CN101787390A - Kit used for detecting soybean diaporthe/phomopsis syndrome and detection method - Google Patents
Kit used for detecting soybean diaporthe/phomopsis syndrome and detection method Download PDFInfo
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Abstract
The invention provides a kit used for detecting soybean diaporthe/phomopsis syndrome and a detection method. The kit comprises real-time fluorescence reaction mixed liquor TaqMan Universal PCR Master Mix including Buffer, Mgcl2, dNTP and Taq enzyme, a primer and a probe. Compared with the prior art, the invention has the advantage that the molecular biology identification and detection of the south-north diaporthe phaseolorum and soybean phomopsis corynesbacterium Michiganense can be carried out on disease tissues found in the processes of actual quarantine inspection and field survey, so as to realizing the significant meaning for the entry-exit inspection and quarantine of China.
Description
Technical field
The present invention relates to a kind of test kit and detection method that is used to detect the soybean diaporthe/phomopsis syndromes.
Background technology
Soybean north stem canker germ Diaporthe phaseolorum var.caulivora (DPC), soybean south stem canker germ Diaporthe phaseolorum var.meridionalis (DPM) is very similar on form to the allied species soybean plan stem rotten germ Phomopsis longicolla of dibbling (PL), on the soybean of being everlasting, cause compound invading so, cause diaporthe/phomopsis syndrome, it is the destructive disease on soybean produces, can cause the soybean in vegetative period and ripening stage dead in flakes, once make the soybean yields loss of Middle West serious in early days in late period nineteen forties and the fifties, caused the world soybean production loss to be about more than 200 ten thousand tons in 1994; The big generation in South America again of the stem canker 1980s, the production loss that causes susceptible soybean varieties is up to 80%, the loss that nineteen eighty-three causes at southeastern US is 37,000,000 dollars, 1996 to 2006 U.S. is because of 1,550,000 tons of Peptic Ulcers loss soybean, and Argentina, Bolivia, Brazil, Canada, Italy, Paraguay, the U.S. lost 19.1 ten thousand tons of soybean altogether because of the stem canker disease in 1998.Soybean is intended the rotten germ of stem dibbling and can cause soybean root rot and influence the soybean seeds quality, causes soybean quality to descend and seed germination rate reduces, and can cause 90% seed not sprout when serious, and economic impact is very big.These germs mainly are distributed in ground such as the U.S., Argentina, Brazil, Canada, Italy, Former Yugoslavia, Croatia, Paraguay, Europe at present.These germs seed of all can causing harm, and on the residual body of soybean plant strain, survive the winter with mycelia and perithecium, pycnidial form, resistance is very strong, and long-distance communications are mainly propagated by fall ill seed and invalid body in the trade of soybean raw grain.China is the big producing country of soybean, at present domestic also do not have the soybean north and southern stem canker disease and intend the generation of the rotten germ of stem dibbling, preceding two kinds of germs still are the inward Plant Quarantine harmful organism of China, artificial and the natural propagation of these germs has constituted great potential threat to soybean in China, and detection technique is the effective means that stops germ to be imported into quickly and accurately.Methods such as RAPD, PCR-RFLP, serology successively are applied to the evaluation of southern and northern stem canker germ of soybean and the rotten germ of soybean plan stem dibbling in recent years, but there are shortcomings such as the strong or repeatability of the not high or specificity of complicated operation or detection sensitivity is bad in these methods, are difficult to apply in actual detected work.Identify the profuse experience that needs by morphology, erroneous judgement easily, and also sense cycle is long.
Summary of the invention
Technical problem to be solved by this invention is: propose southern and northern stem canker germ of quick, reliable, highly sensitive, high specificity and low-cost detection soybean and soybean and intend primer, probe, test kit and the real-time fluorescence PCR detection method of the rotten germ of stem dibbling.
The objective of the invention is to be achieved through the following technical solutions:
A kind of test kit of soybean diaporthe/phomopsis syndromes comprises: contain Buffer, Mgcl
2, dNTP, real-time fluorescence reaction mixture TaqMan Universal PCR Master Mix, primer and the probe of Taq enzyme.
Furthermore, the test kit of described soybean diaporthe/phomopsis syndromes, its component is: the described Buffer that contains, Mgcl
2, dNTP, the per 10 μ L of real-time fluorescence reaction mixture TaqManUniversal PCR Master Mix of Taq enzyme, described primer 0.9 μ M, described probe 0.2 μ M.
