CN102559900A

CN102559900A - PCR (polymerase chain reaction) method of specifically detecting sclerophthora macrospora on gramineous plants

Info

Publication number: CN102559900A
Application number: CN2012100150014A
Authority: CN
Inventors: 高必达; 夏花
Original assignee: Hunan Agricultural University
Current assignee: Hunan Agricultural University
Priority date: 2012-01-18
Filing date: 2012-01-18
Publication date: 2012-07-11
Anticipated expiration: 2032-01-18
Also published as: CN102559900B

Abstract

The invention relates to a PCR (polymerase chain reaction) method of specifically detecting sclerophthora macrospora on gramineous plants. The PCR method is characterized in that a pair of primers are designed according to nucleotide sequences of sclerophthora macrospora HUH 892 strain large submit ribosomal RNA (ribonucleic acid) genes (GenBank: EU826119.1 846 basic groups in total), and a BLAST result shows that the pair of primers have good specificity. The pair of primers can detect sclerophthora macrospora on gramineous plants precisely in PCR detection, so that the sclerophthora macrospora can be prevented and treated at an early date.

Description

Big spore refers to the PCR method of phytophthora on the specific detection grass

Technical field

The present invention relates to the detection method that a kind of big spore refers to phytophthora (Sclerophthora macrospora), be specifically related on a kind of specific detection grass PCR (polymerase chain reaction) technology that big spore refers to phytophthora.

Background technology

Big spore refers to that phytophthora is meant a kind of eucaryon bacterium of phytophthora, belongs to Oomycete, and the member's of this guiding principle perfect stage produces oospore.This bacterium parasitizes multiple grass, is prone to cause oidium.Paddy rice receives oidium harm yellowish, curling, the difficult extraction of lobus cardiacus afterwards, and bottom Lao Ye is withered gradually, and plant stunts, and can not ear or the floral organ deformity, causes heavy losses in partial area.

The diagnosis of paddy rice oidium is generally at first tentatively judged on symptom, uses the microscopy diseased tissues again, promptly confirms as the positive if be checked through the germ oospore.But the medication control is late when being checked through oospore, even if plant can ear, and can not be normally solid.And early stage symptom is difficult for distinguishing mutually with herbicide damage and virus disease.Therefore be necessary to set up a kind of technique for detection, microscopy is not diagnosed to the doubtful diseased plant of oospore.

In the detection of the phytopathy original, PCR be a kind of accurately, efficient, use the widest technology, but be applied to the context of detection that big spore refers to phytophthora, do not appear in the newspapers as yet.

Summary of the invention

The present invention wants the technical solution problem to be: to the deficiency of above-mentioned prior art; Provide on a kind of specific detection grass big spore to refer to the PCR method of phytophthora; It refers to that according to big spore the large ribosomal subunit rna gene sequence that phytophthora has been reported comes designs specificity PCR primer; Set up PCR diagnostic techniques system, the big spore that can detect on multiple grass such as paddy rice, the corn etc. refers to phytophthora, solves the difficult problem of paddy rice oidium early detection.

In order to solve the problems of the technologies described above; The technical scheme that the present invention adopted is: big spore refers to the PCR method of phytophthora on a kind of specific detection grass; This method is to refer to that with big spore the phytophthora Auele Specific Primer carries out pcr amplification to testing sample, and last electrophoresis identifies that this Auele Specific Primer is:

Forward primer: 5 '-AGAATGCAGCGCTAAGC-3 ' (shown in SEQ ID No:1)

(corresponding to 207-223 the base of EU826119.1),

Reverse primer: 5 '-ATGCGCATCCACTCAGC-3 ' (shown in SEQ ID No:2)

(corresponding to 629-613 the base of EU826119.1).

Details are as follows:

One, primer design:

The contriver is according to the specificity of eukaryote ribosomal RNA gene base sequence; From NCBI/GenBank search and download big spore and refer to phytophthora HUH 892 fungus strain large ribosomal subunit rna gene base sequences (GenBank:EU826119.1 is totally 846 bases), have to a sequence.

