CN104120169B - Phytophthora infestans mitochondrial gene type and heterogeneous detection kit and detection method - Google Patents
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Abstract
The invention belongs to phytophthora infestans mitochondrial gene type and heterogeneous identification technology field.It is an object of the invention to provide a kind of detection phytophthora infestans mitochondrial gene type quick, accurate based on polymerase chain reaction (PCR) and the test kit of heterogeneity and detection method.Test kit comprise PCR amplification required for two pairs of specific primers to and Mix, wherein, two pairs of primers are to being respectively as follows: InDel F:5'GTG GGT TCG AGT CCC ACT AA 3', InDel R:5'CTC ACC CGT TCG CTA TGT TT 3', with RSU F:5'ACG GAA TTA TCG GAA GAT TT 3', RSU R:5'AAA TCC CTT TTA TAC TGT AAT TTG AT 3'.Detection process is as follows: extract the genomic DNA of phytophthora infestans bacterial strain；Utilize two specific primers and test kit are carried out PCR amplification；The agarose gel electrophoresis testing result of pcr amplification product；The observation of band and the name of band pattern；The determination of mitochondrial gene type；The judgement that mitochondrion is heterogeneous.This test kit can be used for country's animals and plants inspection and quarantine department and monitor external phytophthora infestans and agricultural sector to zones of different phytophthora infestans colony it can also be used to the monitoring that determines for base phytophthora infestans feature and dynamically change of Ge great potato seed manufacturing enterprise.
Description
Technical field
The invention belongs to pathogenic oomycetes mitochondrial gene type and the technical field of heterogeneous detection.
Background technology
On Rhizoma Solani tuber osi, the most destructive late blight is caused by phytophthora infestans, this disease once in the mid-40 in 19th century in Europe
Being very popular in continent, causes internationally recognizable " Ireland big famine ", and the research of this disease has been started the beginning of Plant Pathology.When
The present, this disease is still the first big disease on Rhizoma Solani tuber osi, and the annual whole world causes economic loss to be 6,700,000,000 dollars, does not include using pesticide
Expense.Molecular Detection multifarious for pathogen for pest and disease risk assessment and prevention and control significant, and mitochondrion by
Then maternal inheritance, can report as the reliable method of phytophthora infestans population surveillance the most to the mensuration of its genotype
The road assay method of 2 kinds of phytophthora infestans mitochondrial gene types, and do not have the heterogeneous detection of phytophthora infestans mitochondrion in the world
Method.Carter proposes the RFLP method of phytophthora infestans mitochondrial gene type first in nineteen ninety, is caused by separation, purification
Sick phytophthora mitochondrial genome DNA, then carry out restriction enzyme digestion and electrophoresis with multiple restricted enzyme, it was found that 4 kinds of mitochondrial genomies
Polymorphism, and be respectively designated as four kinds of mitochondrial gene types such as Ia, Ib, IIa and IIb, also referred to as mitochondrion list times
Type.But the shortcoming of the method is to need to separate and purification mitochondrial genes group DNA, operates complicated, time-consuming, and experimental cost is very
Height, determination efficiency is extremely low.Subsequently, after the mitochondrial genome sequence of phytophthora infestans Ib genotype comes forth, 1998
Griffith etc. located the position of the enzyme action mitochondrial genomies such as Carter on genome, conservative in the both sides of restriction enzyme site
District devises specific primer, and this fragment gene carries out PCR amplification, then carries out enzyme action, thus establishes simplicity, efficient
The detection method of the mitochondrial genome genotype of PCR-RFLP,.But due to Griffith in 1998 PCR-RFLP method not
Consider that phytophthora infestans I type and II this qualitative difference of type are the insertion and deletion of 2kb, thus cause the inspection using PCR-RFLP method
Survey result sometimes the most inconsistent with the RFLP method testing result that Carter proposes.Additionally, what both the above method was detected
The Limited Number of mitochondrial genome genotype only 4 kinds, and fail its heterogeneity is detected.Therefore, the present invention is to 4
On the basis of phytophthora infestans mitochondrial genome sequence analysis, it was found that 2 height region of variability can be used for distinguishing 4 kinds of mitochondrion bases
Because of type, 2 hypervariable regions are separately designed specific primer pair, utilized polymerase chain reaction (PCR), invented pathogenic epidemic disease
Mould mitochondrial gene type detection kit；Meanwhile, can determine that whether it has by 2 hypervariable region amplicon band numbers heterogeneous
Property.The method set up for country quarantine departments to external phytophthora infestans and agricultural sector the prison to zones of different pathogenic bacterium colony
Survey it can also be used to the monitoring that dynamically changes for base phytophthora infestans of Ge great potato seed manufacturing enterprise.