Furthermore, described primer comprises:
A6PL 1F sequence is 5 '-soybean of GAGCGTCATTTCAACCCTCAA-3 ' intends the rotten germ upstream primer of stem dibbling, and,
A6PL 1R sequence is 5 '-soybean of GGCCTTCCAGAGCGAGATATG-3 ' intends the rotten germ downstream primer of stem dibbling;
Described probe comprises:
The A6PL1M sequence is 5 '-soybean of FAM-TTCGTCCAGAAAGC-MGB-3 ' intends the rotten germ probe of stem dibbling.
Furthermore, described primer comprises:
The DP1F sequence is 5 '-the general upstream primer of soybean diaporthe/phomopsis syndromes of AGCCAGGCTTGAGGGTTGA-3 ', and
The DP1R sequence is 5 '-the general downstream primer of soybean diaporthe/phomopsis syndromes of GAATTCAGTGAATCATCGAATCTTTG-3 ';
Described probe comprises:
The DP1P sequence is 5 '-
The soybean diaporthe/phomopsis syndromes general probe of FAM-ACATTGCGCCCTCTGGTATTCCGG-TAMAR-3 '.
Furthermore, described primer comprises:
The DM1F sequence is 5 '-the soybean south stem canker germ upstream primer of AGGCGCACCCAGAAACC-3 ', and,
The DM1R sequence is 5 '-the soybean south stem canker germ downstream primer of TTTTGCTCAGAGTTTCGGTGTAAA-3 ';
Described probe comprises:
The DM1M sequence is 5 '-the soybean south stem canker germ probe of FAM-TGAACTCATACCTTACTGTTG-MGB-3 '.
Furthermore, described primer comprises:
The DC2F sequence is 5 '-the soybean north stem canker germ upstream primer of AGAACCAAGAGATCCGTTGTTGAA-3 ', and,
The DC2R sequence is 5 '-the soybean north stem canker germ downstream primer of CCGGCGGCCAAGCTA-3 ';
Described probe comprises:
The DC2M sequence is the soybean north stem canker germ probe of 5 '-FAM-CATTTATGTTTATTTCTCAGAGTTT-MGB-3 '.
Adopt the detection method of the test kit of described soybean diaporthe/phomopsis syndromes, it is characterized in that, comprise the steps:
A, in determinand, add described primer, probe;
B, amplification step;
The Quality Control step: adopt the test kit of soybean diaporthe/phomopsis syndromes, wherein, described primer comprises:
The DP1F sequence is 5 '-the general upstream primer of soybean diaporthe/phomopsis syndromes of AGCCAGGCTTGAGGGTTGA-3 ', and
The DP1R sequence is 5 '-the general downstream primer of soybean diaporthe/phomopsis syndromes of GAATTCAGTGAATCATCGAATCTTTG-3 ';
Described probe comprises:
The DP1P sequence is 5 '-
The soybean diaporthe/phomopsis syndromes general probe of FAM-ACATTGCGCCCTCTGGTATTCCGG-TAMAR-3 ';
Carry out described steps A and B; And,
Determination step.
Described amplification step comprises the steps:
50 ℃ continue 2 minutes;
95 ℃ continue 10 minutes;
D, 95 ℃ continue 15 seconds;
63 ℃ continue 1 minute, carry out described step D then, till described step D has carried out 40 circulations.
Compared with prior art the invention has the advantages that, can carry out the southern and northern stem canker germ of soybean, the rotten germ molecular biology identification of soybean plan stem dibbling or detection to diseased tissues and the germ of being found in reality quarantine and the field investigation process, significant to the inspection and quarantining for import/export of China.
Description of drawings
The specific detection synoptic diagram of Fig. 1 embodiment that to be the test kit that adopts soybean diaporthe/phomopsis syndromes of the present invention detect for soybean south stem canker germ (Diaporthe phaseolorum var.meridionalis);
The specific detection synoptic diagram of Fig. 2 embodiment that to be the test kit that adopts soybean diaporthe/phomopsis syndromes of the present invention detect for soybean north stem canker germ (Diaporthe phaseolorum var.caulivora);
To be the test kit that adopts soybean diaporthe/phomopsis syndromes of the present invention intend the specific detection synoptic diagram of the embodiment that the rotten germ of stem dibbling detects for soybean to Fig. 3;
Fig. 4 adopts the specific detection synoptic diagram of the test kit of soybean diaporthe/phomopsis syndromes of the present invention for the Quality Control in the soybean diaporthe/phomopsis syndromes testing process.