Following according to this sequence with primerBLAST instrument designs specificity PCR primer sequence:

Forward primer: 5 '-AGAATGCAGCGCTAAGC-3 '

(corresponding to 207-223 the base of EU826119.1)

Reverse primer: 5 '-ATGCGCATCCACTCAGC-3 '

(corresponding to 629-613 the base of EU826119.1);

Above-mentioned primer is synthetic by precious biotechnology (Dalian) ltd.PCR product expection size is 423 bases.

Forward primer and reverse primer are retrieved through BLAST respectively, seen Fig. 1 and Fig. 2.The result shows: cover with forward primer sequence and reverse primer sequence equal 100% and base 100% is identical has only EU826119.1.

Two, the PCR process (describing) of germ with paddy rice:

The extraction of DNA: the big spore of doubtful infection is referred to that the rice plant of phytophthora extracts genomic dna by ordinary method.

To the big spore of doubtful infection that extracts is referred to that the total DNA of rice plant of phytophthora increases, estimate that amplified fragments is about 423bp with above-mentioned designed primer.

Pcr amplification system (25 μ l): DNA cloning template 1 μ l, 10 * PCR Buffer, 2.5 μ l, dNTPs (10mm) 0.5 μ l, Taq enzyme (2.5 μ/μ l) 0.5 μ l, Forward Primer (10mm) 1 μ l, Reverse Primer (10mm) 1 μ l, and dd H ₂O 18.5 μ l.

Amplification condition: preparatory 94 ℃ of 5min of sex change; Loop parameter: 94 ℃ of 30sec; 56 ℃ of 40sec; 72 ℃ of 40sec; Totally 30 circulations; 72 ℃ of 10min extend.

Three, the evaluation of pcr amplification product:

Amplified production is through conventional electrophoresis and with the gel imaging system inspection, and the band that the result occurs conforms to the size of expection, sees Fig. 3.

The PCR product send the order-checking by precious biotechnology (Dalian) ltd, and sequencing result only finds the EU826119.1 as the design of primers basis through BLAST, sees Fig. 4.

The round pcr of mentioning among this paper comprises from all technology of PCR deutero-, like real-time fluorescent PCR.

Compared with prior art; Advantage of the present invention is: a pair of primer that present method is used refers to the design of phytophthora HUH 892 fungus strain large ribosomal subunit rna genes according to big spore; When using this that primer is done the PCR detection, the big spore that can detect on the grass refers to phytophthora, thereby in early days big spore is referred to that phytophthora and its kind cause of disease make a distinction in paddy rice oidium and the crazy top sickness morbidity of corn; Prevent and treat the crazy top sickness of paddy rice oidium and corn early, primer of the present invention is high to specificity.

Description of drawings:

Fig. 1 is the BLAST result of forward primer of the present invention.

Fig. 2 is the BLAST result of reverse primer of the present invention.

Fig. 3 is a rice plant PCR Preliminary detection electrophoresis result.4 swimming lanes from left to right are successively: marker, doubtful diseased plant detects the diseased plant of oospore, healthy paddy rice.

Fig. 4 is paddy rice oidium PCR product sequence B LAST.

Fig. 5 is that the different paddy rice diseased plant PCR of counties and cities detect electrophoresis result.

Among the figure, from left to right each swimming lane be followed successively by Longhui, Shaoyang, Jiang Yong, together with, Shaodong, normal healthy controls, marker, Xinshao, Yanling County, bimodal, Anhua 1, Anhua 2, Hongjiang 1, Hongjiang 2.

Fig. 6 is that originally microscopy is not to oospore but PCR male Xinshao County rice plant, and back microscopy is to oospore.

Fig. 7 is that corn PCR detects electrophoresis result.

