is representative sample AT5-2, AT5-4, AT5-49, AT5-55, AT5-71, AT5-84, AT5-141 respectively; A, B, C be representative sample 10 times, the diluent of 20 times, 100 times respectively; I, II, III three revision tests are represented.
Embodiment
Below in conjunction with specific embodiment, elaboration detailed is further done to the present invention, but embodiments of the present invention are not limited to the scope that embodiment represents.These embodiments only for illustration of the present invention, but not for limiting the scope of the invention.In addition, after reading content of the present invention, those skilled in the art can do various amendment to the present invention, and these equivalent variations fall within appended claims limited range of the present invention equally.
Embodiment 1
One, experiment material
1, vegetable material
7 doubtful yellow twig blade materials of granulated sugar tangerine, all pick up from Guangxi, be numbered AT5-2, AT5-4, AT5-49, AT5-55, AT5-71, AT5-84, AT5-141 respectively, wherein, AT5-2, AT5-4 pick up from Wuming County, and AT5-49, AT5-55 pick up from Cenxi county, and AT5-71 picks up from Fangchenggang City, AT5-84 picks up from Longan County, and AT5-141 picks up from Xilin County.
2, primer
According to the 16SrDNA sequence in Citrus Huanglongbing pathogen Asia kind (CandidantusLiberibacterasiaticus), design primer fd1/fd2(SEQIDNO:1/SEQIDNO:2), OI1/OI2(SEQIDNO:3/SEQIDNO:4), object nucleic acid fragment length is 1160bp, synthesized by Shanghai Jierui Biology Engineering Co., Ltd, primer sequence is as follows:
fd 1：5′-AGAGTTTGATCCTGGCTCAG-3；
fd 2：5′-AAGGAGGTGATCCAGCC-3′。
OI 1：5′-GCGCGTATGCAATACGAGCGGCA-3′；
OI 2：5′-GCCTCGCGACTTCGCAACCCAT-3′。
3, genome DNA extracting reagent kit
Fast-type plant genome DNA extracts cover assembling system, and test kit is purchased from TIANGEN Biotech (Beijing) Co., Ltd., and suit comprises FP 1and FP 2two kinds of damping fluids.
4,2 × TaqPCRMasterMix(is not containing dyestuff), be century (version number: 01182013 of biotechnology (Beijing) company limited purchased from health; Catalog number (Cat.No.): CW0716).
5, RNaseA(10mg/ml), Virahol, ethanol, damping fluid TE, sterilizing distilled water (ddH 2o), RNase-freewater(is purchased from Beijing CoWin Bioscience Co., Ltd.) etc. reagent.
Two, experimental technique
1, DNA sample is extracted
(1) respectively arteries and veins in the doubtful yellow twig blade of clip 7 granulated sugar tangerines, every part of about 100g, by liquid nitrogen grinding to Powdered, it is stand-by to place-20 DEG C of preservations.
(2) get the product that 100mg step (1) is obtained, add the RNaseA(10mg/mL of 400 μ L damping fluid FP1 and 6 μ L), after vortex oscillation 1min, room temperature places 10min, and period gently overturns EP pipe 2 ~ 3 times.
(3) add 130 μ L damping fluid FP2, vortex oscillation 1min makes it fully mix.
(4) the centrifugal 5min of 12000r/min, transfer supernatant liquor is in new EP pipe.
(5) by supernatant liquor recentrifuge 5min, 12000r/min, transfer supernatant liquor is in new EP pipe.
(6) in supernatant liquor, add the Virahol of 0.7 times of volume of precooling, gently overturn EP pipe and make it mix, leave standstill a moment, the centrifugal 2min of 12000r/min, abandons supernatant.
(7) precipitation adds 700 μ L ethanol, and the centrifugal 2min of vortex oscillation 5s, 12000r/min, abandons supernatant.
(8) the 7th step is repeated.
(9) uncap inversion, room temperature 5 ~ 10min, thoroughly dries remaining ethanol.
(10) add appropriate elution buffer TE, put upside down mixing, make the DNA extraction liquid that concentration is 275ng/ μ l, it is stand-by to be placed in-20 DEG C of preservations.
