CN104120169A - Phytophthora infestans mitochondrial genotype and heteroplasmy detection kit and detection method thereof - Google Patents

Phytophthora infestans mitochondrial genotype and heteroplasmy detection kit and detection method thereof Download PDF

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CN104120169A
CN104120169A CN201310145528.3A CN201310145528A CN104120169A CN 104120169 A CN104120169 A CN 104120169A CN 201310145528 A CN201310145528 A CN 201310145528A CN 104120169 A CN104120169 A CN 104120169A
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phytophthora infestans
chondriogen
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杨志辉
朱杰华
徐进
齐明星
秦宇轩
桂秀梅
陶晡
许小虎
张福光
杨毅清
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Hebei Agricultural University
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Abstract

The invention belongs to the technical field of Phytophthora infestans mitochondrial genotype and heteroplasmy identification, and aims to provide a kit for rapidly and accurately detecting Phytophthora infestans mitochondrial genotype and heteroplasmy based on a polymerase chain reaction (PCR), and a detection method thereof. The kit includes two pairs of specific primers needed by PCR amplification, and Mix, wherein the two specific primer pairs comprise InDel-F: 5'-GTGGGTTCGAGTCCCACTAA-3', InDel-R: 5'-CTCACCCGTTCGCTATGTTT-3', RSU-F: 5'-ACGGAATTATCGGAAGATTT-3', and RSU-R: 5'-AAATCCCTTTTATACTGTAATTTGAT-3'. The detection method comprises the following steps: extracting genome DNA of a Phytophthora infestans strain; using the two pairs of the specific primers and the kit to carry out PCR; carrying out agarose gel electrophoresis detection of the obtained PCR amplification product to obtain a detection result; observing a band, and naming a band mode; determining the mitochondrial genotype; and determining the mitochondrial heteroplasmy. The kit can be used for Chinese animal and plant inspection and quarantine department to monitor foreign Phytophthora infestans and the agriculture department to monitor Phytophthora infestans colonies in different areas, and can also be used for various seed tuber production enterprises to determine the characteristics of base Phytophthora infestans and monitor the dynamic change.

