CN105063219A - Guava colletotrichum orbiculare specificity PCR detecting primer and detecting method of guava colletotrichum orbiculare - Google Patents
Guava colletotrichum orbiculare specificity PCR detecting primer and detecting method of guava colletotrichum orbiculare Download PDFInfo
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Abstract
The invention provides a guava colletotrichum orbiculare specificity PCR detecting primer and a detecting method of guava colletotrichum orbiculare. The primer comprises an upstream primer body CGF:5'-CGGGTAGGGTCTCCGTGA-3'and a downstream primer body CGR:5'-CCCAGTGCGAGACGT AAAGT-3', the PCR detecting method of guava colletotrichum orbiculare is built on the basis of the primer, and an amplification product with the fragment of 390 bp can be specifically amplified in guava colletotrichum orbiculare pure DNA and diseased tissue with guava colletotrichum orbiculare by agarose gel electrophoresis. When the detecting primer and the detecting method are used for detecting guava colletotrichum orbiculare, the advantages of being high in accuracy, high in specificity, high in sensitivity, simple and fast in detecting process operation are achieved, the detecting primer and the detecting method can be used for early diagnosis of guava anthracnose and monitoring and authenticating of germs in fields, and reliable technological and theoretical foundations are provided for prevention and treatment of guava anthracnose.
Description
Technical field
The present invention relates to piscidia anthrax bacteria specific PCR and detect primer and detection method thereof, be exclusively used in the Molecular Detection that piscidia anthrax bacteria is quick, sensitive and special, can be used for the early diagnosis of field piscidia anthrax and the monitoring of germ and qualification simultaneously, belong to that corps diseases detects, qualification and Prevention Technique field.
Background technology
By colletotrichum gloeosporioides Penz (
colletotrichumglosoporioides) to infect the piscidia anthrax caused be important disease before subtropical and tropical zones, world piscidia is adopted and after adopting, all over the world, annual piscidia therefore disease damage is lost and reaches 5% ~ 15%.Piscidia is usually in this disease of ripe early infection, but pathogenic bacteria keeps dormancy, until just there is the development of Disease symptoms after fruit maturation, symptom initial in mature fruit is the scab producing dun, circular depressed, and these scabs usually combine and produce large rotten region.In field, anthrax makes prematurity and mellow fruit rot, but mature fruit shows more serious, the form caused between collection period and after adopting is invaded by " wound pathogenic bacteria " usually with physiological damaged points, this a series of pathogenic bacteria causes the destructive post-harvest diseases of most, piscidia anthrax bacteria is exactly one of them, therefore, fast, at premorbid or initial stage and when gathering in the crops, the Dynamics of germ is monitored exactly, take effectively preventing method to the generation of control disease in field and accumulating in time, reduce financial loss tool to be of great significance.
The classification and identification of tradition anthrax bacteria is mainly based on morphological feature and Pathogenicity etc., this authentication method consuming time longer, program is loaded down with trivial details, sensitivity is low and by force empirical, from disease plant, be separated also pathogen identification need time a couple of days, be difficult to accomplish, to disease, monitoring in time and effective propagation and plant disease epidemic controlling pathogenic bacteria occur.Meanwhile, traditional detection method can not specify the situation of anthrax bacteria latent infection before fruit storage and transport, caused and was rotted in a large number by the tangible storage and transport process of the Fructus psidii guajavae immaturus of Colletotrichum gloeosporioides Infection, produce massive losses.Along with the development of Protocols in Molecular Biology, utilize fungi rrna transcribed spacer (internaltranscribedspacer, ITS) conservative property in sequence kind and section belong to variable feature between kind, design Auele Specific Primer carries out polymeric enzyme reaction (polymerasechainreaction, PCR) increase, thus the technology reached pathogenic bacteria carries out rapid detection and qualification is applied widely.Existing researchist utilizes rrna transcribed spacer (ITS) gene to be detection of pathogens target both at home and abroad, have developed the specific detection primer of the Different Kinds of Pathogens fungies such as botrytis cinerea, phytophthora, wilt, rod method, and utilize Auele Specific Primer to carry out pcr amplification, reach pathogenic bacteria and detect fast and accurately and identify.At present, there is not yet relevant piscidia anthrax bacteria (
colletotrichumglosoporioides) report of Molecular Detection.The present invention by colletotrichum (
colletotrichum) ITS sequence compare, design 1 to the primer that can be used for specific detection piscidia anthrax bacteria, for the precise Identification of piscidia anthrax bacteria and rapid detection provide techniques and methods, be conducive to effectively taking prophylactico-therapeutic measures early.