2, pcr amplification reaction system and condition
Utilize the 16SrDNA primer fd in Citrus Huanglongbing pathogen Asia kind (CandidantusLiberibacterasiaticus) 1/ fd 2, OI 1/ OI 2pcr amplification detection is carried out to 7 parts of DNA sample.First round PCR amplification system: i.e. 1.0 μ lDNA templates (DNA extraction liquid is diluted 100 times of volumes, obtained concentration is the DNA profiling of 2.75ng/ μ l), 5 μ l2 × TaqPCRMasterMix, 0.5 μ lfd 1/ fd 2primer, adds ddH 2o mends to 10 μ l, 2 described × TaqPCRMasterMix not containing dyestuff, and wherein dATP, dCTP, dGTP, dTTP concentration is 0.4mmol/L; Mgcl2 concentration is 4mmol/L; Taq enzyme and PfuDNA polysaccharase total concn are 0.05U/ μ L, and described primer fd1, primer fd2 concentration are 5 μm of ol/L, and volume is 0.25 μ l.First round pcr amplification reaction program: 94 DEG C of denaturation 4min, 94 DEG C of sex change 30sec, 58 DEG C of annealing 45sec, 72 DEG C extend 45sec, 35 circulations, and 72 DEG C finally extend 7min, preserve product for 4 DEG C.Second takes turns PCR amplification system: getting first round PCR primer 1.0 μ l respectively dilutes 10 ×, 20 ×, 100 × volume, more therefrom get 1.0 μ l as template, 5 μ l2 × TaqPCRMasterMix, 0.5 μ lOI separately 1/ OI 2primer, adds ddH 2o mends to 10 μ l, described primer OI 1, primer OI 2concentration is 5 μm of ol/L, and volume is 0.25 μ l.Repeat second respectively for three times and take turns pcr amplification, second takes turns pcr amplification reaction program: 94 DEG C of denaturation 4min, 94 DEG C of sex change 45sec, 55 DEG C of annealing 30sec, and 72 DEG C extend 45sec, 35 circulations, and 72 DEG C finally extend 7min, preserve product for 4 DEG C.
3, sepharose electrophoresis detection pcr amplification product
Get 3 μ l6 × Loadingbuffer and second and take turns PCR primer 6.0 μ LDNA sample mix evenly rear loading, with the agarose gel electrophoresis of 1%, deposition condition is: 1 × TAE electrophoretic buffer, voltage 150V, 15min, after electrophoresis terminates, observe under FIREREADERXSD-56-26.M gel imaging system and take pictures.
Three, experimental result
Electrophoresis result display in Fig. 1, the DNA profiling that first round pcr amplification reaction product obtains after diluting 10,20,100 times of volumes all can amplify goal gene.
Embodiment 2
After respectively 7 parts of DNA extraction liquid that embodiment 1 is obtained being diluted 100 times of volumes, obtained concentration is that the DNA profiling of 2.75ng/ μ l is for subsequent use.
Utilize the 16SrDNA primer fd in Citrus Huanglongbing pathogen Asia kind (CandidantusLiberibacterasiaticus) 1/ fd 2, OI 1/ OI 2pcr amplification detection is carried out to 7 parts of DNA sample.First round PCR amplification system: i.e. 1.0 μ lDNA templates (concentration is 2.75ng/ μ l), 5 μ l2 × TaqPCRMasterMix, 0.5 μ lfd 1/ fd 2primer, adds ddH 2o mends to 15 μ l, 2 described × TaqPCRMasterMix not containing dyestuff, and wherein dATP, dCTP, dGTP, dTTP concentration is 0.4mmol/L; Mgcl 2concentration is 4mmol/L; Taq enzyme and PfuDNA polysaccharase total concn are 0.05U/ μ L, described primer fd 1, primer fd 2concentration is 5 μm of ol/L, and volume is 0.25 μ l.First round pcr amplification reaction program: 94 DEG C of denaturation 4min, 94 DEG C of sex change 30sec, 58 DEG C of annealing 45sec, 72 DEG C extend 45sec, 35 circulations, and 72 DEG C finally extend 7min, preserve product for 4 DEG C.Second takes turns PCR amplification system: get first round PCR primer 1.0 μ l RNase-freewater respectively and dilute 15 times of volumes, more therefrom gets 1.0 μ l as template, 5 μ l2 × TaqPCRMasterMix, 0.5 μ lOI separately 1/ OI 2primer, adds ddH 2o mends to 15 μ l, described primer OI 1, primer OI 2concentration is 5 μm of ol/L, and volume is 0.25 μ l.Repeat second respectively for three times and take turns pcr amplification, second takes turns pcr amplification reaction program: 94 DEG C of denaturation 4min, 94 DEG C of sex change 45sec, 55 DEG C of annealing 30sec, and 72 DEG C extend 45sec, 35 circulations, and 72 DEG C finally extend 7min, preserve product for 4 DEG C.
The sepharose deposition condition identical with embodiment 1 is adopted to carry out pcr amplification product detection.
Embodiment 3
After respectively 7 parts of DNA extraction liquid that embodiment 1 is obtained being diluted 100 times of volumes, obtained concentration is that the DNA profiling of 2.75ng/ μ l is for subsequent use.