Summary of the invention
It is an object of the invention to provide a kind of double phytophthora infestans mitochondrial gene type and the heterogeneous detection being detected on one
Test kit, including 2 pairs of PCR primer and Mix, and detection method.
1. technical scheme is as follows:
From GenBank download be complete order-checking Carter 4 kinds of mitochondrial gene types (Ia, Ib, IIa and
IIb) genome sequence.First, with ClustalX, these 4 mitochondrial genome sequences are compared, with BioEdit and
Genome sequence is analyzed by MEGA software, finds that 4 mitochondrial gene type genomes exist 2 kinds of nucleotide polymorphisms, and one
Planting is to be substituted or lack the single nucleotide polymorphism (SNP) caused by mononucleotide, and another is at the 2 of mitochondrial genome
The existence of individual hypervariable region is inserted by large fragment or lacks the length polymorphism caused.In the both sides conserved sequence of these 2 hypervariable regions,
Utilize the Primer-BLAST in NCBI separately design 2 to narrow spectrum PCR primer, respectively primer to InDel-F and
InDel-R, and primer is to RSU-F and RSU-R.It is utilized respectively these 2 pairs of specific primers phytophthora infestans genomic DNA is carried out
PCR expands, by the length polymorphism of agarose gel electrophoresis detection amplicon, by the length of 2 hypervariable region difference amplicons
Degree polymorphism combination determines phytophthora infestans mitochondrial gene type, and according to various combination, proposes a set of for determining different cause
The naming method of sick phytophthora mitochondrial gene type；Meanwhile, phytophthora infestans mitochondrion is judged according to the band number of each hypervariable region
Whether there is heterogeneity.
2. the new method for detection and identification phytophthora infestans mitochondrial gene type and heterogeneity of present invention design is to use
Following steps:
(1) extraction of phytophthora infestans genomic DNA；
(2) amplification of DNA fragments, i.e. carries out polymerase chain reaction, for the DNA of 2 pairs of primers of polymerase chain reaction
Sequence is:
InDel-F:5'-GTGGGTTCGAGTCCCACTAA-3'
InDel-R:5'-CTCACCCGTTCGCTATGTTT-3'
RSU-F:5'-ACGGAATTATCGGAAGATTT-3'
RSU-R:5'-AAATCCCTTTTATACTGTAATTTGAT-3'
(3) agarose gel electrophoresis analysis；
(4) judgement of qualification result.Mitochondrial gene type is judged, specifically by 2 pairs of primer pair amplifies band pattern combinations
As follows；
A. utilize InDel-F with InDel-R primer amplifiable go out the different band of 2 length, its size is respectively 3 kb
With 1 kb, being respectively designated as L and S, the bacterial strain of different mitochondrial gene types there will be 3 kinds of band pattern after amplification: L, S
And LS；
B. utilize RSU-F with RSU-R primer to amplifiable go out 3 kinds of different bands of length, its length be respectively 192 bp,
228 bp and 264 bp, respectively by its named V1、V2And V3, the bacterial strain of different mitochondrial gene types there will be 7 kinds after amplification
Band and combinations thereof pattern, i.e. V1, V2, V3, V1V2, V1V3, V2V3, V1V2V3；
C. the combination of comprehensive above 2 amplifications, amplification has 21 kinds of situations, i.e. corresponding mitochondrial gene type
There are 21 kinds, are respectively as follows: LV1, LV2, LV3, LV1V2, LV1V3, LV2V3, LV1V2V3, SV1, SV2, SV3, SV1V2,
SV1V3, SV2V3, SV1V2V3, LSV1, LSV2, LSV3, LSV1V2, LSV1V3, LSV2V3, LSV1V2V3；
D. the genotype that phytophthora infestans mitochondrion is heterogeneous is: LV1V2, LV1V3, LV2V3, LV1V2V3, SV1V2,
SV1V3, SV2V3, SV1V2V3, LSV1, LSV2, LSV3, LSV1V2, LSV1V3, LSV2V3And LSV1V2V3Deng 15 kinds；
The effect of the present invention is: (1) proposes first and utilizes PCR method to find the side that phytophthora infestans mitochondrion is heterogeneous
Method；(2) the phytophthora infestans mitochondrial gene type detection kit set up, considerably increases the detection number of genotype, by former
Carry out method to can be detected by 4 kinds and increase to the method and can finally determine 21 kinds of mitochondrial gene types；(3) this test kit is easy, high
Effect, accurately, thus overcome that forefathers' phytophthora infestans mitochondrial gene type detection method is complicated, it is high to spend and efficiency is low etc. many asks
Topic.