Summary of the invention
The object of the invention is to detect and qualification piscidia anthrax bacteria in prior art, mainly based on morphological feature, method length consuming time, program is loaded down with trivial details, by force empirical, accuracy is low, be difficult to accomplish the propagation to the timely monitor and forecast pathogenic bacteria that disease occurs, popular and post-harvest diseases causes the problem rotted, provide a kind of piscidia anthrax bacteria specific PCR and detect primer and detection method thereof, PCR of the present invention detection primer and detection method is utilized to detect piscidia anthrax bacteria accuracy high, high specificity, highly sensitive, easy handling, detection time short and reliable results.
Realize object of the present invention to comprise the following steps (technical scheme):
1. by measure piscidia anthrax bacteria (
colletotrichumglosoporioides) and other anthrax bacteria (
colletotrichumspp) rrna transcribed spacer (ITS) gene, compare to colletotrichum not of the same race ITS gene order, design 1 pair of primer piscidia anthrax bacteria to specific amplification effect, namely specific PCR detects the sequence of primer and is:
Upstream primer CGF:5'-CGGGTAGGGTCTCCGTGA-3',
Downstream primer CGR:5'-CCCAGTGCGAGACGTAAAGT-3';
Piscidia anthrax bacteria specific amplification is gone out to the product of 390bp.
2. the foundation of piscidia anthrax bacteria special molecular detection method
(1) extraction of the fruit tissue DNA of piscidia anthrax bacteria is infected.
During for detecting piscidia anthrax bacteria in fruit, NaOH rapid cleavage method is adopted to extract DNA, detailed process is as follows: in 1.0mg falls ill fruit tissue, add 0.5mol/LNaOH10 μ L, by organize fully be milled to paste after proceed in 1.5mL centrifuge tube, 12, the centrifugal 6min of 000rpm, gets supernatant liquor 5 μ l and adds 0.1mol/LTris-HCl(pH=8.0) 495 μ L mix, get 1.0 μ L and increase as pcr template.
(2) DNA extracted with step (1), for template, utilizes this pair of primers of CGF/CGR to carry out pcr amplification.PCR reaction system 25 μ L, comprises 2.5 μ L10 × PCRbuffer(Mg
2+free), 2.0mmol/LMgCl
2, 0.2mmol/LdNTP, 1.0U
taqarchaeal dna polymerase (the precious biotechnology company limited in Takara Dalian), each 0.4 μm of ol/L, the 25ngDNA template of primer CGF/CGR, the aseptic ultrapure water of insufficient section is supplied; Amplification is: 95 DEG C of denaturation 5min, 94 DEG C of sex change 30s, 61 DEG C of annealing 45s, and 72 DEG C extend 30s, totally 35 circulations, and last 72 DEG C extend 10min.
(3) pcr amplification product 5.0 μ L 1.5% sepharose getting step (2) carries out electrophoretic separation, 70-100V, observe under ultraviolet lamp through ethidium bromide staining after 40min, according to the presence or absence of amplified production and clip size thereof, result is judged, if the product of about 390bp can be amplified specifically, can judge to there is piscidia anthrax bacteria in described detection sample, otherwise there is not piscidia anthrax bacteria in described detection sample.
