CN104805211A - PCR (polymerase chain reaction) detection kit for pathogenic bacteria of pokkah boeng disease - Google Patents
PCR (polymerase chain reaction) detection kit for pathogenic bacteria of pokkah boeng disease Download PDFInfo
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Abstract
The invention discloses a PCR (polymerase chain reaction) detection kit for pathogenic bacteria of pokkah boeng disease, and belongs to the technical field of PCR genetic identification in molecular biology. The PCR detection kit comprises a kit body and a kit cover arranged at an opening of the kit body, wherein the kit body comprises an inner kit, an outer kit and a cavity body formed between the inner kit and the outer kit; a shock-absorbing rubber plate is arranged at the bottom of the kit body; a foam pad used for loading reagent bottles and an operating manual are arranged in the inner kit; two reference standard substances and fourteen reagent bottles are arranged on the foam pad; a plurality of ice bags are arranged in the cavity body; one side of the kit cover is rotationally connected with the opening of the kit body, and the other side of the kit cover is bent downwards to form a connecting part; an iron sheet is arranged on a side wall, facing to the kit body, of the connecting part; a magnet corresponding to the iron sheet is arranged on a side wall of the kit body; an electronic thermometer is arranged on the inner surface of the kit cover. The PCR detection kit has the characteristics of simplicity for operation, high sensitivity, strong specificity and the like, and a detection result is accurate, objective and quick.
Description
Technical field
The present invention relates to a kind of Pokkah boeng PCR detection kit, belong to molecular biology PCR Genetic identification technical field.
Background technology
Sugarcane toppers maize ear rot (Pokkah Boeng Disease, PBD) is more general, the global Sugarcane Disease of a kind of generation in sugarcane production, and nineteen twenty-one is in Indonesia's Java reported first.At present, nearly all sugarcane production state and area have this disease to occur.Sugarcane production district of China also often has top rot to occur, and Fujian, Taiwan, Guangxi, Yunnan, Guangdong and Hainan were all once reported, brings massive losses to China's sugarcane production and sugar industry development.
Sugarcane toppers maize ear rot (PBD) is broadcast mainly through gentle the spreading of plant in spite of illness, and susceptible rear sugarcane generally shows as taper and the yellow of spire base portion minus green, and blade tussles or cripetura, has obvious brown wrinkle.Form the tip when falling ill the most serious rotten, the deliquescing of vegetative point surrounding tissue, browning, lobus cardiacus is downright bad, makes whole strain sugarcane withered.Be injured sugarcane strain plant height than healthy tree decreased average 30 ~ 60cm, the average underproduction l ~ 2t in every mu, the sugarcane field that is injured, and cane sugar reduces about 0.56%, and gravity purity reduces about 3%, causes sugarcane yield and quality to decline and Different Varieties degeneration.Sugarcane production the most vigorous 7-9 month is main generation period of top rot, and after hot and humid, arid, Limit irrigation, applied nitrogen too much or after arid run into rainfall, all can cause the extensive generation of top rot.
The pathogenic bacteria of sugarcane toppers maize ear rot (PBD) is Gibberella fujikuroi (Saw.) Wolle, is a kind of fungal disease.In China, the basic research of Sugarcane Disease is weaker. and especially technical in the quick diagnosis of sugarcane breeding for disease resistance and disease, there is larger gap with advanced country.Use for reference or the advanced Defect inspection technology of Introduced From Abroad and Resistance Identification technology, improving sugarcane breeding for disease resistance level and disease screening level, is the Important Problems of sugarcane toppers maize ear rot research.
