CN104726557A - Double-PCR (polymerase chain reaction) molecular detection primer for tobacco phytophthora parasitica and thielaviopsis basicola and detection method - Google Patents
Double-PCR (polymerase chain reaction) molecular detection primer for tobacco phytophthora parasitica and thielaviopsis basicola and detection method Download PDFInfo
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Abstract
The invention relates to a double-PCR (polymerase chain reaction) molecular detection primer for tobacco phytophthora parasitica and thielaviopsis basicola and a detection method. The detection primer comprises YYI-F: TCATTACCACACCTAAAAAACT, YYI-R: ACTTTCGTCCCCACAGTATATT, TB1419-F: GTGTTGGAGGACCCGCGTTTAG and TB1419-R: AGTTGAGGGTTTTTCGGCATGTT. The detection method comprises the steps of performing extraction on total DNA, PCR amplification and gel electrophoresis under certain conditions. According to the detection primer and the detection method provided by the invention, a specific and high-sensitivity double-PCR molecular detection system for the tobacco phytophthora parasitica and thielaviopsis basicola is established, and by detecting the total DNA sequences of materials including tobacco diseased plants, soil and the like, the quick, accurate and ultralow-concentration once double-identification of the tobacco phytophthora parasitica and thielaviopsis basicola is finished.
Description
Technical field
The invention belongs to plant pest quarantine field, be specifically related to double PCR molecular detection primer and the detection method of a kind of tobacco black shank bacterium and root black rot.
Background technology
Tobacco is that Yield and quality is laid equal stress on, and especially payes attention to the crop of quality, and scientific prevention and cure disease and pest ensures that tobacco normal growth is grown, and realizes the key of high-quality, high yield.Black shank and root Black Rotten (the black disease of tobacco two) are global diseases; these two kinds of diseases can occur often together; once morbidity will cause huge financial loss; in Yunnan Province of China, Guizhou, Hubei etc., main Chan Yan district occurs heavier, and serious plot sickness rate can reach more than 30%.Also there is generation in Shandong, Henan, Anhui, Jilin, Deng Chanyan district, Fujian, in recent years have the trend increasing the weight of to endanger.But tobacco black shank bacterium and root black rot, as the two kinds of soil-borne fungus often occurred together, carry out single detection and certainly will cause many troubles, time-consuming, have not been reported at present, do not form specificity and high sensitivity system to its double check.
Summary of the invention
For solving the problem, the invention provides tobacco black shank bacterium and the root black rot double PCR molecular detection primer of a species specificity and high sensitivity, and provide its detection method, establish the tobacco two black germ double PCR Molecular Detection system of specificity and high sensitivity.
The present invention is achieved through the following technical solutions:
Design the double PCR molecular detection primer of a kind of tobacco black shank bacterium and root black rot, comprise following primer pair:
YYI-F：TCATTACCACACCTAAAAAACT，
YYI-R：ACTTTCGTCCCCACAGTATATT；
TB1419-F：GTGTTGGAGGACCCGCGTTTAG，
TB1419-R：AGTTGAGGGTTTTTCGGCATGTT。
Design the double PCR molecular detecting method of a kind of tobacco black shank bacterium and root black rot, comprise the following steps:
(1) STb gene in cigarette strain to be detected, soil or its mixing material is extracted;
(2) with described STb gene for primer described in template claim 1 carries out pcr amplification, adopt 20 μ L reaction systems, comprising: the 5u/ μ L rTaq enzyme of 0.2 μ L, each 0.4 μ L is YYI-F, YYI-R, TB1419-F and TB1419-R primer of 10 μm of ol/L, the STb gene of 1 μ L, the 2.5mmol/L dNTP of 1.5 μ L, the 2.5mmol/L MgCl of 1.0 μ L
2solution, 10 × PCR Buffer of 2 μ L, surplus is ddH
2o; Pcr amplification program: 95 DEG C of denaturation 4min, 95 DEG C of sex change 30s of 35 circulations, 59 DEG C of annealing 50s and 72 DEG C of extension 30s, finally extend 5min at 72 DEG C;
(3) get gained PCR primer and carry out agarose gel electrophoresis, as there is specific band at 208bp place, illustrating that material to be detected is with tobacco black shank bacterium, otherwise then not carrying; As there is specific band at 131bp place, illustrating that material to be detected is with black root of tobacco bacterium, otherwise then not carrying.