3. detailed description of the invention is:
(1) Kit components includes: each 10 μMs of primer AsF and AsR(), Mix [Taq enzyme (0.1-0.2 U/ μ L), dATP
(0.06-0.10 mM)、dTTP (0.06-0.10 mM)、dGTP(0.06-0.10 mM)、dCTP(0.06-0.10 mM)、
MgCl2(0.8-1.3 mM)、KCl(130-160 mM)、Tris-HCl(25-30 mM, pH 9.0-9.3)、1% Triton X-
100 and stabilizer], ddH2O；
(2) extraction of DNA: extract the DNA of phytophthora infestans mycelia according to conventional CTAB method；
(3) polymerase chain reaction:
A. polymerase chain reaction reference hierarchy is 25 μ l, and each reagent dosage is respectively as follows:
Template DNA 1.0 μ L
Primer-AsF 0.5 μ L
Primer-AsR 0.5 μ L
Mix 12.5μL
ddH2O 10.5μL
B. PCR reaction condition: reaction is carried out in PCR instrument, and reaction condition is:
Utilize the amplification that InDel-F and InDel-R primer is carried out, need first 95 DEG C of denaturations 5 minutes, according still further to following condition
Carry out 27 circulations:
95 DEG C of 30-45 seconds of degeneration
Renaturation 65 DEG C 2-4 minute
Extend 72 DEG C 2-4 minute
Keep 9-10 minute at 72 DEG C after loop ends, react after terminating 4 DEG C of preservations；
Utilize the amplification that RSU-F and RSU-R primer is carried out, need first 95 DEG C of denaturations 2 minutes, carry out according still further to following condition
30 circulations:
95 DEG C of 40-50 seconds of degeneration
45 DEG C of 40-50 seconds of renaturation
Extend 72 DEG C of 40-50 seconds
Keep 6-8 minute at 72 DEG C after loop ends, react after terminating 4 DEG C of preservations；
(4) electrophoresis detection:
Utilize the amplification of InDel-F and InDel-R primer: loading 2.5 μ l, the agarose gel electrophoresis with 1.2% detects.
Utilize the amplification of RSU-F and RSU-R primer: loading 3 μ l, the agarose gel electrophoresis with 2.5% detects.At 5 V/cm electric-field strengths
Degree lower electrophoresis 20-50 minute, carries out agarose gel dyeing with EB, observes stripe size；
(5) judgement of testing result:
A. utilizing InDel-F and InDel-R primer can amplify 2 kinds of different size of bands, size is respectively 3 kb
With 1 kb, being respectively designated as L and S, the bacterial strain of different mitochondrial gene types there will be 3 kinds of compound modes after amplification: L, S and
LS；
B. RSU-F with RSU-R primer is utilized can to amplify the band of 3 different sizes, respectively 192 bp, 228
Bp and 264 bp, is respectively designated as V1、V2And V3, the bacterial strain of different mitochondrial gene types there will be 7 kinds of combination sides after amplification
Formula: V1, V2, V3, V1V2, V1V3, V2V3, V1V2V3；
C. the combination of comprehensive above 2 amplifications, amplification has 21 kinds of situations, i.e. corresponding mitochondrial gene type
There are 21 kinds, are respectively as follows: LV1, LV2, LV3, LV1V2, LV1V3, LV2V3, LV1V2V3, SV1, SV2, SV3, SV1V2,
SV1V3, SV2V3, SV1V2V3, LSV1, LSV2, LSV3, LSV1V2, LSV1V3, LSV2V3, LSV1V2V3；
D. heterogeneous judgement: when phytophthora infestans genome is carried out PCR amplification, any 1 district of its 2 hypervariable regions
There is two and above band simultaneously in territory amplicon, then this bacterium is heterogeneous.Genotype totally 15 kinds heterogeneous accordingly, respectively
For LV1V2, LV1V3, LV2V3, LV1V2V3, SV1V2, SV1V3, SV2V3, SV1V2V3, LSV1, LSV2, LSV3,
LSV1V2, LSV1V3, LSV2V3And LSV1V2V3。
Claims (7)
1. double phytophthora infestans mitochondrial gene type and the heterogeneity of detecting is in the test kit of one, it is characterised in that: reagent
Box comprises 2 pairs of specific primers and Mix；
Described 2 pairs of specific primers are respectively as follows: InDel-F:
5'-GTGGGTTCGAGTCCCACTAA-3', InDel-R:5'-CTC ACC CGT TCG CTA TGT TT-3', RSU-
F:5'-ACG GAA TTA TCG GAA GAT TT-3',
RSU-R:5'-AAA TCC CTT TTA TAC TGT AAT TTG AT-3'；
The composition of described Mix is the Taq enzyme of 0.1-0.2U/ μ L, and the concentration of dATP, dTTP, dGTP, dCTP is 0.06-
The MgCl of 0.10mM, 0.8-1.3mM2, the KCl of 130-160mM, the Tris-HCl of 25-30mM, pH 9.0-9.3,1%Triton
X-100 and stabilizer.