The diagnostic techniques of sugarcane toppers maize ear rot (PBD) experienced by from internal sympton observation → phase-contrast microscopy → immunological technique → PCR rapid detection 4 stage.China's research is in this respect relatively backward, and diagnostic method is based on inner observation of symptoms and phase-contrast microscopy, and application immunological technique and PCR detect PBD and be also in the starting stage.Early 1980s, immunological technique obtained widespread use in the detection, diagnosis and treatment of biomedicine, phytopathogenic and animal pathogenic microorganism.Since 1984, immunological technique is applied to sugarcane PBD and diagnoses by external many scholars, with the new platform sugar 22 (ROC) of Guangxi main breed for research object, (Tissue blot DNA hybridization and ITS sequence analyzing and testing sugarcane toppers maize ear rot pathogenetic bacteria Gibberella fujikuroi (Saw.) Wolle, also obtain comparatively ideal effect to utilize DNA hybridization technology.Round pcr is that 20th century the mid-80 one of setting up is external rapidly, the technology of a large amount of amplifying target genes, has been widely used in the subjects such as molecular biology, medical science, agronomy, for clone's genetic analysis of gene and medical diagnosis on disease etc.Because PCR can a certain DNA fragmentation of specific amplified, therefore, in the detection of pathogenic micro-organism, having superiority than traditional method, cultivating as do not needed when round pcr detects organism, there is high sensitivity, quick etc.During the nearly last ten years, round pcr is widely applied in Plant diseases detects, also many scholars are had to be studied sugarcane toppers maize ear rot (PBD) molecular detection technology, as Zhang Yujuan utilizes the ITS zone design of the 18S-28S rDNA of sugarcane PBD pathogenetic bacteria 1 pair of special primer, (forward primer is: 5'-CTCTTGGTTCTGGCATCG-3', reverse primer is: 5'-GTTCAGCGGGTATTCCTA-3') carry out PCR detection, have detected 14 PBD samples, obtaining size is 545-546DNA specific amplified product, ITS sequence and sickle-like bacteria homology are up to 99%.
Isothermal amplification technique (Isothermal Amplification Technology) is Progress of Nucleic Acid Amplification Technologies, its reaction process maintains at a constant temperature all the time, is reached the object of Rapid nucleic acid amplification by the enzyme and respective Auele Specific Primer adding different activities.Ring mediation nucleic acid isothermal amplification technology (loop-mediated isothermal amplification, LAMP), it is exactly the one of isothermal PCR technology, it is the constant temperature nucleic acid amplification method of a kind of novelty of exploitation in 2000, be characterized in 6 zone design 4 species-specific primers for target gene, utilize a kind of strand displacement archaeal dna polymerase to be incubated 30 ~ 60 minutes in isothermal condition (about 63 DEG C), can nucleic acid amplification reaction be completed.Compared with Standard PCR, do not need the processes such as the thermally denature of template, temperature cycle, electrophoresis and ultraviolet visualization.LAMP is a kind of brand-new nucleic acid amplification method, has feature that is simple, quick, high specificity.
But up to now, on the market not yet for the PCR kit of Pokkah boeng detection.The present invention develops and a kind of detects Pokkah boeng PCR kit, for the departments such as China's sugarcane scientific research, quarantine, quality inspection provide quick, sensitive, Defect inspection reagent kit product and technical service accurately.
Summary of the invention
Object of the present invention, is to provide a kind of Pokkah boeng PCR detection kit.This test kit is used for the detection of Pokkah boeng, have the features such as simple to operate, susceptibility is high, high specificity, and detected result is accurate, objective, quick.
The technical scheme that the present invention solves the problems of the technologies described above is as follows: a kind of Pokkah boeng PCR detection kit, comprise box body and the lid being arranged on described box opening place, described box body comprises inner box, outer box, and the cavity between inner box and outer box, the outer bottom surrounding of described box body is provided with vibration reducing rubber plate, described inner box inside is provided with foam pad for loaded reagent bottle and 1 part of working instructions, described foam pad is provided with 2 reference standards and 14 bottles of reagent, 2 reference standards are respectively positive criteria product and negative standards's product, 14 bottles of reagent are respectively 1 bottle of upstream primer, 1 bottle of downstream primer, 1 bottle of dNTPsMixture, 1 bottle of Bst 2.0DNA Polymerase, 2 bottles of 1 × Isothermal AmplificationBuffer and 8 bottle ddH
2o, multiple ice bag is placed with in described cavity, the side of described lid and the opening part of box body are rotationally connected, opposite side is bent to form connection section downwards, described connection section is provided with iron plate towards the sidewall of box body, the sidewall of described box body is provided with the magnet corresponding with iron plate, and the internal surface of described lid is provided with electronic thermometer.