For above-mentioned double PCR molecular detecting method, described STb gene concentration is not less than 50ng/ μ L.
positive beneficial effect of the present invention:
(1) the present invention is in the conserved regions of comprehensive consideration DNA sequence dna, whether form secondary structure, G+C content, base stochastic distribution, length, on the basis of the many factors such as the interference of similar sequences, and through necessary modification and a large amount of verification experimental verifications, final design has also filtered out tobacco black shank bacterium and the root black rot double PCR molecular detection primer of specificity and high sensitivity, and provide its detection method, establish the tobacco two black germ double PCR Molecular Detection system of specificity and high sensitivity, by detecting cigarette strain invalid body, the full DNA sequence of soil or its mixing material, complete the quick and precisely ultralow density once dual qualification of tobacco black shank bacterium and root black rot, and furthermore achieved that the accurate early warning monitoring to pest and disease risk under different tillage and cultivation condition, what significantly improve the two black disease of tobacco prevents and treats level, meet the active demand in leaf tobacco production.
(2) double PCR Molecular Detection of the present invention can be reacted with two pairs of primer pairs, two kinds of To Templates simultaneously, so, higher to the design requirements of primer, stable secondary structure can not be formed between two pairs of primers, therefore, the design of primer, while guarantee primer amplification specificity and susceptibility, also will meet the condition of double PCR.
(3) the present invention be elected to be detect target sequence be the rDNA-ITS of tobacco black shank bacterium and the rDNA-ITS sequence of black root of tobacco bacterium, the rDNA-ITS sequence similarity of these two kinds of pathogenic bacterias is high, when designing primer, difficulty is very big, and the present invention detects primer and detection system and overcomes the technical barrier being difficult to carry out double PCR Molecular Detection because the sequence degree of approximation is high for a long time.
Accompanying drawing explanation
Specificity identification (one) the agarose gel electrophoresis figure of Fig. 1 primer of the present invention;
In figure, M is DL 2000 DNA Marker, and 1 is black root of tobacco bacterium, and 2 is tobacco black shank bacterium, and 3 is sickle-like bacteria, and 4 is tobacco brown spot pathogen, and 5 is tobacco botrytis cinerea, and 6 is normal tobacco leaf, and 7 is Tobacco Angular Leaf Spot Disease bacterium, and 8 is frog-eye leaf spot of tobacco bacterium, and CK is ddH
2o blank, the rDNA-ITS sequence of above-mentioned each pathogenic bacteria all has higher similarity.
Specificity identification (two) the agarose gel electrophoresis figure of Fig. 2 primer of the present invention;
In figure, M is DL 2000 DNA Marker, and 1 ~ 4 is respectively black root of tobacco bacterium and tobacco black shank bacterium mixed bacterium 1, black root of tobacco bacterium and tobacco black shank bacterium mixed bacterium 2, black root of tobacco bacterium, tobacco black shank bacterium, and CK is ddH
2o blank.
Fig. 3 embodiment of the present invention 2 detected result agarose gel electrophoresis figure;
In figure, M is DL 2000 DNA Marker, and 1 ~ 8 hybrid dna being respectively tobacco black shank bacterium and root black rot, soil sample DNA1, soil sample DNA2, soil sample DNA3, soil sample DNA4, soil sample DNA 5, soil sample DNA 6, soil sample DNA7, CK are ddH
2o blank.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in more detail.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
embodiment 1
A double PCR molecular detection primer for tobacco black shank bacterium and root black rot, primer pair is as follows:
YYI-F：TCATTACCACACCTAAAAAACT，
YYI-R：ACTTTCGTCCCCACAGTATATT；
TB1419-F：GTGTTGGAGGACCCGCGTTTAG，
TB1419-R：AGTTGAGGGTTTTTCGGCATGTT。
the specificity identification test of primer of the present invention:
(1) respectively with the genomic dna 50ng/ μ L of black root of tobacco bacterium, the genomic dna 50ng/ μ L of black shank bacterium, and with the tobacco brown spot pathogen of concentration, tobacco ash arrhizus bacteria, frog-eye leaf spot of tobacco bacterium, tobacco leaf, the genomic dna of tobacco Cercospora Sojina Hara and sickle-like bacteria is template, pcr amplification is carried out with primer of the present invention, adopt 20 μ L reaction systems, comprising: the 5u/ μ L rTaq enzyme of 0.2 μ L, each 0.4 μ L is the YYI-F of 10 μm of ol/L, YYI-R, TB1419-F and TB1419-R primer, the STb gene of 1 μ L, the 2.5mmol/L dNTP of 1.5 μ L, the 2.5mmol/L MgCl of 1.0 μ L
2solution, 10 × PCR Buffer of 2 μ L, surplus is ddH
2o, pcr amplification program: 95 DEG C of denaturation 4min, 95 DEG C of sex change 30 s of 35 circulations, 59 DEG C of annealing, 50 s and 72 DEG C of extension 30 s, finally extend 5 min at 72 DEG C.