Guardian technique of the present invention is that the efficient specific PCR of piscidia anthrax bacteria detects the design of primer and the foundation of detection method thereof.In order to the specific PCR obtaining piscidia anthrax bacteria detects primer sequence, sibling species and common several pathogenic fungies are belonged to for for examination material with the 18 strain piscidia anthrax bacterias and multiple anthrax that derive from the different provinces such as China Fujian, Hainan and Guangdong, CTAB method is adopted to extract strains tested genomic dna, concrete grammar is as follows: take a morsel hypha powder (hypha powder had just covered semicircular base is advisable) in 1.5mL centrifuge tube, adds 900 μ L2%CTAB(cetyl trimethylammonium bromides) extracting solution (2%CTAB; 100mmol/LTris-HCl, pH8.0; 20mmol/LEDTA, pH8.0; 1.4mol/LNaCl) He 90 μ LSDS(Sodium dodecylbenzene sulfonatees) [note: CTAB, SDS need 60 DEG C of preheatings], use oscillator vibrates mixing, 60 DEG C of water-bath 1h(DNA are released in damping fluid), 12000r.min
-1centrifugal 15min; Get supernatant liquor 700 μ L, add equal-volume phenol, chloroform, primary isoamyl alcohol mixed solution (three volume ratio 25:24:1), mixing of vibrating gently, 12000r.min
-1centrifugal 9min; Get supernatant liquor 500 μ L, add equal-volume chloroform again extracting once, 12000r.min
-1centrifugal 5min; Get supernatant liquor 350 μ L, add 1/10 volume 3mol.L
-1naAc and 2 times of volume dehydrated alcohol ,-20 DEG C of precipitations 30min, 12000r.min
-1centrifugal 5min; Abandoning supernatant, adds 700 μ L ice 70% ethanol and carries out washing (centrifugal; Incline and fall supernatant liquor), Bechtop dries alcohol-free taste, adds 30 ~ 60 μ LTE(10mmol/LTris-HCl, 0.1mmol/LEDTA, pH8.0) solution dissolves, and obtains DNA solution, by UV spectrophotometer measuring DNA concentration and to be diluted to 25ng/ μ L stand-by.With fungi Internal Transcribed Spacer (ITS) universal primer ITS1:5'-TCCGTAGGGAACCTGCGG-3' and ITS4:5'-TCCTCCGCTTATTGATATGC-3' to for examination piscidia anthrax bacteria (
c.glosoporioides) ITS gene increase, PCR reaction system 50 μ L, comprises 2.5 μ L10 × PCRbuffer(Mg
2+free), 2.0mmol/LMgCl
2, 0.2mmol/LdNTP, 1.0U
taqarchaeal dna polymerase (the precious biotechnology company limited in Takara Dalian), each 0.4 μm of ol/L, the 25ngDNA template of primer TIS1/ITS4, the aseptic ultrapure water of insufficient section is supplied.Amplified reaction program is: 94 DEG C of denaturation 5min; 94 DEG C of sex change 1min, 55 DEG C of annealing 30S, 72 DEG C of extension 1min, 35 circulations, last 72 DEG C extend 10min.Pcr amplification product is delivered to Shanghai Sheng Gong biotechnology company limited to check order, by check order obtain piscidia anthrax bacteria (
c.glosoporioides) ITS sequence and GenBank in
colletotrichumbelong to 18 ITS gene orders not of the same race and carry out tetraploid rice analysis, according to the difference site (in BioEdit comparison) between piscidia anthrax bacteria and other kind, the piscidia anthrax bacteria by PrimerPrimer5 software design
c.glosoporioidesauele Specific Primer, primer sequence is: upstream primer CGF:5'-CGGGTAGGGTCTCCGTGA-3', downstream primer CGR:5'-CCCAGTGCGAGACGTAAAGT-3', primer synthesis synthesized by Shanghai Sheng Gong biotechnology company limited.Designing on the basis of special primer, by the optimization of PCR reaction system and Amplification, the piscidia anthrax bacteria detection method set up, genomic dna for the piscidia anthrax bacteria tried and other pathogenic bacteria is template, the specificity of piscidia anthrax bacteria Auele Specific Primer CGF/CGR is verified, result shows, primer CGF/CGR can only amplify the band that size is about 390bp specifically from 18 piscidia anthrax bacteria DNA for examination, and other anthrax-bacilus, pathogenic fungi and negative control are all without amplified band.Illustrate that piscidia anthrax bacteria and other anthrax-bacilus and pathogenic fungi can make a distinction primer by this, the specificity to have kind, can be used for the fast and reliable detection of piscidia anthrax bacteria and qualification.
The present invention compared with prior art, has following technical superiority and beneficial effect:
1. high specificity, accuracy is high: the present invention is according to fungi rrna transcribed spacer (internaltranscribedspacer, ITS) conservative property in sequence kind and section belong to variable feature between kind, devise PCR primer piscidia anthrax bacteria to specific amplified effect, and with the fruit tissue carrying piscidia anthrax bacteria, testing authentication has been carried out to the piscidia anthrax bacteria of different geographic origin, only have piscidia anthrax bacteria and carry this germ fruit in can amplify the electrophoretic band of a 390bp specifically, illustrate that the primer designed by the present invention has very strong specificity and accuracy.
