CN107012245A - The dizzy epidemic disease bacterium real-time fluorescent PCR reagent case of one kind detection Kidney bean and its detection method - Google Patents

The dizzy epidemic disease bacterium real-time fluorescent PCR reagent case of one kind detection Kidney bean and its detection method Download PDF

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CN107012245A
CN107012245A CN201710331889.5A CN201710331889A CN107012245A CN 107012245 A CN107012245 A CN 107012245A CN 201710331889 A CN201710331889 A CN 201710331889A CN 107012245 A CN107012245 A CN 107012245A
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kidney bean
epidemic disease
disease bacterium
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pcr
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CN107012245B (en
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尤佳
王溪桥
王军平
文朝慧
满岳
周小平
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Inspection & Quarantine Technology Center Of Gansu Entry-Exit Inspection & Quarantine Bureau
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Abstract

It is used to detect the dizzy epidemic disease bacterium Taqman Real Time PCR kits of Kidney bean and its detection method the invention provides one kind, kit includes amplification premixed liquid, fluorescence correction liquid, positive control, negative control, upstream and downstream primer, probe, RNase free ddH2O.This kit utilizes special amplimer and Taqman probes, using Real Time round pcrs, carries out qualitative analysis to measuring samples by Ct values, as a result accurate and visual, time saving and energy saving, detection time is shortened to 50 minutes；And the sensitivity than regular-PCR is high, available for the dizzy epidemic disease bacterium sample of Kidney bean for detecting humble content, detection DNA threshold value reaches 180 fg/ μ l；Reagent consumables cost is relatively low, it is adaptable to the detection research of entry and exit port quarantine departments mass detection business and scientific research institutions.

Description

The dizzy epidemic disease bacterium real-time fluorescent PCR reagent case of one kind detection Kidney bean and its detection method
Technical field
It is specifically a kind of to be used to detect the dizzy epidemic disease bacterium Taqman of Kidney bean the present invention relates to microorganism detection field Real-Time PCR kits and its detection method.
Background technology
Kidney bean swoon epidemic disease bacterium (Pseudomonas savastanoiPv.phaseolicola) be China inward plant Quarantine harmful organisms.Its host range is mainly legume, including soybean, Kidney bean, pea, mung bean etc..Invaded by the germ The dizzy epidemic disease of beans caused by dye has generation in Europe, Asia, America, Africa, Oceania, legume crop can be caused seriously to damage Lose, even have no harvest sometimes.The dizzy epidemic disease bacterium of Kidney bean mainly makees long-distance communications by infected seed, and the optimum growth temperature of germ is 25-30 DEG C, Chinese each legume producing region is provided with its condition colonized.This germ once incoming China, will be to China Soybean Industry is produced and had a strong impact on.In recent years, with the growth of domestic consumption demand, China's imported soybean amount is in blowout situation, Import volume in 2016 is even more to reach 83,910,000 tons.External soybean main production country majority is that Kidney bean is swooned the epidemic-stricken area of epidemic disease bacterium, therefore the disease Bacterium is very risky with the incoming China of import legume material.
The dizzy epidemic disease bacterium detection method of Kidney bean includes Physiology and biochemistry method, fatty acid analysis method, pathogenic test combination slide and coagulated Collect experiment, ELISA etc., but these conventional method qualification times are long, and sensitivity is not high, therefore accelerates to make import pulse family The Fast Detection Technique research of the dizzy epidemic disease bacterium of Kidney bean is carried in thing, it has also become the problem of current more and more in the urgent need to address.
TaqMan Real-time PCR are the probes that a fluorescence labeling is added on the basis of regular-PCR method, The probe is an oligonucleotides, two ends one reporter fluorescence group of mark and a quenching fluorescence group respectively.When probe is complete, The fluorescence signal of reporter group transmitting is quenched group absorptions；When PCR is expanded, the 5'-3' 5 prime excision enzyme activities of Taq enzyme are by probe enzyme Degraded is cut, reporter fluorescence group and quenching fluorescence group is separated, so that fluorescence monitoring system can receive fluorescence signal, i.e., it is every A DNA is expanded, just has a fluorescence molecule to be formed, accumulation and the PCR primer formation Complete Synchronization of fluorescence signal is realized. Accumulated using fluorescence signal, whole PCR processes are monitored in real time, whole process is only needed one hour, common molecular life is eliminated in addition Thing various disadvantages.Both at home and abroad there is not yet the report that the dizzy epidemic disease bacterium real-time fluorescence method of quarantine harmful organisms Kidney bean is set up.