Get gained 8 groups of PCR primer respectively and carry out agarose gel electrophoresis, only have the 1st group to occur specific band at 131bp place, illustrate that this group is with black root of tobacco bacterium; Only have the 2nd group to occur specific band at 208bp place, illustrate that this group is with tobacco black shank bacterium, sees Fig. 1, show that the specificity of system of the present invention detection single culture is stronger.
(2) respectively with black root of tobacco bacterium and tobacco black shank bacterium mixed bacterium 1, black root of tobacco bacterium and tobacco black shank bacterium mixed bacterium 2, black root of tobacco bacterium, the genomic dna of tobacco black shank bacterium is template, concentration is 50ng/ μ L, pcr amplification is carried out with primer of the present invention, adopt 20 μ L reaction systems, comprising: the 5u/ μ L rTaq enzyme of 0.2 μ L, each 0.4 μ L is the YYI-F of 10 μm of ol/L, YYI-R, TB1419-F and TB1419-R primer, the STb gene of 1 μ L, the 2.5mmol/L dNTP of 1.5 μ L, the 2.5mmol/L MgCl of 1.0 μ L
2solution, 10 × PCR Buffer of 2 μ L, surplus is ddH
2o, pcr amplification program: 95 DEG C of denaturation 4min, 95 DEG C of sex change 30s of 35 circulations, 59 DEG C of annealing 50s and 72 DEG C of extension 30s, finally extend 5min at 72 DEG C.
Get gained 4 groups of PCR primer respectively and carry out agarose gel electrophoresis, the 1st, 2 group all there is specific band at 208bp place and 131bp place, illustrates that this group is with black root of tobacco bacterium and tobacco black shank bacterium; 3rd group there is specific band at 131bp place, illustrates that this group is with black root of tobacco bacterium, and does not carry tobacco black shank bacterium; 4th group there is specific band at 208bp place, illustrates that this group is with tobacco black shank bacterium, and does not carry black root of tobacco bacterium, see Fig. 2, shows that the specificity of system of the present invention detection single culture and dual bacterial classification is all stronger.
embodiment 2
A double PCR molecular detecting method for tobacco black shank bacterium and root black rot, comprises the following steps:
(1) from the soil of different vega, soil sample STb gene is extracted respectively: soil sample DNA1, soil sample DNA2, soil sample DNA3, soil sample DNA4, soil sample DNA 5, soil sample DNA 6, soil sample DNA7;
(2) with the hybrid dna 50ng/ μ L of tobacco black shank bacterium and root black rot, and above-mentioned each STb gene is that template embodiment 1 primer carries out pcr amplification, adopt 20 μ L reaction systems, comprising: the 5u/ μ L rTaq enzyme of 0.2 μ L, each 0.4 μ L is YYI-F, YYI-R, TB1419-F and TB1419-R primer of 10 μm of ol/L, the STb gene of 1 μ L, the 2.5mmol/L dNTP of 1.5 μ L, the 2.5mmol/L MgCl of 1.0 μ L
2solution, 10 × PCR Buffer of 2 μ L, surplus is ddH
2o; Pcr amplification program: 95 DEG C of denaturation 4min, 95 DEG C of sex change 30 s of 35 circulations, 59 DEG C of annealing, 50 s and 72 DEG C of extension 30 s, finally extend 5 min at 72 DEG C.
(3) get gained 8 groups of PCR primer and carry out agarose gel electrophoresis, 1st, 6,7,8 groups all there is specific band at 208bp place and 131bp place, illustrate that this group is with black root of tobacco bacterium and tobacco black shank bacterium, otherwise 2nd, 3,4,5 groups there is not specific band, illustrate that this group does not carry two black germs, see Fig. 3.
The present invention relates to the method extracting STb gene in bacterial strain, soil or its mixing material and be routine techniques, do not repeat them here.
The present embodiment tobacco black shank bacterium used, root black rot bacterial strain are provided by University Of Science and Technology Of He'nan, are that main Chan Yan district, the Henan such as 2012 ~ 2013 years Luoyang from Henan, Nanyang, Zhengzhou gathers; The agriculture university's plant protection institute microbial collection room present of Alternaria alternate bacterial strain, frog-eye leaf spot of tobacco bacterial strain, tobacco gray mold bacterial strain effluent south, sickle-like bacteria bacterial strain is presented by academy of agricultural sciences of Henan Province, and tobacco leaf is laboratory plantation gained.Soil sample picks up from Yiyang, Luoyang vega.
