CN101857894A - Primer, probe, kit and method for detecting Monilinia fructicola (Winter) Honey - Google Patents
Primer, probe, kit and method for detecting Monilinia fructicola (Winter) Honey Download PDFInfo
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Abstract
The invention discloses a primer, oligonucleotide probe, kit and detection method for detecting Monilinia fructicola (Winter) Honey through real-time PCR detection. The primer contains the sequences of SEQ ID NO:1 and SEQ ID NO:2; the oligonucleotide probe contains the sequence of SEQ ID NO:3; and the detection method in that the primer and the oligonucleotide probe are used to perform real-time PCR to detect Monilinia fructicola (Winter) Honey. By adopting the method of the invention, the simple, fast, specific and sensitive identification or detection of Monilinia fructicola (Winter) Honey can be performed to the found diseased tissues in actual quarantine process and field investigation process and the method is suitable to be used by the departments of port inspection and quarantine, agricultural production, plant protection and the like.
Description
Technical field
The present invention relates to a kind of biotechnology, specifically, relate to and a kind of Monilinia fructicola (Winter) Honey (Monilinia fructicola (Winter) Honey) is carried out simple, quick, special, probe for real-time fluorescence PCR detection method and test kit thereof delicately; Being suitable for departments such as Check and Examination of Port quarantine, agriculture production, plant protection uses.
Background technology
Monilinia fructicola (Winter) Honey (Monilinia fructicola (Winter) Honey) has been listed " the dangerous venereal disease worm of the inward Plant Quarantine of the People's Republic of China (PRC) weeds register " that China announces in May, 2007 in, also lists European Union's quarantine harmful organisms register simultaneously in.This pathogenic bacteria hosts such as multiple fruit such as plum, apricot, peach, nectarine, cherry, plum Lee, apple, pears and grape and Chaenomeles, hawthorn, loquat genus of causing harm cause that fruit is rotten, spend corruption and leaf withered etc.It can cause harm in the orchard, the shelf time fruit of causing harm again, also can be in fruit latent infection.This germ is distributed in the geographic many countries in North America, Oceania, Europe, Asia, Africa, Sino-U.S. and the Caribbean Sea in the world, also there is report in the part peach garden, Beijing and the Taiwan of China.China is from fruit such as a large amount of plums of national import, apple such as the U.S., it is very big that Monilinia fructicola (Winter) Honey enters China's risk with trade fruit, the agriculture production of China constituted potential threat, therefore, need prevent importing into of this pathogenic fungi in the strict reinforcement quarantine of port quarantine department.
Because Monilinia fructicola (Winter) Honey is similar at aspects such as pathogenic bacteria morphological specificity, cultivation proterties, hazard symptoms with european race a kind of fruit, such as apple, pear, etc. brown rot germ (M.fructigena) to two other kind drupe brown rot germ (M.laxa) of this genus, be not easily distinguishable with morphological method, need abundant evaluation experience, generally need veteran fungi connoisseur.And need separation and Culture, time-consuming longer.
Real-time fluorescence PCR detection method is a kind of a kind of detection method that just grew up in 1996 on the qualitative PCR basis, this method is because added one section TaqMan probe in PCR, thereby can avoid unavoidable non-specific amplification of qualitative PCR and false positive phenomenon more effectively, this method is used more widely medically obtaining, and this method was applied to the detection by quantitative of transgenic product again in recent years.Abroad, the real-time fluorescence PCR first Application is in 2000 in the fungal diseases of plants detection range.In China, people such as Zhang Guiming, Cheng Yinghui are applied to real-time fluorescence PCR detection method in the detection of tilletia indica mitra and allied species thereof in calendar year 2001 first.
TaqMan MGB detecting probe method is the method for updating that produces after the TaqMan method, and the full name of MGB is Minor Groove Binder, is pushed out in calendar year 2001.Compare with the TaqMan probe, its principle be TaqMan MGB probe one be probe 3 ' end mark self non-luminous cancellation fluorescence molecule, to replace the TAMRA fluorescent mark that routine can be luminous; The 2nd, 3 ' end of probe combines Minor Groove binder binding substances in addition, and the Tm value of probe is improved, and has increased the hybrid stability of probe greatly.
But, the real-time fluorescence PCR detection method of TaqMan MGB probe is applied to the detection of pathogenic fungi, no matter abroad still at the early-stage still at home, need do more exploratory development work.
Summary of the invention
That purpose of the present invention is intended to propose is quick, reliable, highly sensitive, high specificity and low price ground detect Monilinia fructicola (Winter) Honey primer, probe, test kit and real-time fluorescence PCR detection method.