Exploitation is strictly quarantined suitable for the quick diagnosis technology of port quarantine to the dizzy epidemic disease bacterium of Kidney bean, to preventing such Pathogen it is incoming most important.However, the detection method of authority is the China of national quality supervision and inspection general bureau issue at present People's republic's inspection and quarantining for import/export professional standard（SN/T1586.2-2008）Wrapped in the dizzy epidemic disease bacterium quarantine identification method of Kidney bean Include：Physiological and biochemical index detection, pathogenic detection, common molecular method.The detection of biochemical indicator is wherein carried out to suspicious bacterial strain At least four must be completed：Contact enzyme positive, oxidase negative, PEARLITOL 25C feminine gender, glutarate feminine gender and exclude suspicious bacterium colony Next step experiment is carried out again, and experimental period is long；Pathogenic detection can not often make auxiliary identification of means separately as detection method； The sensitivity of regular-PCR method is general, and needs into performing PCR amplification, two steps of agarose gel electrophoresis, and the time is at least 3 Hour, the nucleic acid dye pollution environment used in electrophoresis experiment, with high carcinogenic, there is certain influence on health.In addition, For other phytopathogens, the primer that the conventional 16S rDNA universal primers in laboratory can be used as conventional PCR method enters performing PCR Amplification is sequenced to identify, but 16S rDNA universal primers are directed to the specific extreme difference of Bacteria Identification under Pseudomonas, it is difficult to identify To kind.Therefore, for port quarantine, daily import and export detection portfolio is big, and bacterial strain effect is identified not using commonsense method Good, detection efficiency is low, meanwhile, detection cycle length can increase importers or exporters's operation cost.
It is used to detect the dizzy epidemic disease bacterium Taqman Real-Time PCR examinations of Kidney bean therefore, the present patent application aims to provide one kind Agent box and its detection method.
The content of the invention
In view of the deficiencies in the prior art, are used to detect the dizzy epidemic disease bacterium Taqman Real- of Kidney bean the invention provides one kind Time PCR kits and its detection method, using special amplimer and Taqman probes, using Real-Time PCR skills Measuring samples are carried out the dizzy epidemic disease bacterium qualitative analysis of Kidney bean by art by Ct values.
In order to achieve the above object, present invention employs following technical scheme：
One kind is used to detect the dizzy epidemic disease bacterium Taqman Real-Time PCR kits of Kidney bean, including reagent box main body and such as table 1 Listed reagent；Wherein reagent box main body includes：Box body, the box body is hollow, includes freezing liquid；Reagent is placed in box；In box body Side is provided with lid；Box body side is provided with the DNA that testing sample is placed in drawer, drawer.Kit side is additionally provided with narrow meshed Cavity, cavity is separated with freezing liquid in kit using flexible plastic sheet, volumetric expansion when freezing liquid solidifies, and plastic sheet is to cavity Curving, extenuates the pressure of expansion, so as to avoid kit from being burst.
Reagent contained by the kit of table 1
Include reagent Specification
PCR Forward Primer 2OD
PCR Reverse Primer 2OD
Taqman Probe 2OD
Expand premixed liquid 1ml
RNase-free ddH2O 1.5ml
Fluorescence correction liquid 1ml
Positive control 100μl
Negative control 100μl
Preferably, it is a kind of to be used to detect PCR Forward in the dizzy epidemic disease bacterium Taqman Real-Time PCR kits of Kidney bean Primer sequences are：5’-GTGGGTACATCTGGAATC -3’；Obtained using the dizzy epidemic disease bacterium argK genes of Kidney bean as stencil design.
Preferably, it is a kind of to be used to detect PCR in the dizzy epidemic disease bacterium Taqman Real-Time PCR kits of Kidney bean Reverse Primer sequences are：5’-CTGACATCCTCCGTATTG -3’；Set using the dizzy epidemic disease bacterium argK genes of Kidney bean as template Meter is obtained.
Preferably, it is a kind of to be used to detect Taqman in the dizzy epidemic disease bacterium Taqman Real-Time PCR kits of Kidney bean Probe sequences are：5’-(FAM) ATCACCGTCGCACCATACGA (TAMRA)-3’；Using Kidney bean swoon epidemic disease bacterium argK genes as Stencil design is obtained.