Embodiment
The invention will be further described below in conjunction with accompanying drawing and preferred embodiment.
The invention discloses the test kit that is used for the southern and northern stem canker germ of soybean, the rotten germ real-time fluorescence PCR detection of soybean plan stem dibbling, described test kit comprises above-mentioned primer, probe.
The invention also discloses the southern and northern stem canker germ of soybean, the rotten germ real-time fluorescence PCR of soybean plan stem dibbling detection method, comprise the steps:
(1) earlier the southern and northern stem canker germ of soybean, soybean are intended the rotten germ of stem dibbling and allied species carries out sequencing and comparative analysis, find out the southern and northern stem canker germ of soybean respectively, soybean is intended the exclusive base site that the rotten germ of stem dibbling is different from other allied species; (2) according to the principle of design and the method for design of TaqMan MGB probe, the southern and northern stem canker germ of design soybean, soybean are intended special TaqMan MGB probe, primer and general probe, the primer that the rotten germ real-time fluorescence PCR of stem dibbling detects; (3) designed probe is screened and the optimization of reaction system and reaction conditions, filter out Auele Specific Primer, probe and universal primer, the probe of optimization, and suitable reaction system and the reaction conditions of optimization; (4) add above-mentioned primer, probe and carry out the southern and northern stem canker germ of soybean, the rotten germ real-time fluorescence PCR reaction of soybean plan stem dibbling.
The invention discloses the oligonucleotide probe and the primer that are used for southern and northern stem canker germ of soybean and the rotten germ real-time fluorescence PCR detection of soybean plan stem dibbling, described oligonucleotide probe comprises TaqMan MGB primer, the probe of specific detection soybean south stem canker germ; TaqManMGB probe of the TaqMan MGB primer of detection soybean north stem canker germ, probe and the rotten germ of detection soybean plan stem dibbling and detection soybean diaporthe/phomopsis syndromes Quality Control primer, probe see the following form:
Example 1, soybean south stem canker germ TaqMan MGB probe for real-time fluorescence PCR detect and test kit
As shown in Figure 1, be used for primer, probe and test kit that soybean south stem canker germ (Diaporthe phaseolorum var.meridionalis) real-time fluorescence PCR detects, be used for the specific detection of soybean south stem canker germ.
Its test kit comprises: use the real-time fluorescence PCR assay kit that soybean south stem canker germ detects that is used for that this primer, probe make.
Example 2, soybean north stem canker germ TaqMan MGB probe for real-time fluorescence PCR detect and test kit
As shown in Figure 2, be used for primer, probe and test kit that soybean north stem canker germ (Diaporthe phaseolorum var.caulivora) real-time fluorescence PCR detects, be used for the specific detection of soybean north stem canker germ.
Its test kit comprises: use the real-time fluorescence PCR assay kit that soybean north stem canker germ detects that is used for that this primer, probe make.
Example 3, soybean are intended the rotten germ TaqMan of stem dibbling MGB probe for real-time fluorescence PCR and detect and test kit
As shown in Figure 3, be used for soybean and intend primer, probe and the test kit that rotten germ (Phomopsis longicolla) real-time fluorescence PCR of stem dibbling detects, be used for the specific detection that soybean is intended the rotten germ of stem dibbling.
Test kit comprises: use the soybean that is used for that this primer, probe make and intend the real-time fluorescence PCR assay kit that the rotten germ of stem dibbling detects.
Example 4, soybean diaporthe/phomopsis syndromes TaqMan probe for real-time fluorescence PCR detect and test kit
As shown in Figure 4, be used for universal primer, probe and test kit that soybean diaporthe/phomopsis syndromes real-time fluorescence PCR detects, be used for the Quality Control of soybean diaporthe/phomopsis syndromes testing process.The Quality Control step is promptly carried out definite once more process to the result of check, and this step also can be in the initial period of whole check, so that the object that is detected is screened certainly.
More than all test kits the embodiment of following component is all arranged:
Test kit comprises: use the real-time fluorescence PCR assay kit that the soybean diaporthe/phomopsis syndromes detects that is used for that this primer, probe make.The system cumulative volume of its detection reaction is 10 μ L, and each composition is: real-time fluorescence reaction mixture TaqMan Universal PCR Master Mix (contains Buffer, Mgcl
2, dNTP, Taq enzyme), primer 0.9 μ M, probe 0.2 μ M.