For achieving the above object, the invention provides a primer, its sequence is SEQ ID NO:1:SZMfc-F 5 '-CCTTCGGGCCTTGTATGCT-3 '.
Another primer also is provided, and its sequence is SEQ ID NO:2:
SZMfc-R 5’-CCAAGAGATCCGTTGTTGAAAGT-3’。
An oligonucleotide probe is provided again, and its sequence is SEQ ID NO:3:5 '-FAM-CCAGAGGATAATTAAAC-MGB-3 ', and wherein FAM is a fluorescence molecule, and MGB refers to combine in addition Minor Groove binder binding substances.
Also provide to be used for primer and the oligonucleotide probe that the Monilinia fructicola (Winter) Honey real-time fluorescence PCR detects, described oligonucleotide probe comprises the TaqManMGB probe of specific detection Monilinia fructicola (Winter) Honey and the oligonucleotide sequence of specific combination.
Preferably, described primer has following nucleotide sequence:
SZMfc-F：5’-CCTTCGGGCCTTGTATGCT-3’SEQ?ID?NO：1
SZMfc-R：5’-CCAAGAGATCCGTTGTTGAAAGT-3’SEQ?ID?NO：2。
Preferably, described oligonucleotide probe has following nucleotide sequence:
SZMfc-Pb：5’-FAM-CCAGAGGATAATTAAAC-MGB-3’SEQ?ID?NO：3。
The present invention provides a kind of test kit that the Monilinia fructicola (Winter) Honey real-time fluorescence PCR detects that is used in addition, and described test kit comprises above-mentioned primer and oligonucleotide probe.
The present invention also provides a kind of Monilinia fructicola (Winter) Honey real-time fluorescence PCR detection method, comprises the steps:
(1) earlier Monilinia fructicola (Winter) Honey, drupe brown rot germ and european race a kind of fruit, such as apple, pear, etc. brown rot germ internal transcribed spacer district (ITS) are carried out sequencing and comparative analysis, find out the exclusive base site that Monilinia fructicola (Winter) Honey is different from other allied species respectively;
(2) primer and the oligonucleotide probe of the detection of design Monilinia fructicola (Winter) Honey real-time fluorescence PCR;
(3) designed probe is screened and the optimization of reaction system and reaction conditions, filter out the primer and the probe of optimization, and suitable reaction system and the reaction conditions of optimization;
(4) add above-mentioned primer, probe and carry out the reaction of Monilinia fructicola (Winter) Honey real-time fluorescence PCR.
Adopt the criterion as a result of this detection method to be: to be that the positive control report fluorescence of template has clear signal to increase by 1) if the quantitative PCR instrument detects with Monilinia fructicola (Winter) Honey DNA, 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, and represents that then the fungi that is detected is a Monilinia fructicola (Winter) Honey; 2) be that the positive control report fluorescence of template has clear signal to increase if the quantitative PCR instrument detects with Monilinia fructicola (Winter) Honey DNA, 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, and represents that then the fungi that is detected is not a Monilinia fructicola (Winter) Honey.
Adopt technique scheme, the technical progress that the present invention gives prominence to is: 1, designed at the primer that is applicable to Monilinia fructicola (Winter) Honey real-time fluorescence PCR rapid detection, oligonucleotide probe and test kit, overcome the shortcoming that existing morphology authentication method sense cycle is long, need rich experiences, also overcome the not high or shortcoming such as specificity is strong or repeatability is bad of existing molecular detecting method complicated operation or detection sensitivity.2, the present invention is fit to the detection of Monilinia fructicola (Winter) Honey, and suitable fruit host passes the detection of the Monilinia fructicola (Winter) Honey of band.3, because the present invention detects fast, high specificity, be specially adapted to the strong especially occasion uses of ageing requirement such as port quarantine.
In sum, adopt the present invention to carry out Monilinia fructicola (Winter) Honey molecular biology identification or detection to the diseased tissues of being found in reality quarantine and the field investigation process, it is significant.
Description of drawings
Fig. 1 is Auele Specific Primer probe amplification result among the specific embodiment of the invention embodiment 2, and the right is the numbering of test sample; 812672 is Monilinia fructicola (Winter) Honey M.fructicola bacterial strain, 816263 is Monilinia fructicola (Winter) Honey M.fructicola bacterial strain, 815702 is Monilinia fructicola (Winter) Honey M.fructicola bacterial strain, MFC-nb is a Monilinia fructicola (Winter) Honey M.fructicola bacterial strain, P4236-nb is a Monilinia fructicola (Winter) Honey M.fructicola bacterial strain, 10923 is Monilinia fructicola (Winter) Honey M.fructicola bacterial strain, 10921 is drupe brown rot germ M.laxa bacterial strain, 10922 is european race a kind of fruit, such as apple, pear, etc. brown rot germ M.fructigena bacterial strain, and Pd-1 is a palm mould germ Phytophthora palmivora bacterial strain.