Preferably, it is a kind of to expand premixed liquid for detecting in the dizzy epidemic disease bacterium Taqman Real-Time PCR kits of Kidney bean Including：Archaeal dna polymerase, 2 × PCR buffer solutions（500mM/L KCl、100 mM/L Tris-HCl、150mM/L MgCl2、1mg/ Ml gelatin）, dNTP stock solutions, RNase H.
Preferably, it is a kind of to be used to detect fluorescence correction liquid in the dizzy epidemic disease bacterium Taqman Real-Time PCR kits of Kidney bean Including：ROX fluorescent dyes.
Preferably, it is a kind of to be used to detect that the positives control of the dizzy epidemic disease bacterium Taqman Real-Time PCR kits of Kidney bean is adopted With the DNA of the dizzy epidemic disease bacterium reference culture institute extraction purification of Kidney bean.
Preferably, it is a kind of to be used to detect that negative control to be adopted in the dizzy epidemic disease bacterium Taqman Real-Time PCR kits of Kidney bean With healthy soya seeds extraction purification DNA.
Preferably, it is a kind of to be used to detect Kidney bean -20 DEG C of guarantors of dizzy epidemic disease bacterium Taqman Real-Time PCR kits use Deposit, it is closed to preserve, prevent pollution.
One kind is used to detect the dizzy epidemic disease bacterium Taqman Real-Time PCR detection methods of Kidney bean, including following operation step Suddenly：
1st, probe and primer dilution；
2nd, PCR reaction solutions are configured；
3rd, Real-time PCR reactions are carried out；
4th, analysis of experimental results.
Preferably, it is a kind of to be used to detect the dizzy epidemic disease bacterium Taqman Real-Time PCR detection methods of Kidney bean, wherein step 1 Probe and primer dilution include：Probe and primer centrifuge 5min before uncapping, and then according to nmol numbers, use RNase-free ddH2It is X μM of Oligo solution that O, which is configured to concentration,.Compound concentration is that X μM of Oligo solution adds RNase-free ddH2O bodies Product：
V (ml)=nmol numbers/X
Preferably, a kind of to be used to detect the dizzy epidemic disease bacterium Taqman Real-Time PCR detection methods of Kidney bean, wherein step 2 is configured Prepared during PCR reaction solutions by the methods described of table 2：
The compound method of table 2PCR reaction solutions
Reagent Usage amount
Expand premixed liquid 10 μl
PCR Forward Primer(10μM) 0.4 μl
PCR Reverse Primer(10μM) 0.4 μl
Taqman Probe 0.2 μl
RNase-free ddH2O 6.6 μl
DNA 2μl
Fluorescence correction liquid 0.4μl
Total 20 μl
Preferably, a kind of to be used to detect the dizzy epidemic disease bacterium Taqman Real-Time PCR detection methods of Kidney bean, wherein step 3 is carried out Two-step method PCR amplifications standardization program, specifically includes following steps when Real-time PCR react：
Hold Stage:Pre-degeneration
Reps:1
95 DEG C 30 seconds
PCR Stage:PCR reacts
Reps：40
95 DEG C 5 seconds
60 DEG C 30 seconds.
Preferably, it is a kind of to be used to detect the dizzy epidemic disease bacterium Taqman Real-Time PCR detection methods of Kidney bean, wherein step 4 The specific method of analysis of experimental results is：
Real-time PCR reaction amplification curves and CT values are confirmed after the completion of reaction.
If 2 Duplicate Samples testing result CT >=40 of testing sample, simultaneously there is typical case in positive control detection 20≤CT≤36 Amplification curve, negative control and blank control result be normal, now can be determined that the sample does not detect Kidney bean and swooned epidemic disease bacterium.
If 2 Duplicate Samples testing result CT≤36 of testing sample, simultaneously there is typical case in positive control detection 20≤CT≤36 Amplification curve, negative control and blank control result be normal, now can be determined that sample detection Kidney bean is swooned epidemic disease bacterium.
If simultaneously there is typical amplification curve, positive control in the ＜ CT ＜ 40 of 2 Duplicate Samples testing results of testing sample 36 Simultaneously there is typical amplification curve in detection 20≤CT≤36, and negative control and blank control result are normal, now should suitably increase Real-time PCR detection is reformed after DNA profiling amount.