Its detection reaction condition is an amplification step, comprises the steps:
50 ℃ continue 2 minutes;
95 ℃ continue 10 minutes;
D, 95 ℃ continue 15 seconds;
63 ℃ continue 1 minute, carry out described step D then, till described step D has carried out 40 circulations.
Its criterion as a result is: 1) if the positive control report fluorescence that it is template that the quantitative PCR instrument detects with the southern and northern stem canker germ of soybean, the rotten germ DNA of soybean plan stem dibbling has clear signal to increase, the report fluorescence of negative control and blank does not have fluorescent signal to increase, and test result of samples is to report that fluorescent signal rises appreciably, report fluorescence positive control, negative control and the sample of Quality Control all has clear signal to increase, and represents that then the fungi that is detected is that the southern and northern stem canker germ of soybean, soybean are intended the rotten germ of stem dibbling; 2) if the positive control report fluorescence that it is template that the quantitative PCR instrument detects with the southern and northern stem canker germ of soybean, the rotten germ DNA of soybean plan stem dibbling has fluorescent signal to increase, the report fluorescence of negative control and blank does not have fluorescent signal to increase, and test result of samples is to report that fluorescence does not have fluorescent signal to increase yet, the growth that the report fluorescence positive control of Quality Control, negative control have fluorescent signal represents that then the fungi that is detected is not the soybean diaporthe/phomopsis syndromes.
The experimental example of a check is as follows:
The quarantine of imported soybean north stem canker germ is identified.From the import U.S. soybean, find the suspicious beans bar of falling ill, detect that the real-time fluorescence PCR detected result is according to the method described in embodiment 2 and the embodiment 4:
Be positive with soybean diaporthe/phomopsis syndromes universal primer, probe DP1F/DP1R, DP1P real-time fluorescence PCR result, soybean north stem canker germ Auele Specific Primer, probe DC2F/DC2R, DC2M real-time fluorescence PCR result are positive.
Adopt same procedure to verify all the other kinds (soybean south stem canker germ, soybean are intended the rotten germ of stem dibbling), the result all proves and adopts method of the present invention to detect the soybean diaporthe/phomopsis syndromes than traditional form method more fast, accurately, and is easy to apply.
Above content be in conjunction with concrete preferred implementation to further describing that the present invention did, can not assert that concrete enforcement of the present invention is confined to these explanations.For the general technical staff of the technical field of the invention, without departing from the inventive concept of the premise, can also make some simple deduction or replace, all should be considered as belonging to protection scope of the present invention.
Claims (8)
1. a test kit that is used to detect the soybean diaporthe/phomopsis syndromes is characterized in that, comprising: contain Buffer, Mgcl
2, dNTP, real-time fluorescence reaction mixture TaqManUniversal PCR Master Mix, primer and the probe of Taq enzyme.
2. the test kit that is used to detect the soybean diaporthe/phomopsis syndromes as claimed in claim 1 is characterized in that, its component is: the described Buffer that contains, Mgcl
2, dNTP, the per 10 μ L of real-time fluorescence reaction mixture TaqMan Universal PCR Master Mix of Taq enzyme, described primer 0.9 μ M, described probe 0.2 μ M.
3. the test kit that is used to detect the soybean diaporthe/phomopsis syndromes as claimed in claim 1 is characterized in that,
Described primer comprises:
A6PL 1F sequence is 5 '-soybean of GAGCGTCATTTCAACCCTCAA-3 ' intends the rotten germ upstream primer of stem dibbling, and,
A6PL 1R sequence is 5 '-soybean of GGCCTTCCAGAGCGAGATATG-3 ' intends the rotten germ downstream primer of stem dibbling;
Described probe comprises:
A6PL 1M sequence is 5 '-soybean of FAM-TTCGTCCAGAAAGC-MGB-3 ' intends the rotten germ probe of stem dibbling.