On the basis of technique scheme, the present invention can also do following improvement.
Further, described positive criteria product are the DNA that Pokkah boeng extracts, described negative controls is the DNA of the sample extraction not containing Pokkah boeng, the primer sequence of described upstream primer is: 5'-CTCTTGGTTCTGGCATCG-3', and the primer sequence of described downstream primer is: 5'-GTTCAGCGGGTATTCCTA-3'.
Further, described foam pad comprises and is positioned at inner the first top foam pad of inner box, be positioned at the second foam pad of inner box interior central and be positioned at inner the 3rd foam pad on the lower of inner box, and described first foam pad, second foam pad and the 3rd foam pad be arranged in parallel up and down, described first foam pad has 6 and place positive control standard substance respectively, negative control standard substance, upstream primer, downstream primer, the groove of the circle of dNTPsMixture and Bst 2.0DNA Polymerase reagent bottle, described first foam pad is also provided with the through hole of two circular put in fingers for taking out the first foam pad, described second foam pad has 5 and place two 1 × Isothermal Amplification Buffer and three ddH respectively
2the groove of the circle of O reagent bottle, described second foam pad is also provided with the through hole of two circular put in fingers, for taking out the second foam pad, described 3rd foam pad having 5 and placing five ddH respectively
2the groove of the circle of O reagent bottle.
Further, described positive criteria product are the white PE plastics tubing of white cap, described negative controls is the red PE plastics tubing of red cap, described upstream primer is the white PE Plastic Bottle of black caps, described downstream primer is the white PE plastics tubing of black caps, and described dNTPs Mixture is the blue PE plastics tubing of blue cap, and described Bst2.0DNA Polymerase is the white PE Plastic Bottle of purple cap, described 10 × PCR Buffer is the white PE plastics tubing of white cap, described ddH
2o is the white PE Plastic Bottle of yellow cap.
Further, described positive criteria product are 1 pipe, 20 μ L, described negative controls is 1 pipe, 20 μ L, and described upstream primer is 1 pipe, 30 μ L, described downstream primer is 1 pipe, 30 μ L, described dNTPsMixture is 1 pipe, 30 μ L, and described Bst 2.0DNA Polymerase is 1 pipe, 10 μ L, described 1 × Isothermal Amplification Buffer is 2 pipes, often pipe 125 μ L, described ddH
2o is 8 pipes, often pipe 125 μ L.
A PCR detection method for Pokkah boeng, it utilizes test kit mentioned above, carries out PCR method detection, comprise the steps: sample DNA
(1) extraction of template DNA: the template of DNA as PCR extracting sample;
(2) isothermal PCR reaction system is built: reaction system cumulative volume is 25 μ L, wherein:
Isothermal PCR amplified reaction: testing sample, positive control and negative control are carried out pcr amplification respectively, the parameter of described isothermal PCR amplified reaction is: 65 DEG C of isothermal 45min, 80 DEG C of inactivation 20min ,-20 DEG C of maintenances;
(3) pcr amplification product of testing sample, positive control and negative control is obtained respectively, and in-20 DEG C of preservations;
(4) isothermal PCR product carries out electrophoresis detection: each 5 μ L of pcr amplification product getting step (3) gained testing sample, positive control and negative control respectively, amplification is detected with 1% agarose gel electrophoresis, single amplified band is there is in described positive control at 550bp place, then there is not single amplified band at 550bp in described negative control, if testing sample is consistent with the electrophoresis result of positive control, it is then Pokkah boeng, if testing sample is consistent with the electrophoresis result of negative control, then it not Pokkah boeng.