Preferably, a kind of to be used to detect the dizzy epidemic disease bacterium Taqman Real-Time PCR detection methods of Kidney bean, this method is removed Above-mentioned detection Kidney bean is needed to swoon outside epidemic disease bacterium Taqman Real-Time PCR kits, in addition it is also necessary to which Real-time PCR are expanded Instrument, autoclaving centrifuge tube and micropipette rifle and autoclaving pipette tips.
Beneficial effect：There is toxin resistance encoding gene argK in the dizzy epidemic disease bacterium of Kidney bean, the gene exists only in Kidney bean in itself Dizzy epidemic disease bacterium and Pseudomonas syringae pv.actinidiae (P.syringaePv.actinidiae) in two kinds of bacteriums；Adopt There is the specificity of height with argK as the target gene of detection.And conventional method is used as detection using 16S rDNA universal primers Target gene, it is low for cloves Pseudomonas specificity, at least 23 strains are retrieved in genebank paired, it is difficult to reflect Fixed arrive is planted.The present patent application designs Taqman probes and amplimer, and optimization system obtains Optimal system configuration and amplification program Condition, is produced based on the dizzy epidemic disease bacterium Real-time PCR detection kits of Taqman probes Kidney bean, test of many times result is proved The kit can quick from strains tested, specifically detect that Kidney bean is swooned epidemic disease bacterium, and kit sensitivity in addition is far above Standard PCR, detection DNA threshold value reaches 180 fg/ μ L.
Being designed to further facilitate of kit has been provided with freezing liquid in test, kit, reagent and sample DNA are being surveyed Low temperature can be kept during examination, makes test result more accurate.
The present invention can greatly save the dizzy epidemic disease bacterium detection time cost of Kidney bean, and whole experiment is only needed 50 minutes；Detection is used Reagent asepsis environment-protecting.Test experience process operation is simple, has configured machine in system, full name is visual；Seminal plasma fructose detection kit consumes Material cost is low, it is adaptable to port quarantine department mass detection business；Kit sensitivity is far above Standard PCR, detection DNA's Threshold value reaches 180 fg/ μ L；Once there is positive findings in kit high specificity, sample, you can judge to contain China's quarantine Harmful organism Kidney bean is swooned epidemic disease bacterium.
Brief description of the drawings
Fig. 1 is a kind of amplification for being used to detect the dizzy epidemic disease bacterium Taqman Real-Time PCR detection methods of Kidney bean of the present invention Curve.
Fig. 2 is of the invention a kind of for detecting the dizzy epidemic disease bacterium Taqman Real-Time PCR detection method probes pair of Kidney bean The sensitivity technique amplification curve of the dizzy epidemic disease bacterium of Kidney bean.
Fig. 3 is of the invention a kind of for detecting the dizzy epidemic disease bacterium Taqman Real-Time PCR detection method positive bacterias of Kidney bean The pathogenic detection figure of strain.
Fig. 4 is of the invention a kind of for detecting the dizzy epidemic disease bacterium Taqman Real-Time PCR detection kits signal of Kidney bean Figure.
Fig. 5 is of the invention a kind of for detecting the dizzy epidemic disease bacterium Taqman Real-Time PCR detection kit tangent planes of Kidney bean Schematic diagram.
Embodiment
To make the purpose, technical scheme and advantage of invention clearer, below in conjunction with the accompanying drawings to the specific implementation of the present invention Mode is described in detail.The example of these preferred embodiments is illustrated in the accompanying drawings.Shown in accompanying drawing and according to attached What the embodiments of the present invention of figure description were merely exemplary, and the present invention is not limited to these embodiments.
Here, it should also be noted that, in order to avoid having obscured technical scheme because of unnecessary details, It illustrate only in accompanying drawing and according to the solution of the present invention closely related structure and/or process step, and eliminate relation not Big other details.
Embodiment 1
As shown in figs. 4 and 5, it is a kind of to be used to detect the dizzy epidemic disease bacterium Taqman Real-Time PCR kits of Kidney bean, including examination Agent box main body and as listed in table 3 reagent；Wherein reagent box main body includes：Box body 3, the box body 3 is hollow, includes freezing liquid 8；Box Interior placement reagent 2；Lid 1 is provided with above box body；The side of box body 3 is provided with the DNA 5 that testing sample is placed in drawer 4, drawer 4. Kit side is additionally provided with narrow meshed cavity 6, and cavity 6 is separated with freezing liquid in kit 8 using flexible plastic sheet 7, is freezed Volumetric expansion when liquid 8 solidifies, plastic sheet 7 extenuates the pressure of expansion to the curving of cavity 6, so as to avoid kit from being burst.