2. highly sensitive: traditional detection of pathogens method is by steps such as separation, purifying and Morphological Identifications, the success of this traditional method needs the pathogenic agent ability success running up to q.s in incidence tissue.And after special primer and the ITS gene universal primer (ITS1/ITS4) of design joined together to carry out nested PCR amplification by the present invention, 1fg can be reached to the detection sensitivity of piscidia anthrax bacteria on DNA level, higher 10000 times than Standard PCR detection;
3. practicality is good: the rapid detection of piscidia anthrax bacteria, has important actual application value.The detection method of traditional piscidia anthrax bacteria is generally after symptom appears in plant, by its disease symptom, pathogenic bacteria is separated, purifying, a series of loaded down with trivial details process such as qualification, required time is longer, difficulty is added to detecting pathogen fast and accurately, traditional method is monitored dynamically and detects due to the pathogenic bacteria that can not carry out field in time early stage in disease morbidity, misses an opportunity because of a delay often to the control of agriculture production.Whether the present invention to being with in plant tissue piscidia anthrax bacteria can detect, if the electrophoretic band of 390bp can be amplified specifically, illustrate in plant tissue to there is piscidia anthrax bacteria, therefore the present invention can be used for piscidia anthrax bacteria cause disease show disease before early monitoring, for determining that the formulation of disease control best period and formulation control strategy provides scientific basis, therefore the present invention has good practicality;
4. easy and simple to handle, quick: application the inventive method, result of determination is got final product after the agarose electrophoresis of DNA extraction, pcr amplification and routine is carried out to the plant tissue of band piscidia anthrax bacteria, whole testing process adopts DNA rapid extracting method, simple to operate, without the need to carrying out separation and Culture to pathogenic bacteria, substantially reduce detection time, general whole testing process can complete in 6 hours.
Accompanying drawing explanation
The specific PCR amplification figure of the piscidia anthrax bacteria that Fig. 1 will detect for the present invention, in figure: swimming lane 1,13 is 2000bpMarker, swimming lane 2 is positive control, swimming lane 3-6 is piscidia anthrax bacteria, and swimming lane 7 is Glorosprium musarum Cookeet Mass, and swimming lane 8 is watermelon anthrax bacteria, swimming lane 9 is Colletotrichum truncatum, swimming lane 10 is piscidia alternaria, and swimming lane 11 is Phytophthora cactorum bacterium, and swimming lane 12 is negative control.
Fig. 2 is that the susceptibility of piscidia anthrax bacteria of the present invention detects amplification figure, Fig. 2-a is the susceptibility detected result of substance PCR to piscidia anthrax bacteria, Fig. 2-b is the susceptibility detected result of nest-type PRC to piscidia anthrax bacteria, in figure, swimming lane 1 is 2000bpMarker, swimming lane 2 is 10ng, swimming lane 3 is 1ng, swimming lane 4 is 100pg, and swimming lane 5 is 10pg, and swimming lane 6 is 1pg, swimming lane 7 is 100fg, swimming lane 8 is 10fg, and swimming lane 9 is 1fg, and swimming lane 10 is 100ag, swimming lane 11 is negative control, and swimming lane 12 is positive control.
Fig. 3 is the detected result figure of incidence tissue of the present invention, in figure, swimming lane 1,13 is 2000bpMarker, and swimming lane 2-3,5-6 are the guava fruit of natural occurrence, and swimming lane 8-9 is the guava fruit of artificial inoculation morbidity, swimming lane 4,7,10,12 is negative control, and swimming lane 11 is positive control.
Embodiment
Below in conjunction with specific embodiment, the present invention is further elaborated, but be not used for limiting the scope of the invention.Following examples are experiment condition all conveniently, or has delivered the operative technique code described in pertinent literature, or according to the experiment condition that manufacturer advises.
embodiment 1:PCR detects the design of primer sequence and the specific amplification of primer pair piscidia anthrax bacteria
1. the Design and synthesis of primer
Is checked order in this laboratory obtain 10 strain piscidia anthrax bacterias (
c.glosoporioides) ITS sequence and GenBank in
colletotrichumbelong to 18 ITS gene orders not of the same race and carry out tetraploid rice analysis, according to the difference site (in BioEdit comparison) between piscidia anthrax bacteria and other kind, the piscidia anthrax bacteria by PrimerPrimer5 software design
c.glosoporioidesauele Specific Primer, primer sequence is: upstream primer CGF:5'-CGGGTAGGGTCTCCGTGA-3', downstream primer CGR:5'-CCCAGTGCGAGACGTAAAGT-3', primer synthesis synthesized by Shanghai Sheng Gong biotechnology company limited.