Embodiment
The present invention is described in further detail below by specific embodiment.
Example example 1 Monilinia fructicola (Winter) Honey TaqMan MGB probe for real-time fluorescence PCR detects and test kit
A kind ofly be used for primer, oligonucleotide probe and the test kit that Monilinia fructicola (Winter) Honey (Monilinia fructicola) real-time fluorescence PCR detects, be used for the specific detection of Monilinia fructicola (Winter) Honey.
Described primer has following nucleotide sequence:
SZMfc-F：5’-CCTTCGGGCCTTGTATGCT-3’SEQ?ID?NO：1
SZMfc-R：5’-CCAAGAGATCCGTTGTTGAAAGT-3’SEQ?ID?NO：2，
Described oligonucleotide probe has following nucleotide sequence:
SZMfc-Pb：5’-FAM-CCAGAGGATAATTAAAC-MGB-3’SEQ?IDNO：3。
Described test kit is for using the real-time fluorescence PCR assay kit that is used for the Monilinia fructicola (Winter) Honey detection that above-mentioned primer and oligonucleotide probe are made.
Embodiment 2 utilizes Monilinia fructicola (Winter) Honey TaqMan MGB probe for real-time fluorescence PCR detection method that the quarantine of Monilinia fructicola (Winter) Honey among bright Lee of import is identified
The primer and oligonucleotide probe are primer and the oligonucleotide probes among the embodiment 1.
The system cumulative volume of its detection reaction is 10 μ L, each composition is: real-time fluorescence reaction mixture TaqMan Universal PCR Master Mix 5 μ L (ABI company, product type 4304437), primer 0.9 μ M, probe 0.2 μ M, dna profiling 1 μ L (template concentrations 10-100ng/ μ L).
Its detection reaction condition sees Table 1.
Table 1
But from bright Lee of the import U.S., find the hypochondriasis fruit, detect according to above-mentioned method and condition, the real-time fluorescence PCR detected result is: with Monilinia fructicola (Winter) Honey DNA is that the positive control report fluorescence of template has clear signal to increase, the report fluorescence of negative control and blank does not have fluorescent signal to increase, and from the detected result positive (detected result as shown in Figure 1) of dna profiling among bright Lee of import.Show that bright Lee of the import that is detected carries Monilinia fructicola (Winter) Honey.
Adopt method of the present invention to detect Monilinia fructicola (Winter) Honey more fast, accurately, and be easy to apply than traditional form method.
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 (10)
1. primer, its sequence is SEQ ID NO:1:
5’-CCTTCGGGCCTTGTATGCT-3’。
2. primer, its sequence is SEQ ID NO:2:
5’-CCAAGAGATCCGTTGTTGAAAGT-3’。
3. a pair of primer, its sequence is:
SZMfc-F：5’-CCTTCGGGCCTTGTATGCT-3’ SEQ?ID?NO：1
SZMfc-R：5’-CCAAGAGATCCGTTGTTGAAAGT-3’ SEQ?ID?NO：2。
4. oligonucleotide probe, its sequence is SEQ ID NO:3:
5’-FAM-CCAGAGGATAATTAAAC-MGB-3’。
5. a pair of primer and an oligonucleotide probe, the sequence of described a pair of primer is:
SZMfc-F：5’-CCTTCGGGCCTTGTATGCT-3’ SEQ?ID?NO：1
SZMfc-R：5’-CCAAGAGATCCGTTGTTGAAAGT-3’ SEQ?ID?NO：2，
The sequence of a described oligonucleotide probe is:
SZMfc-Pb：5’-FAM-CCAGAGGATAATTAAAC-MGB-3’SEQ?IDNO：3。
6. be used for a pair of primer and an oligonucleotide probe that Monilinia fructicola (Winter) Honey detects.