Reagent contained by the kit of table 3
Include reagent Specification
PCR Forward Primer 2OD
PCR Reverse Primer 2OD
Taqman Probe 2OD
Expand premixed liquid 1ml
RNase-free ddH2O 1.5ml
Fluorescence correction liquid 1ml
Positive control 100μl
Negative control 100μl
Further, it is a kind of to be used to detect PCR Forward in the dizzy epidemic disease bacterium Taqman Real-Time PCR kits of Kidney bean Primer sequences are：5’-GTGGGTACATCTGGAATC -3’.
Further, it is a kind of to be used to detect PCR in the dizzy epidemic disease bacterium Taqman Real-Time PCR kits of Kidney bean Reverse Primer sequences are：5’-CTGACATCCTCCGTATTG -3’.
Further, it is a kind of to be used to detect Taqman in the dizzy epidemic disease bacterium Taqman Real-Time PCR kits of Kidney bean Probe sequences are：5’-(FAM) ATCACCGTCGCACCATACGA (TAMRA)-3’.
Further, it is a kind of to be used to detect amplification premix in the dizzy epidemic disease bacterium Taqman Real-Time PCR kits of Kidney bean Liquid includes：Archaeal dna polymerase, 2 × PCR buffer solutions（500mM/L KCl、100 mM/L Tris-HCl、150mM/L MgCl2、 1mg/ml gelatin）, dNTP stock solutions, RNase H.
Further, it is a kind of to be used to detect fluorescence correction in the dizzy epidemic disease bacterium Taqman Real-Time PCR kits of Kidney bean Liquid includes：ROX fluorescent dyes.
Further, it is a kind of to be used to detect the positives control of the dizzy epidemic disease bacterium Taqman Real-Time PCR kits of Kidney bean Using the DNA of the dizzy epidemic disease bacterium reference culture institute extraction purification of Kidney bean.
Further, it is a kind of to be used to detect negative control in the dizzy epidemic disease bacterium Taqman Real-Time PCR kits of Kidney bean Using healthy soya seeds extraction purification DNA.
Further, it is a kind of to be used to detect dizzy -20 DEG C of the epidemic disease bacterium Taqman Real-Time PCR kits use of Kidney bean Preserve, it is closed to preserve, prevent pollution.
Embodiment 2
Present embodiments providing one kind is used to detect the dizzy epidemic disease bacterium Taqman Real-Time PCR detection methods of Kidney bean, including such as Lower operating procedure：
1st, probe and primer dilution；
2nd, PCR reaction solutions are configured；
3rd, Real-time PCR reactions are carried out；
4th, analysis of experimental results；
Preferably, it is a kind of to be used to detect the dizzy epidemic disease bacterium Taqman Real-Time PCR detection methods of Kidney bean, wherein step 1 probe And primer dilution includes：Probe and primer centrifuge 5min before uncapping, and then according to nmol numbers, use RNase-free ddH2O It is X μM of Oligo solution to be configured to concentration.Compound concentration is that X μM of Oligo solution adds RNase-free ddH2O volumes：
V (ml)=nmol numbers/X
Preferably, a kind of to be used to detect the dizzy epidemic disease bacterium Taqman Real-Time PCR detection methods of Kidney bean, wherein step 2 is configured Prepared during PCR reaction solutions by the methods described of table 4：
The compound method of table 4PCR reaction solutions
Reagent Usage amount
Expand premixed liquid 10 μl
PCR Forward Primer(10μM) 0.4 μl
PCR Reverse Primer(10μM) 0.4 μl
Taqman Probe 0.2 μl
RNase-free ddH2O 6.6 μl
DNA 2μl
Fluorescence correction liquid 0.4μl
Total 20 μl
Preferably, a kind of to be used to detect the dizzy epidemic disease bacterium Taqman Real-Time PCR detection methods of Kidney bean, wherein step 3 is carried out Two-step method PCR amplifications standardization program, specifically includes following steps when Real-time PCR react：
Hold Stage:Pre-degeneration
Reps:1
95 DEG C 30 seconds
PCR Stage:PCR reacts
Reps：40
95 DEG C 5 seconds
60 DEG C 30 seconds.
Preferably, it is a kind of to be used to detect the dizzy epidemic disease bacterium Taqman Real-Time PCR detection methods of Kidney bean, wherein step 4 The specific method of analysis of experimental results is：
Real-time PCR reaction amplification curves and CT values are confirmed after the completion of reaction.