4. the test kit that is used to detect the soybean diaporthe/phomopsis syndromes as claimed in claim 1 is characterized in that,
Described primer comprises:
The DP1F sequence is 5 '-the general upstream primer of soybean diaporthe/phomopsis syndromes of AGCCAGGCTTGAGGGTTGA-3 ', and
DP 1R sequence is 5 '-the general downstream primer of soybean diaporthe/phomopsis syndromes of GAATTCAGTGAATC ATCGAATCTTTG-3 ';
Described probe comprises:
DP 1P sequence is 5 '-the soybean diaporthe/phomopsis syndromes general probe of FAM-ACATTGCGCCCTCTGGTATTCCGG-TAMAR-3 '.
5. the test kit that is used to detect the soybean diaporthe/phomopsis syndromes as claimed in claim 1 is characterized in that,
Described primer comprises:
The DM1F sequence is 5 '-the soybean south stem canker germ upstream primer of AGGCGCACCCAGAAACC-3 ', and,
The DM1R sequence is 5 '-the soybean south stem canker germ downstream primer of TTTTGCTCAGAGTTTCGGTGTAAA-3 ';
Described probe comprises:
DM 1M sequence is 5 '-the soybean south stem canker germ probe of FAM-TGAACTCATACCTTACTGTTG-MGB-3 '.
6. the test kit that is used to detect the soybean diaporthe/phomopsis syndromes as claimed in claim 1 is characterized in that,
Described primer comprises:
The DC2F sequence is 5 '-the soybean north stem canker germ upstream primer of AGAACCAAGAGATCCGTTGTTGAA-3 ', and,
The DC2R sequence is 5 '-the soybean north stem canker germ downstream primer of CCGGCGGCCAAGCTA-3 ';
Described probe comprises:
The DC2M sequence is the soybean north stem canker germ probe of 5 '-FAM-CATTTATGTTTATTTCTCAGAGTTT-MGB-3 '.
7. adopt as the described detection method that is used to detect the test kit of soybean diaporthe/phomopsis syndromes of arbitrary claim in the claim 1,2,3,5 or 6, it is characterized in that, comprise the steps:
A, in determinand, add described primer, probe;
B, amplification step;
Quality Control step: adopt the test kit of soybean diaporthe/phomopsis syndromes as claimed in claim 4, carry out described steps A and B; And,
Determination step.
8. detection method as claimed in claim 7 is characterized in that described amplification step comprises the steps:
50 ℃ continue 2 minutes;
95 ℃ continue 10 minutes;
D, 95 ℃ continue 15 seconds;
63 ℃ continue 1 minute, carry out described step D then, till described step D has carried out 40 circulations.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154501A (en) * | 2011-04-15 | 2011-08-17 | 中华人民共和国连云港出入境检验检疫局 | PCR (Polymerase Chain Reaction) detection kit and method for two soybean germs |
| CN103388026A (en) * | 2013-07-17 | 2013-11-13 | 南京农业大学 | Phomopsis longicolla Hobbs detection target, PCR primer composition thereof and applications of the detection target and the PCR primer composition |
| CN105713962A (en) * | 2014-12-03 | 2016-06-29 | 南京农业大学 | LAMP primer composition for detection of phomopsis longicolla hobbs and application thereof |
| CN105713960A (en) * | 2014-12-02 | 2016-06-29 | 南京农业大学 | Specific LAMP primer composition used for detection of phomopsis longicolla and application thereof |
-
2009
- 2009-01-23 CN CN2009101052820A patent/CN101787390B/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154501A (en) * | 2011-04-15 | 2011-08-17 | 中华人民共和国连云港出入境检验检疫局 | PCR (Polymerase Chain Reaction) detection kit and method for two soybean germs |
| CN102154501B (en) * | 2011-04-15 | 2012-12-05 | 中华人民共和国连云港出入境检验检疫局 | PCR (Polymerase Chain Reaction) detection kit and method for two soybean germs |
| CN103388026A (en) * | 2013-07-17 | 2013-11-13 | 南京农业大学 | Phomopsis longicolla Hobbs detection target, PCR primer composition thereof and applications of the detection target and the PCR primer composition |
| CN103388026B (en) * | 2013-07-17 | 2016-01-13 | 南京农业大学 | The detection target of soybean Phomopsis seed decay pathogen and PCR primer composition thereof and application |
| CN105713960A (en) * | 2014-12-02 | 2016-06-29 | 南京农业大学 | Specific LAMP primer composition used for detection of phomopsis longicolla and application thereof |
| CN105713962A (en) * | 2014-12-03 | 2016-06-29 | 南京农业大学 | LAMP primer composition for detection of phomopsis longicolla hobbs and application thereof |
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