7. according to a pair of primer and the oligonucleotide probe that Monilinia fructicola (Winter) Honey detects that be used for of claim 6, described primer sequence is:
SZMfc-F：5’-CCTTCGGGCCTTGTATGCT-3’ SEQ?ID?NO：1
SZMfc-R：5’-CCAAGAGATCCGTTGTTGAAAGT-3’ SEQ?ID?NO：2。
8. according to a pair of primer and the oligonucleotide probe that Monilinia fructicola (Winter) Honey detects that be used for of claim 6, described sequence oligonucleotide probe is:
SZMfc-Pb：5’-FAM-CCAGAGGATAATTAAAC-MGB-3’SEQ?IDNO：3。
9. one kind is used for the test kit that the Monilinia fructicola (Winter) Honey real-time fluorescence PCR detects, and described test kit comprises a pair of primer and an oligonucleotide probe, and the sequence of described a pair of primer is:
SZMfc-F：5’-CCTTCGGGCCTTGTATGCT-3’ SEQ?ID?NO：1
SZMfc-R：5’-CCAAGAGATCCGTTGTTGAAAGT-3’ SEQ?ID?NO：2，
The sequence of a described oligonucleotide probe is:
SZMfc-Pb：5’-FAM-CCAGAGGATAATTAAAC-MGB-3’SEQ?IDNO：3。
10. a soybean fungal disease real-time fluorescence PCR detection method comprises the steps:
(1) earlier Monilinia fructicola (Winter) Honey, drupe brown rot germ and european race a kind of fruit, such as apple, pear, etc. brown rot germ internal transcribed spacer district are carried out sequencing and comparative analysis, find out the exclusive base site that Monilinia fructicola (Winter) Honey is different from other allied species respectively;
(2) primer and the oligonucleotide probe of the detection of design Monilinia fructicola (Winter) Honey real-time fluorescence PCR, the sequence of described a pair of primer is:
SZMfc-F：5’-CCTTCGGGCCTTGTATGCT-3’ SEQ?ID?NO：1
SZMfc-R：5’-CCAAGAGATCCGTTGTTGAAAGT-3’ SEQ?ID?NO：2，
The sequence of a described oligonucleotide probe is:
SZMfc-Pb：5’-FAM-CCAGAGGATAATTAAAC-MGB-3’SEQ?IDNO：3；
(3) add above-mentioned primer, probe and carry out the reaction of Monilinia fructicola (Winter) Honey real-time fluorescence PCR.
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| CN 200910106535 CN101857894B (en) | 2009-04-10 | 2009-04-10 | Primer, probe, kit and method for detecting Monilinia fructicola (Winter) Honey |
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| CN 200910106535 CN101857894B (en) | 2009-04-10 | 2009-04-10 | Primer, probe, kit and method for detecting Monilinia fructicola (Winter) Honey |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110031438A (en) * | 2019-04-19 | 2019-07-19 | 深圳出入境检验检疫局动植物检验检疫技术中心 | A kind of activity test method of Monilinia fructicola (Winter) Honey |
| RU2751248C2 (en) * | 2019-12-09 | 2021-07-12 | Федеральное государственное бюджетное научное учреждение "Всероссийский научно-исследовательский институт цветоводства и субтропических культур" (ФГБНУ ВНИИЦиСК) | Primers for detecting the species monilinia laxa, monilinia fruticola, monilinia fructigena |
| CN114703310A (en) * | 2022-01-17 | 2022-07-05 | 福建农业职业技术学院 | Monilinia fructicola LAMP (loop-mediated isothermal amplification) detection primers and application thereof |
-
2009
- 2009-04-10 CN CN 200910106535 patent/CN101857894B/en not_active Expired - Fee Related
Non-Patent Citations (3)
| Title |
|---|
| Y. LUO. Z ET AL: "Quantification of airborne spores of Monilinia fructicola in stone fruit orchards of California using real-time PCR", 《EUR J PLANT PATHOL》 * |
| 吴品珊等: "美澳型核果褐腐病菌的检疫鉴定方法", 《植物检疫》 * |
| 樊锦艳等: "褐腐病菌三种分子鉴定方法的比较", 《植物保护学报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110031438A (en) * | 2019-04-19 | 2019-07-19 | 深圳出入境检验检疫局动植物检验检疫技术中心 | A kind of activity test method of Monilinia fructicola (Winter) Honey |
| RU2751248C2 (en) * | 2019-12-09 | 2021-07-12 | Федеральное государственное бюджетное научное учреждение "Всероссийский научно-исследовательский институт цветоводства и субтропических культур" (ФГБНУ ВНИИЦиСК) | Primers for detecting the species monilinia laxa, monilinia fruticola, monilinia fructigena |
| CN114703310A (en) * | 2022-01-17 | 2022-07-05 | 福建农业职业技术学院 | Monilinia fructicola LAMP (loop-mediated isothermal amplification) detection primers and application thereof |
| CN114703310B (en) * | 2022-01-17 | 2024-03-26 | 福建农业职业技术学院 | LAMP (loop-mediated isothermal amplification) detection primer for brown rot of peach and application thereof |
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