the extraction of strains tested genomic dna
CTAB method is adopted to extract strains tested genomic dna, concrete grammar is as follows: take a morsel hypha powder (hypha powder had just covered semicircular base is advisable) in 1.5mL centrifuge tube, adds 900 μ L2%CTAB(cetyl trimethylammonium bromides) extracting solution (2%CTAB; 100mmol/LTris-HCl, pH8.0; 20mmol/LEDTA, pH8.0; 1.4mol/LNaCl) He 90 μ LSDS(Sodium dodecylbenzene sulfonatees) [note: CTAB, SDS need 60 DEG C of preheatings], use oscillator vibrates mixing, 60 DEG C of water-bath 1h(DNA are released in damping fluid), 12000r.min
-1centrifugal 15min; Get supernatant liquor 700 μ L, add equal-volume phenol, chloroform, primary isoamyl alcohol mixed solution (each volume ratio 25:24:1), mixing of vibrating gently, 12000r.min
-1centrifugal 9min; Get supernatant liquor 500 μ L, add equal-volume chloroform again extracting once, 12000r.min
-1centrifugal 5min; Get supernatant liquor 350 μ L, add 1/10 volume 3mol.L
-1naAc and 2 times of volume dehydrated alcohol ,-20 DEG C of precipitations 30min, 12000r.min
-1centrifugal 5min; Abandoning supernatant, adds 700 μ L ice 70% ethanol and carries out washing (centrifugal; Incline and fall supernatant liquor), Bechtop dries alcohol-free taste, adds 30 ~ 60 μ LTE(10mmol/LTris-HCl, 0.1mmol/LEDTA, pH8.0) solution dissolves, and obtains DNA solution, by UV spectrophotometer measuring DNA concentration and to be diluted to 25ng/ μ L stand-by.
primer specificity PCR verifies
Genomic dna for the piscidia anthrax bacteria tried and other pathogenic bacteria is template, the specificity of piscidia anthrax bacteria Auele Specific Primer (upstream primer CGF:5'-CGGGTAGGGTCTCCGTGA-3', downstream primer CGR:5'-CCCAGTGCGAGACGTAAAGT-3') is verified.PCR reaction system 25 μ L, comprises 2.5 μ L10 × PCRbuffer(Mg
2+free), 2.0mmol/LMgCl
2, 0.2mmol/LdNTP, 1.0U
taqarchaeal dna polymerase (the precious biotechnology company limited in Takara Dalian), each 0.4 μm of ol/L, the 25ngDNA template of primer CGF/CGR, the aseptic ultrapure water of insufficient section is supplied.Amplified reaction program is: 94 DEG C of denaturation 5min; 94 DEG C of sex change 1min, 61 DEG C of annealing 45S, 72 DEG C of extension 30s, 35 circulations, last 72 DEG C extend 10min.Get 5 μ LPCR products and carry out 1.5% agarose electrophoresis detection, observe under ultraviolet lamp after ethidium bromide staining, having that it's too late the specificity of size to piscidia anthrax bacteria Auele Specific Primer is verified according to DNA band.
primer specificity the result
Pcr amplification result shows, primer CGF/CGR can only amplify the band (Fig. 1) that size is about 390bp specifically from 18 piscidia anthrax bacteria DNA for examination, and other anthrax-bacilus, pathogenic fungi and negative control are all without amplified band.Illustrate that piscidia anthrax bacteria and other anthrax-bacilus and pathogenic fungi all can make a distinction primer by this, the specificity to have kind, can be used for the fast and reliable detection of piscidia anthrax bacteria and qualification.