Description

Phytophthora infestans chondriogen type and heterogeneous detection kit and detection method
Technical field
The invention belongs to pathogenic oomycetes chondriogen type and the heterogeneous technical field detecting.
Background technology
On potato, the destructive late blight of tool is caused by phytophthora infestans, and this disease was once very popular in Europe in the mid-40 in 19th century, caused internationally recognizable " being hard up greatly in Ireland ", the research of this disease had been started to the beginning of plant pathology.Now, this disease is still first disease on potato, and it is 6,700,000,000 dollars that financial loss is caused in the annual whole world, does not comprise the expense of using agricultural chemicals.Significant for pest and disease risk assessment and prevention and control for the multifarious Molecular Detection of pathogenic bacteria, and plastosome is owing to being matrilinear inheritance, its genotypic mensuration be can be used as to the reliable method of phytophthora infestans population surveillance, report in the world at present the measuring method of 2 kinds of phytophthora infestans chondriogen types, and do not had in the world the method for the detection of phytophthora infestans plastosome heterogeneity.Carter has proposed the RFLP method of phytophthora infestans chondriogen type first in nineteen ninety, by separated, purifying phytophthora infestans Mitochondrial Genome Overview DNA, with multiple restriction enzyme, carry out restriction enzyme digestion and electrophoresis again, found the polymorphism of 4 kinds of Mitochondrial Genome Overview, and distinguished called after Ia, Ib, four kinds of chondriogen types such as IIa and IIb, also referred to as plastosome haplotype.But the shortcoming of the method is to need separated and purifying plastochondria genomic dna, complicated operation, time-consuming, and experimental cost is very high, and determination efficiency is extremely low.Subsequently, after the genotypic Mitochondrial Genome Overview sequence of phytophthora infestans Ib comes forth, Griffith in 1998 etc. have located the genomic positions of enzyme tangent line plastochondria such as Carter on genome, in the both sides of restriction enzyme site conserved regions design Auele Specific Primer, this fragment gene is carried out to pcr amplification, then carry out enzyme and cut, thereby set up the genotypic detection method of Mitochondrial Genome Overview easy, efficient PCR-RFLP.But because the PCR-RFLP method of Griffith in 1998 is not considered phytophthora infestans I type and this qualitative difference of II type and is the insertion and deletion of 2kb, thereby cause using the detected result of PCR-RFLP method sometimes sometimes inconsistent with the RFLP method detected result that Carter proposes.In addition, only 4 kinds of the genotypic Limited Numbers of Mitochondrial Genome Overview that above two kinds of methods detect, and fail its heterogeneity to detect.Therefore, the present invention is on to the basis of 4 phytophthora infestans Mitochondrial Genome Overview sequential analysis, found that 2 height region of variability can be used for distinguishing 4 kinds of chondriogen types, 2 hypervariable regions have been designed respectively Auele Specific Primer pair, utilize polymerase chain reaction (PCR), invented phytophthora infestans chondriogen type detection kit; Meanwhile, whether tool is heterogeneous by 2 hypervariable region amplicon band numbers, can to determine it.The method is set up for the monitoring to different zones pathogenic bacterium colony to external phytophthora infestans and agricultural sector of national sanitary authority, also can be used for Ge great potato seed manufacturing enterprise for the monitoring of base phytophthora infestans dynamic change.
Summary of the invention
The object of this invention is to provide a kind of double phytophthora infestans chondriogen type and the heterogeneous detection kit that is detected on one, comprise 2 pairs of PCR primers and Mix, and detection method.
1. technical scheme of the present invention is as follows:
From GenBank, download the genome sequence of 4 kinds of chondriogen types (Ia, Ib, IIa and IIb) of the Carter that has completed order-checking.First, with ClustalX, these 4 Mitochondrial Genome Overview sequences are compared, with BioEdit and MEGA software, genome sequence is analyzed, find that 4 chondriogen type genomes exist 2 kinds of nucleotide polymorphisms, a kind of is to substitute or lack by mononucleotide the single nucleotide polymorphism (SNP) causing, and another is to exist and insert or lack by large fragment the length polymorphism causing in genomic 2 hypervariable regions of plastosome.In the conserved sequence of the both sides of these 2 hypervariable regions, utilize the Primer-BLAST in NCBI to design respectively 2 pairs of narrow spectrum PCR primers, be respectively primer pair InDel-F and InDel-R, and primer pair RSU-F and RSU-R.Utilize respectively these 2 pairs of Auele Specific Primers to carry out pcr amplification to phytophthora infestans genomic dna, by agarose gel electrophoresis, detect the length polymorphism of amplicon, length polymorphism by the different amplicons in 2 hypervariable regions combines determines phytophthora infestans chondriogen type, and according to various combination, propose a set of for determining the naming method of different phytophthora infestans chondriogen types; Meanwhile, according to the band number of each hypervariable region, judge whether phytophthora infestans plastosome has heterogeneity.