If 2 Duplicate Samples testing result CT >=40 of testing sample, simultaneously there is typical case in positive control detection 20≤CT≤36 Amplification curve, negative control and blank control result be normal, now can be determined that the sample does not detect Kidney bean and swooned epidemic disease bacterium.
If 2 Duplicate Samples testing result CT≤36 of testing sample, simultaneously there is typical case in positive control detection 20≤CT≤36 Amplification curve, negative control and blank control result be normal, now can be determined that sample detection Kidney bean is swooned epidemic disease bacterium.
If simultaneously there is typical amplification curve, positive control in the ＜ CT ＜ 40 of 2 Duplicate Samples testing results of testing sample 36 Simultaneously there is typical amplification curve in detection 20≤CT≤36, and negative control and blank control result are normal, now should suitably increase Real-time PCR detection is reformed after DNA profiling amount.
Preferably, a kind of to be used to detect the dizzy epidemic disease bacterium Taqman Real-Time PCR detection methods of Kidney bean, this method is removed Above-mentioned detection Kidney bean is needed to swoon outside epidemic disease bacterium Taqman Real-Time PCR kits, in addition it is also necessary to which Real-time PCR are expanded Instrument, autoclaving centrifuge tube and micropipette rifle and autoclaving pipette tips.
Embodiment 3
A kind of specific method for being used to detect the dizzy epidemic disease bacterium Taqman Real-Time PCR detections of Kidney bean is present embodiments provided, Comprise the following steps：
1.1 strains tested
The dizzy epidemic disease bacterium of different plants individual specimen separation Kidney bean（Pseudomonas savastanoipv phaseolicola）Bacterium 3 parts of strain, Kidney bean bacterial wilting germ（Curtobacterium flaccumfaciens pv .flaccumfaciens）, dish Beans common bacteria epidemic disease（Xanthomonas axonopodis pv.phaseoli）, tomato bacterial tikka （Pseudomonas syringaepv.tomato）, watermelon bacterial fruit blotch（Acidovorax avenae subsp.Citrulli）, tomato bacterial ulcer（Clavibacter michiganensis subsp. michiganensis）, tomato Bacillary scab（Xanthomonas campestris pv.vesicatoria）Each 1 part of bacterial strain sample.
It is prepared by 1.2 nucleic acid
Picking single bacterium colony, liquid nitrogen grinding extracts bacterial strain DNA using TIANGEN DP320 plant genome DNAs extracts kit, DNA concentration, -20 DEG C of preservations are extracted with the measurement of ultramicron nucleic acid-protein instrument.
The specific test of 1.3 probes
Epidemic disease bacterium different plants individual specimen isolated strains DNA and other strains testeds DNA is swooned as template using Kidney bean, to the spy Pin carries out the specific detection of real-time fluorescence PCR.PCR reaction solutions are configured by kit specification, while setting positive control, the moon Property control and blank control.Reaction system such as table 5 below：
The PCR of table 5 specific detection reaction system
Reagent Usage amount
Expand premixed liquid 10 μl
PCR Forward Primer(10μM) 0.4 μl
PCR Reverse Primer(10μM) 0.4 μl
Taqman Probe 0.2 μl
RNase-free ddH2O 6.6 μl
DNA 2μl
Fluorescence correction liquid 0.4μl
Total 20 μl
The sensitivity test of 1.4 probes
Positive sample DNA concentration is measured with ultramicron nucleic acid-protein instrument, then DNA will be extracted and carries out 10 times of gradient dilutions, dilution 10 Pipe：1.8μg/ml、1.8×10-1μg/ml、1.8×10-2μg/ml、1.8×10-3μg/ml、1.8×10-4μg/ml、1.8×10-5μg/ml、1.8×10-6μg/ml、1.8×10-7μg/ml、1.8×10-8μg/ml、1.8×10-9μg/ml.Respectively take 2 μ l conducts Template carries out real-time fluorescent PCR amplification, and reaction system is with 1.3.Positive criteria threshold value is used as using Ct≤36.