The present invention is not limited to above-mentioned embodiment, and those skilled in the art also can make multiple change accordingly, but to be anyly equal to the present invention or similar change all should be encompassed in the scope of the claims in the present invention.
Claims (3)
1. a double PCR molecular detection primer for tobacco black shank bacterium and root black rot, is characterized in that, comprises following primer pair:
YYI-F：TCATTACCACACCTAAAAAACT，
YYI-R：ACTTTCGTCCCCACAGTATATT；
TB1419-F：GTGTTGGAGGACCCGCGTTTAG，
TB1419-R：AGTTGAGGGTTTTTCGGCATGTT。
2. a double PCR molecular detecting method for tobacco black shank bacterium and root black rot, is characterized in that, comprise the following steps:
(1) STb gene in cigarette strain to be detected, soil or its mixing material is extracted;
(2) with described STb gene for primer described in template claim 1 carries out pcr amplification, adopt 20 μ L reaction systems, comprising: the 5u/ μ L rTaq enzyme of 0.2 μ L, each 0.4 μ L is YYI-F, YYI-R, TB1419-F and TB1419-R primer of 10 μm of ol/L, the STb gene of 1 μ L, the 2.5mmol/L dNTP of 1.5 μ L, the 2.5mmol/L MgCl of 1.0 μ L
2solution, 10 × PCR Buffer of 2 μ L, surplus is ddH
2o; Pcr amplification program: 95 DEG C of denaturation 4min, 95 DEG C of sex change 30s of 35 circulations, 59 DEG C of annealing 50s and 72 DEG C of extension 30s, finally extend 5min at 72 DEG C;
(3) get gained PCR primer and carry out agarose gel electrophoresis, as there is specific band at 208bp place, illustrating that material to be detected is with tobacco black shank bacterium, otherwise then not carrying; As there is specific band at 131bp place, illustrating that material to be detected is with black root of tobacco bacterium, otherwise then not carrying.
3. for double PCR molecular detecting method according to claim 2, it is characterized in that: described STb gene concentration is not less than 50ng/ μ L.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105274241A (en) * | 2015-11-21 | 2016-01-27 | 云南省烟草公司大理州公司 | Molecular detection primer and quick detection method for thielaviopsis basicola |
| CN105543392A (en) * | 2016-02-19 | 2016-05-04 | 河南农业大学 | Duplex-PCR detection method for maize kernel rot pathogen |
| CN111961744A (en) * | 2020-08-31 | 2020-11-20 | 中国烟草总公司郑州烟草研究院 | Tobacco black shank early warning gene Ntab0278480 and application thereof |
| CN112143825A (en) * | 2020-09-28 | 2020-12-29 | 华南农业大学 | Dual PCR detection primer for distinguishing and detecting peanut black rot and peanut-based rot and application |
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| CN104232782A (en) * | 2014-09-29 | 2014-12-24 | 云南省烟草农业科学研究院 | PCR (polymerase chain reaction) primer for detecting tobacco soil-borne fungal pathogens as well as application and method of PCR primer |
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Patent Citations (1)
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| CN104232782A (en) * | 2014-09-29 | 2014-12-24 | 云南省烟草农业科学研究院 | PCR (polymerase chain reaction) primer for detecting tobacco soil-borne fungal pathogens as well as application and method of PCR primer |
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| 张丽芳等: "3 对引物快速鉴定烟草黑胫病菌的比较研究", 《云南农业大学学报》 * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105274241A (en) * | 2015-11-21 | 2016-01-27 | 云南省烟草公司大理州公司 | Molecular detection primer and quick detection method for thielaviopsis basicola |
| CN105543392A (en) * | 2016-02-19 | 2016-05-04 | 河南农业大学 | Duplex-PCR detection method for maize kernel rot pathogen |
| CN111961744A (en) * | 2020-08-31 | 2020-11-20 | 中国烟草总公司郑州烟草研究院 | Tobacco black shank early warning gene Ntab0278480 and application thereof |
| CN111961744B (en) * | 2020-08-31 | 2022-06-03 | 中国烟草总公司郑州烟草研究院 | Tobacco black shank early warning gene Ntab0278480 and application thereof |
| CN112143825A (en) * | 2020-09-28 | 2020-12-29 | 华南农业大学 | Dual PCR detection primer for distinguishing and detecting peanut black rot and peanut-based rot and application |
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