2. the present invention's design is to adopt following steps for detection of discriminating phytophthora infestans chondriogen type and heterogeneous novel method:
(1) extraction of phytophthora infestans genomic dna;
(2) amplification of DNA fragments, carries out polymerase chain reaction, for the DNA sequence dna of 2 pairs of primers of polymerase chain reaction, 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.Chondriogen type is by the incompatible judgement of 2 pairs of primer pair amplified band type series, specific as follows;
A. utilize InDel-F and InDel-R primer can amplify 2 bands that length is different, its size is respectively 3 kb and 1 kb, is distinguished called after L and S, and the bacterial strain of different chondriogen types there will be 3 kinds of band pattern: L, S and LS after amplification;
B. utilize RSU-F and RSU-R primer pair can amplify 3 kinds of different bands of length, its length is respectively 192 bp, 228 bp and 264 bp, respectively by its called after V 1, V 2and V 3, the bacterial strain of different chondriogen types there will be 7 kinds of bands and integrated mode, i.e. V after amplification 1, V 2, V 3, V 1v 2, V 1v 3, V 2v 3, V 1v 2v 3;
C. the combination of comprehensive above 2 amplifications, amplification has 21 kinds of situations, and corresponding chondriogen type has 21 kinds, is respectively: LV 1, LV 2, LV 3, LV 1v 2, LV 1v 3, LV 2v 3, LV 1v 2v 3, SV 1, SV 2, SV 3, SV 1v 2, SV 1v 3, SV 2v 3, SV 1v 2v 3, LSV 1, LSV 2, LSV 3, LSV 1v 2, LSV 1v 3, LSV 2v 3, LSV 1v 2v 3;
D. the genotype of phytophthora infestans plastosome heterogeneity is: LV 1v 2, LV 1v 3, LV 2v 3, LV 1v 2v 3, SV 1v 2, SV 1v 3, SV 2v 3, SV 1v 2v 3, LSV 1, LSV 2, LSV 3, LSV 1v 2, LSV 1v 3, LSV 2v 3and LSV 1v 2v 3deng 15 kinds;
Effect of the present invention is: (1) has proposed to utilize PCR method to find the method for phytophthora infestans plastosome heterogeneity first; (2) the phytophthora infestans chondriogen type detection kit of setting up, has increased genotypic detection number greatly, can detect 4 kinds be increased to the method and can finally determine 21 kinds of chondriogen types by original method; (3) this test kit is easy, efficient, accurate, thus overcome forefathers' phytophthora infestans chondriogen type detection method complicated, spend the problems such as high and efficiency is low.
3. embodiment is:
Each 10 μ M 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), MgCl (1) test kit composition comprises: 2(0.8-1.3 mM), KCl (130-160 mM), Tris-HCl (25-30 mM, pH 9.0-9.3), 1% Triton X-100 and stablizer], ddH 2o;
(2) extraction of DNA: the DNA that extracts 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:
Template DNA 1.0 μ L
Primer-AsF 0.5 μ L
Primer-AsR 0.5 μ L
Mix 12.5μL
ddH 2O 10.5μL
B. PCR reaction conditions: reaction is carried out on PCR instrument, and reaction conditions is:
The amplification that utilizes InDel-F and InDel-R primer to carry out, needs first 95 ℃ of denaturations 5 minutes, then carries out 27 circulations according to following condition:
95 ℃ of sex change 30-45 second
65 ℃ of 2-4 minute of renaturation
Extend 72 ℃ of 2-4 minute
After loop ends, at 72 ℃, keep 9-10 minute, after reaction finishes 4 ℃ of preservations;
The amplification that utilizes RSU-F and RSU-R primer to carry out, needs first 95 ℃ of denaturations 2 minutes, then carries out 30 circulations according to following condition:
95 ℃ of sex change 40-50 second
45 ℃ of renaturation 40-50 second
Extend 72 ℃ of 40-50 seconds
After loop ends, at 72 ℃, keep 6-8 minute, after reaction finishes 4 ℃ 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.Electrophoresis 20-50 minute under 5 V/cm strength of electric field, carries out sepharose dyeing with EB, observes stripe size;
(5) judgement of detected result:
A. utilize InDel-F and InDel-R primer can amplify 2 kinds of Bu Tong bands of size, size is respectively 3 kb and 1 kb, difference called after L and S, and the bacterial strain of different chondriogen types there will be 3 kinds of array mode: L, S and LS after amplification;
B. utilize RSU-F and RSU-R primer can amplify 3 different big or small bands, be respectively 192 bp, 228 bp and 264 bp, respectively called after V 1, V 2and V 3, the bacterial strain of different chondriogen types there will be 7 kinds of array mode: V after amplification 1, V 2, V 3, V 1v 2, V 1v 3, V 2v 3, V 1v 2v 3;
C. the combination of comprehensive above 2 amplifications, amplification has 21 kinds of situations, and corresponding chondriogen type has 21 kinds, is respectively: LV 1, LV 2, LV 3, LV 1v 2, LV 1v 3, LV 2v 3, LV 1v 2v 3, SV 1, SV 2, SV 3, SV 1v 2, SV 1v 3, SV 2v 3, SV 1v 2v 3, LSV 1, LSV 2, LSV 3, LSV 1v 2, LSV 1v 3, LSV 2v 3, LSV 1v 2v 3;
D. heterogeneous judgement: when phytophthora infestans genome is carried out to pcr amplification, two and above band appear in any 1 region amplicon of its 2 hypervariable regions simultaneously, this bacterium is heterogeneous.Heterogeneous genotype is totally 15 kinds accordingly, is respectively LV 1v 2, LV 1v 3, LV 2v 3, LV 1v 2v 3, SV 1v 2, SV 1v 3, SV 2v 3, SV 1v 2v 3, LSV 1, LSV 2, LSV 3, LSV 1v 2, LSV 1v 3, LSV 2v 3and LSV 1v 2v 3.