2. phytophthora infestans mitochondrial gene type and heterogeneous detection method, it is characterised in that utilize the examination described in claim 1
Agent box, with the genomic DNA of phytophthora infestans as template, utilizes 2 pairs of specific primers in test kit to carry out pcr amplification reaction, root
The band pattern of amplification and combination according to 2 PCR, it is judged that whether the mitochondrial gene type of phytophthora infestans and mitochondrion have heterogeneous
Property；Wherein InDel-F Yu InDel-R pairing carries out PCR, RSU-F and RSU-R pairing and carries out PCR.
3. detection method as claimed in claim 2, it is characterised in that: the extraction of the genomic DNA of phytophthora infestans uses CTAB
Method, with deionized water by DNA concentration regulation to 5-10ng/ μ l.
4. detection method as claimed in claim 2, it is characterised in that: utilize InDel-F and InDel-R specific primers to entering
During performing PCR, amplifiable go out the different band of 2 length, its size is respectively 3kb and 1kb, is respectively designated as L and S, no
After amplification, there will be 3 kinds of mitochondrial gene type compound modes: L, S and LS with the bacterial strain of mitochondrial gene type.
5. detection method as claimed in claim 2, it is characterised in that: utilize RSU-F and RSU-R specific primers to carry out PCR
Time, amplifiable go out 3 bands varied in size, its length is respectively 192bp, 228bp and 264bp, is respectively designated as V1、V2With
V3, the bacterial strain of different mitochondrial gene types there will be 7 kinds of band pattern i.e. after amplification: V1,V2,V3,V1V2,V1V3,V2V3,
V1V2V3。
6. the detection method as described in claim 2-5 is arbitrary, it is characterised in that: the band pattern combination of 2 PCR amplifications
Having 21 kinds altogether, namely corresponding phytophthora infestans mitochondrial gene type has 21 kinds, it is respectively as follows: LV1,LV2,LV3,LV1V2,
LV1V3,LV2V3,LV1V2V3,SV1,SV2,SV3,SV1V2,SV1V3,SV2V3,SV1V2V3,LSV1,LSV2,LSV3,LSV1V2,
LSV1V3,LSV2V3,LSV1V2V3。
7. the detection method as described in claim 2-5 is arbitrary, it is characterised in that: the genotype of phytophthora infestans heterogeneity has
LV1V2,LV1V3,LV2V3,LV1V2V3,SV1V2,SV1V3,SV2V3,SV1V2V3,LSV1,LSV2,LSV3,LSV1V2,LSV1V3,
LSV2V3And LSV1V2V3。
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Non-Patent Citations (4)
| Title |
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| Polymorphisms in Phytophthora infestans: Four Mitochondrial Haplotypes Are Detected after PCR Amplification of DNA from Pure Cultures or from Host Lesions;Griffith G W等;《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》;19981031;第64卷(第10期);4007-4014 * |
| 深部念珠菌的基因诊断、分子分型及耐药机制研究;李劲松;《中国博士学位论文全文数据库医药卫生科技辑》;20041215(第4期);E059-113页 * |
| 线粒体DNA 的异质性与检测方法;刘艳等;《动物学杂志》;20061231;第41卷(第5期);120-126 * |
| 线粒体DNA异质性检测方法的研究进展;翟仙敦和莫耀楠;《河南科技大学学报（医学版）》;20060331;第24卷(第1期);71-73 * |
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