1.5 Real-time PCR react
Method PCR expands standardization program and expanded in two steps：95℃ 30s；95 DEG C of 5s, 60 DEG C of 30s, 40 circulations. Real-time PCR instruments device sets probes report fluorophor FAM and quenching fluorescence group TAMRA.The positive is used as using CT≤36 Level threshold value.
1.6 positive strains are inoculated with
Real-time PCR react the bacterial strain for obtaining positive findings, on the legume for inoculating health, are placed in 24 DEG C of weathers Incubator, observes disease symptom.
2 interpretations of result
The specificity of 2.1 probes
The probe provided using the kit, to the isolated Kidney bean of different plants individual specimen swoon epidemic disease bacteria strain and other Strains tested carries out the specific detection of real-time fluorescence PCR.As a result show probe to the dizzy epidemic disease bacteria strain sample of 3 parts of Kidney beans and sun Property control performance specific positive amplification, fluorescence signal substantially increases, rather than aimed strain and negative control, blank control do not have Detect fluorescence signal（Such as accompanying drawing 1）.Show that the kit probe has extraordinary specificity to the dizzy epidemic disease bacterium of Kidney bean.
The sensitivity test of 2.2 probes
Ultramicron nucleic acid-protein instrument measurement positive sample DNA concentration is 18 μ g/ml, by stoste DNA profiling and 10 times of gradient dilutions DNA profiling afterwards carries out real-time fluorescent PCR amplification, 18 μ g/ml, 1.8 μ g/ml, 1.8 × 10 simultaneously-1μg/ml、1.8×10-2μ g/ml、1.8×10-3μg/ml、1.8×10-4μ g/ml have obvious amplification curve, 1.8 × 10-5μ g/ml CT values are more than 36 （Such as accompanying drawing 2）, therefore the mixed probe sensitivity is 1.8 × 10-4μ g/ml, i.e. 180fg/ μ l, significantly larger than regular-PCR are sensitive Degree.
The pathogenic detection of 2.3 positive strains
Real-time PCR are reacted to the bacterial strain for obtaining positive findings, on the legume for inoculating health, 24 DEG C of gas are placed in Incubator is waited, cotyledon incidence is observed after three days, obvious foxiness and haloing occurs in cotyledon shrinkage deformation（Such as accompanying drawing 3）, with dish The dizzy epidemic disease bacterium Disease symptom of beans is identical.
In summary, it is used to detect the dizzy epidemic disease bacterium Taqman Real-Time of Kidney bean the embodiments of the invention provide one kind PCR kit and its detection method, using special amplimer and Taqman probes, using Real-Time round pcrs, lead to Cross Ct values and measuring samples are carried out with the dizzy epidemic disease bacterium qualitative analysis of Kidney bean.
Described above is only the embodiment of the application, it is noted that for the ordinary skill people of the art For member, on the premise of the application principle is not departed from, some improvements and modifications can also be made, these improvements and modifications also should It is considered as the protection domain of the application.
<110>Inspection and Quarantine Combined Technology Center, Gansu Entry-Exit Inspection and Quarantine Bureau
<120>One kind is used to detect the dizzy epidemic disease bacterium Taqman Real-Time PCR kits of Kidney bean and its detection method
<160> 3
<210> 1
<211> 111
<212> DNA
<213>Kidney bean is swooned epidemic disease bacterium (Pseudomonas savastanoi pv.phaseolicola)
<400> 1
gtgggtacat ctggaatc 18
<210> 2
<211> 114
<212> DNA
<213>Kidney bean is swooned epidemic disease bacterium (Pseudomonas savastanoi pv.phaseolicola)
<400> 2
ctgacatcct ccgtattg 18
<210> 3
<211> 20
<212> DNA
<213>Kidney bean is swooned epidemic disease bacterium (Pseudomonas savastanoi pv.phaseolicola)
<400> 3
atcaccgtcg caccatacga 20

Claims (10)

1. one kind is used to detect the dizzy epidemic disease bacterium Taqman Real-Time PCR kits of Kidney bean, it is characterised in that kit bag Include reagent box main body and reagent；Wherein reagent box main body includes：Box body, the box body is hollow, includes and examination is placed in freezing liquid, box Lid is provided with above agent, box body, box body side is provided with the DNA that testing sample is placed in drawer, drawer；Reagent includes：PCR It is Forward Primer, PCR Reverse Primer, Taqman Probe, amplification premixed liquid, RNase-free ddH2O, glimmering Light correcting fluid, positive control and negative control；Kit side is additionally provided with narrow meshed cavity, cavity and freezing liquid in kit Separated using flexible plastic sheet.