embodiment 2: the sensitivity technique of primer pair piscidia anthrax bacteria genomic dna
1. standard PCR amplification
Dilute piscidia anthrax bacteria genomic dna with aseptic ultrapure water, the series concentration being mixed with 10 times of orders of magnitude is for subsequent use.Primer CGF/CGR of the present invention is used to carry out pcr amplification to the genomic dna of different series concentration, assess the susceptibility that this primer pair piscidia anthrax bacteria genomic dna detects, amplification reaction system and response procedures as follows: PCR reaction system 25 μ L, comprises 2.5 μ L10 × PCRbuffer(Mg
2+free), 2.0mmol/LMgCl
2, 0.2mmol/LdNTP, 1.0U
taqarchaeal dna polymerase (the precious biotechnology company limited in Takara Dalian), each 0.4 μm of ol/L, the 25ngDNA template of primer CGF/CGR, the aseptic ultrapure water of insufficient section is supplied.Amplified reaction program is: 94 DEG C of denaturation 5min; 94 DEG C of sex change 1min, 61 DEG C of annealing 45S, 72 DEG C of extension 1min, 35 circulations, last 72 DEG C extend 10min.
nested PCR amplification
Dilute piscidia anthrax bacteria genomic dna with aseptic ultrapure water, the series concentration being mixed with 10 times of orders of magnitude is for subsequent use.With fungi Internal Transcribed Spacer (ITS) universal primer ITS1:5'-TCCGTAGGGAACCTGCGG-3' and ITS4:5'-TCCTCCGCTTATTGATATGC-3' for outer primer, special primer CGF/CGR of the present invention is that the genomic dna of outer primer to piscidia anthrax bacteria different series concentration carries out nested PCR amplification, assess the susceptibility that primer CGF/CGR of the present invention is detected piscidia anthrax bacteria genomic dna by nest-type PRC
first round pcr amplification:the genomic dna reacting primer pair different series concentration using ITS universal primer ITS1:5'-TCCGTAGGGAACCTGCGG-3' and ITS4:5'-TCCTCCGCTTATTGATATGC-3' as the first round carries out pcr amplification, amplification reaction system and response procedures as follows: PCR reaction system 25 μ L, comprises 2.5 μ L10 × PCRbuffer(Mg
2+free), 2.0mmol/LMgCl
2, 0.2mmol/LdNTP, 1.0U
taqarchaeal dna polymerase (the precious biotechnology company limited in Takara Dalian), each 0.4 μm of ol/L, the 25ngDNA template of primer TIS1/ITS4, the aseptic ultrapure water of insufficient section is supplied.Amplified reaction program is: 94 DEG C of denaturation 5min; 94 DEG C of sex change 1min, 55 DEG C of annealing 30S, 72 DEG C of extension 1min, 35 circulations, last 72 DEG C extend 10min.
second takes turns pcr amplification:after first round pcr amplification result, getting 1 μ L first round PCR primer is that template and primer CGF/CGR combine and carry out nested PCR amplification.PCR reaction system 25 μ L, comprises 2.5 μ L10 × PCRbuffer(Mg
2+free), 2.0mmol/LMgCl
2, 0.2mmol/LdNTP, 1.0U
taqarchaeal dna polymerase (the precious biotechnology company limited in Takara Dalian), each 0.4 μm of ol/L, the 25ngDNA template of primer CGF/CGR, the aseptic ultrapure water of insufficient section is supplied.Amplified reaction program is: 94 DEG C of denaturation 5min; 94 DEG C of sex change 1min, 61 DEG C of annealing 45S, 72 DEG C of extension 1min, 35 circulations, last 72 DEG C extend 10min.
Standard PCR and nest-type PRC sensitivity comparative result show, when carrying out standard PCR amplification with primer CGF/CGR of the present invention, reaction sensitivity can reach 10pgDNA25 μ L
-1reaction system (in Fig. 2 a).Carry out the first round using ITS gene universal primer ITS1/ITS4 further and increase the PCR primer that obtains as template, take turns amplimer using CGF/CGR as second and carry out nested PCR amplification, can find out that the specific amplification band of sleeve type PCR is much brighter than Standard PCR from electrophorogram, can make originally to can't see band sample (1pg, 100fg, 10fg, 1pg/25 μ L reaction system) produce bands visible (b in Fig. 2), sensitivity reaches 1fgDNA25 μ L
-1reaction system, will improve about 10000 times than Standard PCR.
embodiment 3: the detection of piscidia anthrax bacteria in morbidity fruit
the extraction of piscidia anthrax bacteria DNA in morbidity fruit: adopt NaOH rapid cleavage method to extract DNA, detailed process is as follows: in 1.0mg falls ill fruit, add 0.5mol/LNaOH10 μ L, by organize fully be milled to paste after proceed in 1.5mL centrifuge tube, 12, the centrifugal 6min of 000rpm, getting supernatant liquor 5 μ L and add 0.1mol/LTris-HCl(pH=8.0) 495 μ L mix, get 1.0 μ L and increase as pcr template.