Claims (10)

1. double phytophthora infestans chondriogen type and a heterogeneous test kit that is detected on one, is characterized in that: test kit comprises a pair of Auele Specific Primer DNA sequence dna and Mix.
2. detection phytophthora infestans chondriogen type as claimed in claim 1 and heterogeneous test kit, it is characterized in that: test kit comprises 2 pairs of PCR specific primers pair, be respectively: 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'.
3. detection phytophthora infestans chondriogen type as claimed in claim 1 and heterogeneous test kit, is characterized in that: the composition of test kit Mix is 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), MgCl 2(0.8-1.3 mM), KCl (130-160 mM), Tris-HCl (25-30 mM, pH 9.0-9.3), 1% Triton X-100 and stablizer.
4. test kit as claimed in claim 1, it is characterized in that: the detection method of test kit is, the genomic dna of phytophthora infestans of take is template, utilize 2 pairs of specific primers of designed, designed to carrying out pcr amplification reaction, according to the band pattern of the amplification of 2 PCR and combination, judge whether the chondriogen type of phytophthora infestans and plastosome have heterogeneity.
5. kit test method as claimed in claim 4, is characterized in that: CTAB method is used in the extraction of the genomic dna of phytophthora infestans, with deionized water by DNA concentration adjustment to 5-10 ng/ μ l.
6. reagent test method as claimed in claim 4, is characterized in that: in PCR primer used, PCR is carried out in InDel-F and InDel-R pairing, and RSU-F matches and carries out PCR with RSU-R.
7. the kit test method as described in claim 4 and 6, it is characterized in that: utilize InDel-F and InDel-R specific primers when carrying out PCR, can amplify 2 bands that length is different, its size is respectively 3 kb and 1 kb, distinguished called after L and S, the bacterial strain of different chondriogen types there will be 3 kinds of chondriogen type array modes after amplification: L, S and LS.
8. the detection method as described in claim 4 and 6, is characterized in that: while utilizing RSU-F and RSU-R specific primers to carry out PCR, can amplify 3 bands that vary in size, its length is respectively 192 bp, 228 bp and 264 bp, respectively called after V 1, V 2and V 3, the bacterial strain of different chondriogen types there will be 7 kinds of band pattern after amplification: V 1, V 2, V 3, V 1v 2, V 1v 3, V 2v 3, V 1v 2v 3.
9. the detection method as described in claim 7 and 8, is characterized in that: the band pattern combination of 2 pcr amplification results has 21 kinds altogether, is also that corresponding phytophthora infestans chondriogen type has 21 kinds, and it is respectively: LV 1, LV 2, LV 3, LV 1v 2, LV 1v 3, LV 2v 3, LV 1v 2v 3, SV 1, SV 2, SV 3, SV 1v 2, SV 1v 3, SV 2v 3, SV 1v 2v 3, LSV 1, LSV 2, LSV 3, LSV 1v 2, LSV 1v 3, LSV 2v 3, LSV 1v 2v 3.
10. the detection method as described in claim 1 and 7, is characterized in that: the genotype of phytophthora infestans heterogeneity has LV 1v 2, LV 1v 3, LV 2v 3, LV 1v 2v 3, SV 1v 2, SV 1v 3, SV 2v 3, SV 1v 2v 3, LSV 1, LSV 2, LSV 3, LSV 1v 2, LSV 1v 3, LSV 2v 3and LSV 1v 2v 3.
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Citations (1)

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CN101921832A (en) * 2010-04-13 2010-12-22 南京农业大学 Primer, kit and detection method for detecting phytophthora infestans

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CN101921832A (en) * 2010-04-13 2010-12-22 南京农业大学 Primer, kit and detection method for detecting phytophthora infestans

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李劲松: "深部念珠菌的基因诊断、分子分型及耐药机制研究", 《中国博士学位论文全文数据库医药卫生科技辑》, no. 4, 15 December 2004 (2004-12-15), pages 059 - 113 *
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