2. it is according to claim 1 a kind of for detecting the dizzy epidemic disease bacterium Taqman Real-Time PCR kits of Kidney bean, Characterized in that, the PCR Forward Primer sequences are：5’-GTGGGTACATCTGGAATC -3’.
3. it is according to claim 1 a kind of for detecting the dizzy epidemic disease bacterium Taqman Real-Time PCR kits of Kidney bean, Characterized in that, the PCR Reverse Primer sequences are：5’-CTGACATCCTCCGTATTG -3’.
4. it is according to claim 1 a kind of for detecting the dizzy epidemic disease bacterium Taqman Real-Time PCR kits of Kidney bean, Characterized in that, the Taqman Probe sequences are：5’-(FAM) ATCACCGTCGCACCATACGA (TAMRA)-3’.
5. one kind is used to detect the dizzy epidemic disease bacterium Taqman Real-Time PCR detection methods of Kidney bean, it is characterised in that this method Including：（1）Probe and primer dilution；（2）Configure PCR reaction solutions；（3）Carry out Real-time PCR reactions；（4）Experimental result Analysis.
6. a kind of it is used to detecting that Kidney bean to be swooned epidemic disease bacterium Taqman Real-Time PCR detection sides according to claim 5 Method, it is characterised in that step 1 probe and primer dilution include：Probe and primer centrifuge 5min before uncapping, then according to nmol Number, uses RNase-free ddH2It is X μM of Oligo solution that O, which is configured to concentration,；Compound concentration is that X μM of Oligo solution is added RNase-free ddH2O volumes：V (ml)=nmol numbers/X.
7. a kind of it is used to detecting that Kidney bean to be swooned epidemic disease bacterium Taqman Real-Time PCR detection sides according to claim 5 Method, it is characterised in that step 2 configuration PCR reaction solutions include：Expand the μ l of premixed liquid 10, PCR Forward Primer (10 μM) 0.4μl、PCR Reverse Primer(10μM) 0.4μl、Taqman Probe 0.2μl、RNase-free ddH2O 6.6μ The μ l of l, DNA 2, the μ l of fluorescence correction liquid 0.4.
8. a kind of it is used to detecting that Kidney bean to be swooned epidemic disease bacterium Taqman Real-Time PCR detection sides according to claim 5 Method, it is characterised in that step 3 carries out two-step method PCR amplification standardization programs during Real-time PCR reactions, specifically includes as follows Step：Pre-degeneration：95 DEG C 30 seconds；PCR react 95 DEG C 5 seconds, 60 DEG C 30 seconds, 40 times circulation.
9. a kind of it is used to detecting that Kidney bean to be swooned epidemic disease bacterium Taqman Real-Time PCR detection sides according to claim 5 Method, it is characterised in that the specific method of step 4 analysis of experimental results is：Real-time PCR reactions are confirmed after the completion of reaction Amplification curve and CT values；If 2 Duplicate Samples testing result CT >=40 of testing sample, positive control detection 20≤CT≤36 simultaneously go out Existing typical amplification curve, negative control and blank control result are normal, now can be determined that the sample does not detect Kidney bean and swooned epidemic disease Germ；If 2 Duplicate Samples testing result CT≤36 of testing sample, simultaneously there is typical expand in positive control detection 20≤CT≤36 Increase curve, negative control and blank control result are normal, now can be determined that the dizzy epidemic disease bacterium of sample detection Kidney bean；If to be measured Simultaneously there is typical amplification curve in the ＜ CT ＜ 40 of 2 Duplicate Samples testing results of sample 36, and positive control detection 20≤CT≤36 are simultaneously There is typical amplification curve, negative control and blank control result are normal, and reality is reformed after now should suitably increasing DNA profiling amount When fluorescent PCR detect.
Application of primer of the epidemic disease bacterium argK genes for template in Kidney bean port quarantine 10. Kidney bean is swooned, it is characterised in that described Primer sequence such as sequence table SEQ ID NO：1、SEQ ID NO：2 and SEQ ID NO：3.
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