pcr amplification detects:primer CGF/CGR of the present invention is utilized to carry out pcr amplification.PCR reaction system 25 μ L, comprises 2.5 μ L10 × PCRbuffer(Mg
2+free), 2.0mmol/LMgCl
2, 0.2mmol/LdNTP, 1.0U
taqarchaeal dna polymerase (the precious biotechnology company limited in Takara Dalian), each 0.4 μm of ol/L, the 25ngDNA template of primer CGF/CGR, the aseptic ultrapure water of insufficient section is supplied; Amplification is: 95 DEG C of denaturation 5min, 94 DEG C of sex change 30s, 61 DEG C of annealing 45s, and 72 DEG C extend 30s, totally 35 circulations, and last 72 DEG C extend 10min.
result detects:get pcr amplification product 5.0 μ L 1.5% agarose electrophoresis to be separated, voltage is 70-100V, electrophoresis terminates to observe under ultraviolet lamp after ethidium bromide staining, according to the presence or absence of amplified production and clip size thereof, result is judged, if the product of about 390bp can be amplified specifically, can judge in morbidity plant tissue with piscidia anthrax bacteria.Detected result (Fig. 3) shows, all piscidia anthrax bacteria can be detected in the guava fruit of the typical fruit of piscidia anthrax disease symptom and artificial inoculation, healthy plant tissue and negative control then occur without specific band, illustrate that this cover technology can be used for the rapid molecular detection of piscidia anthrax bacteria in guava fruit.
The foregoing is only preferred embodiment of the present invention, all equalizations done according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.
SEQUENCELISTING
<110> Inst. of Plant Protection, fujian Academy of Agricultural Science
<120> piscidia anthrax bacteria specific PCR detects primer and detection method thereof
<130>4
<160>4
<170>PatentInversion3.3
<210>1
<211>18
<212>DNA
<213> artificial sequence
<400>1
cgggtagggtctccgtga18
<210>2
<211>20
<212>DNA
<213> artificial sequence
<400>2
cccagtgcgagacgtaaagt20
<210>3
<211>18
<212>DNA
<213> artificial sequence
<400>3
tccgtagggaacctgcgg18
<210>4
<211>20
<212>DNA
<213> artificial sequence
<400>4
tcctccgcttattgatatgc20
Claims (3)
1. piscidia anthrax bacteria specific PCR detects primer, it is characterized in that: primer sequence is:
Upstream primer CGF:5'-CGGGTAGGGTCTCCGTGA-3',
Downstream primer CGR:5'-CCCAGTGCGAGACGTAAAGT-3';
Piscidia anthrax bacteria specific amplification is gone out to the product of 390bp.
2. utilize a piscidia anthrax bacteria detection method for primer described in claim 1, it is characterized in that: comprise the following steps:
(1) from the plant tissue infecting piscidia anthrax bacteria, DNA is extracted;
(2) DNA extracted with step (1) is for template, and utilize this pair of primers of CGF/CGR to carry out pcr amplification, the condition of pcr amplification is: PCR reaction system 25 μ L, comprises 2.5 μ L10 × PCRbuffer, 2.0mmol/LMgCl
2, 0.2mmol/LdNTP, 1.0U
taqarchaeal dna polymerase, each 0.4 μm of ol/L, the 25ngDNA template of primer CGF/CGR, the aseptic ultrapure water of insufficient section is supplied; Amplification is 95 DEG C of denaturation 5min, 94 DEG C of sex change 30s, 61 DEG C of annealing 45s, and 72 DEG C extend 30s, totally 35 circulations, and last 72 DEG C extend 10min;
(3) pcr amplification product getting 5.0 μ L steps (2) carries out electrophoretic separation with 1.5% sepharose, voltage is 70-100V, observe under ultraviolet lamp through ethidium bromide staining after 40min, according to the presence or absence of amplified production and clip size thereof, result is judged, if the product of 390bp can be amplified specifically, can judge to there is piscidia anthrax bacteria in described detection sample.
3. the application of primer as claimed in claim 1 in the early diagnosis of field piscidia anthrax and the monitoring of germ and qualification.
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