Utilize the 16SrDNA primer fd in Citrus Huanglongbing pathogen Asia kind (CandidantusLiberibacterasiaticus) 1/ fd 2, OI 1/ OI 2pcr amplification detection is carried out to 7 parts of DNA sample.First round PCR amplification system: i.e. 1.0 μ lDNA templates (concentration is 2.75ng/ μ l), 5 μ l2 × TaqPCRMasterMix, 0.5 μ lfd 1/ fd 2primer, adds ddH 2o mends to 20 μ l, 2 described × TaqPCRMasterMix not containing dyestuff, and wherein dATP, dCTP, dGTP, dTTP concentration is 0.4mmol/L; Mgcl 2concentration is 4mmol/L; Taq enzyme and PfuDNA polysaccharase total concn are 0.05U/ μ L, described primer fd 1, primer fd 2concentration is 5 μm of ol/L, and volume is 0.25 μ l.First round pcr amplification reaction program: 94 DEG C of denaturation 4min, 94 DEG C of sex change 30sec, 58 DEG C of annealing 45sec, 72 DEG C extend 45sec, 35 circulations, and 72 DEG C finally extend 7min, preserve product for 4 DEG C.Second takes turns PCR amplification system: get first round PCR primer 1.0 μ l respectively and dilute 20 volumes, more therefrom gets 1.0 μ l as template, 5 μ l2 × TaqPCRMasterMix, 0.5 μ lOI separately 1/ OI 2primer, adds ddH 2o mends to 20 μ l, described primer OI 1, primer OI 2concentration is 5 μm of ol/L, and volume is 0.25 μ l.Repeat second respectively for three times and take turns pcr amplification, second takes turns pcr amplification reaction program: 94 DEG C of denaturation 4min, 94 DEG C of sex change 45sec, 55 DEG C of annealing 30sec, and 72 DEG C extend 45sec, 35 circulations, and 72 DEG C finally extend 7min, preserve product for 4 DEG C.
The sepharose deposition condition identical with embodiment 1 is adopted to carry out pcr amplification product detection.

Claims (2)

1. a Citrus Huanglongbing pathogen Asia kind Chao Shi pcr amplification detection system, comprise first round PCR amplification system and second and take turns PCR amplification system, it is characterized in that: described first round PCR amplification system and second is taken turns PCR amplification system final volume and is 10 ~ 20 μ L, wherein include DNA profiling 1.0 μ L, 2 × TaqPCRMasterMix5 μ L, primer 0.5 μ L, surplus is sterilizing distilled water; It is obtained after wherein the second DNA profiling of taking turns PCR amplification system dilutes 10 ~ 20 times of volumes by first round pcr amplification product;
The DNA profiling concentration 2.75ng/ μ L of described first round PCR amplification system;
2 described × TaqPCRMasterMix, it is by dATP, dCTP, dGTP, dTTP, Mgcl 2, Taq enzyme and PfuDNA polysaccharase composition; DATP, dCTP, dGTP, dTTP of 0.4mmol/L is comprising concentration; Concentration is the Mgcl of 4mmol/L 2; Total concn is Taq enzyme and the PfuDNA polysaccharase of 0.05U/ μ L;
Described primer comprises upstream primer and downstream primer, and described upstream primer is fd 1: 5 '-AGAGTTTGATCCTGGCTCAG-3; Fd 2: 5 '-AAGGAGGTGATCCAGCC-3 '; Described downstream primer is OI 1: 5 '-GCGCGTATGCAATACGAGCGGCA-3 '; OI 2: 5 '-GCCTCGCGACTTCGCAACCCAT-3 '; Described upstream primer and downstream primer concentration are 5 μm of ol/L, and volume ratio is 1:1.
2. utilize the Citrus Huanglongbing pathogen Asia kind Chao Shi pcr amplification detection system described in claim 1 to carry out pcr amplification reaction, it is characterized in that: this amplified reaction comprises first round pcr amplification and second and takes turns pcr amplification, second DNA profiling of taking turns pcr amplification is obtained first round pcr amplification product dilution, first round pcr amplification reaction condition is 94 DEG C of denaturation 4min, 94 DEG C of sex change 30sec, 58 DEG C of annealing 45sec, 72 DEG C extend 45sec, 35 circulations, 72 DEG C finally extend 7min; Second to take turns pcr amplification reaction condition be 94 DEG C of denaturation 4min, and 94 DEG C of sex change 45sec, 55 DEG C of annealing 45sec, 72 DEG C extend 45sec, and 35 circulations, 72 DEG C finally extend 7min.
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EP3643788A4 (en) * 2017-06-20 2021-01-06 MGI Tech Co., Ltd. Pcr primer pair and application thereof
EP3643787A4 (en) * 2017-06-20 2021-01-06 MGI Tech Co., Ltd. Pcr primer pair and application thereof
EP3643789A4 (en) * 2017-06-20 2021-01-06 MGI Tech Co., Ltd. Pcr primer pair and application thereof

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