Among the figure, each swimming lane is respectively from left to right: the negative contrast 1 in the 1st road (not adding dna profiling); The negative contrast 2 in the 2nd road (healthy corn); The 3-5 road is the corn diseased plant; The negative contrast 3 in the 6th road (paddy rice healthy tree); The positive contrast in the 7th road (paddy rice diseased plant); The 8th road is marker.

Fig. 8 is the result who is BLAST of 1 paddy rice downy mildew fungus strain PCR product sequence, has shown that 8 PCR product base sequences are delivered to the accession number that obtains behind the GenBank.

" strain " expression fungus strain among the figure, 7 last 2 letters of paddy rice downy mildew fungus strain name are SD, 1 last 2 letter of the crazy top of corn fungus strain name is YM.Fungus strain HUH892 refers to the phytophthora fungus strain for the big spore of Germany's report.

Embodiment:

Embodiment one paddy rice downy mildew diseased plant detects

With primer of the present invention to picking up from Longhui, Shaoyang, Jiang Yong, being PCR together with the rice plant of the doubtful infection paddy rice oidium in, Shaodong, Xinshao, Yanling County, bimodal, Anhua, Hongjiang and detecting.Electrophoresis result is seen Fig. 5.The result shows that diseased plant all is positive, and normal healthy controls is negative.Every at a distance from 5 days microscopies once to wherein not detecting oospore and planting into Xinshao County diseased plant alive, microscopy is seen Fig. 6 to oospore when diseased plant lobus cardiacus flavescence white.

Choose in the above-mentioned PCR product 7 brighter PCR products of electrophoresis spot and send the order-checking of precious biotechnology (Dalian) ltd, sequence is submitted NCBI/GenBank, obtains accession number (HQ641401.1; HQ641399.1; HQ641402.1, HQ641395.1, HQ641398.1; HQ641397.1, HQ641394.1).Fig. 8 demonstration is the result that BLAST obtains with HQ641401.1, proves the existence of above-mentioned accession number.

The crazy top sickness strain of embodiment two corns detects

With primer of the present invention to having done the PCR detection through the milpa that microscopy confirm to infect crazy top sickness to picking up from the Chenxi County, Hunan.Electrophoresis result is seen Fig. 7.The result shows, 3 corn diseased plant samples and positive control (paddy rice diseased plant) all are positive, and 3 negative controls are all negative.Wherein 1 the crazy top of corn PCR product send the order-checking of precious biotechnology (Dalian) ltd, and sequence is submitted NCBI/GenBank, obtains accession number HQ641400.1, sees Fig. 8.

Fig. 8 shows that what do that sequence that BLAST obtains comes the front with 1 paddy rice downy mildew PCR product all is to find the sequence of this patent primer PCR product and refer to the phytophthora sequence as the big spore of the Germany of template; Proved that the PCR product is consistent with the template sequence height, proved that the PCR product of embodiment one and embodiment two refers to phytophthora for big spore entirely.

Can know that by above embodiment of the present invention this refers to that to big spore phytophthora is special to Auele Specific Primer, adopt this primer that big spore is referred to that the disease that phytophthora causes makes PCR, can obtain result accurately and reliably.

Claims

1. big spore refers to the PCR method of phytophthora on the specific detection grass, and it is characterized in that: this method is to refer to that with big spore the phytophthora Auele Specific Primer carries out pcr amplification to testing sample, and last electrophoresis identifies that this Auele Specific Primer is:

Forward primer: 5 '-AGAATGCAGCGCTAAGC-3 ',

Reverse primer: 5 '-ATGCGCATCCACTCAGC-3 '.

2. big spore refers to the PCR method of phytophthora on the specific detection grass as claimed in claim 1; It is characterized in that: said Auele Specific Primer is to refer to that according to big spore the nucleotide sequence of phytophthora HUH 892 fungus strain large ribosomal subunit rna genes (GenBank:EU826119.1 is totally 846 bases) designs, and PCR product size is 423 bases.