The invention has the beneficial effects as follows:
1. security is high: PCR detection kit of the present invention, and box body is divided into inner box, outer box and cavity, in cavity, place ice bag, cold storage effect is good, and the cooling period is long, is more convenient to the preservation of reagent, and ice bag does not directly contact with the reagent of inner box, avoid reagent contaminated or lost efficacy.
2. good airproof performance: PCR detection kit of the present invention, is provided with magnetic lock locking device in the junction of lid and box body, the good airproof performance of test kit, is convenient to mobile and transport.
3. practical: PCR detection kit of the present invention, be provided with electrical thermometer at lid internal surface, intuitively can distinguish temperature residing for the reagent in box body.
4. be convenient to transport: PCR detection kit of the present invention, is provided with damping rubber bottom box body, ensure that test kit is in mobile and transportation, can not because of excessive concussion make reagent bottle impaired with break, ensure that the safety storing of test kit and reagent.
5. wide market: PCR detection kit of the present invention; when being applied to the detection of Pokkah boeng; there is simple to operate, the feature such as susceptibility is high, high specificity; and detected result is accurate, objective, quick; greatly can improve detection efficiency; and structure simple, reasonable in design, easily manufacture, the huge market demand, be suitable for large-scale production.
Accompanying drawing explanation
Fig. 1 is the structural representation after the present invention opens the lid.
Fig. 2 is the electrophorogram of Pokkah boeng of the present invention after the qualification of isothermal PCR detection method.
In accompanying drawing, the list of parts representated by each label is as follows:
1, box body, 2, lid, 3, inner box, 4, outer box, 5, cavity, 6, vibration reducing rubber plate, 7, foam pad, 8, positive criteria product, 9, negative standards's product, 10, upstream primer, 11, downstream primer, 12, dNTPs Mixture, 13, Bst 2.0DNA Polymerase, 14,1 × IsothermalAmplification Buffer, 15, ddH
2o, 16, connection section, 17, iron plate, 18, magnet, 19, electronic thermometer, 20, through hole.
Embodiment
Be described principle of the present invention and feature below in conjunction with accompanying drawing, example, only for explaining the present invention, is not intended to limit scope of the present invention.
As shown in Figure 1, a kind of Pokkah boeng PCR detection kit, comprise box body 1 and the lid 2 being arranged on described box body 1 opening part, described box body 1 comprises inner box 3, outer box 4, and the cavity 5 between inner box 3 and outer box 4, the outer bottom surrounding of described box body 1 is provided with vibration reducing rubber plate 6, the foam pad 7 for loaded reagent bottle and 1 part of working instructions are provided with in described inner box 3, described foam pad 7 is provided with 2 reference standards and 14 bottles of reagent, 2 reference standards are respectively positive criteria product 8 and negative standards's product 9, 14 bottles of reagent are respectively 1 bottle of upstream primer 10, 1 bottle of downstream primer 11, 1 bottle of dNTPs Mixture 12, 1 bottle of Bst2.0DNA Polymerase 13, 2 bottles of 1 × IsothermalAmplification Buffer 14 and 8 bottles of ddH
2o 15, multiple ice bag is placed with in described cavity 5, the side of described lid 2 and the opening part of box body 1 are rotationally connected, opposite side is bent to form connection section 16 downwards, described connection section 16 is provided with iron plate 17 towards the sidewall of box body 1, the sidewall of described box body 1 is provided with the magnet 18 corresponding with iron plate 17, and the internal surface of described lid 2 is provided with electronic thermometer 19.
The DNA that described positive criteria product 8 extract for Pokkah boeng, described negative controls 9 is the DNA of the sample extraction not containing Pokkah boeng, the primer sequence of described upstream primer 10 is: 5'-CTCTTGGTTCTGGCATCG-3', and the primer sequence of described downstream primer 11 is: 5'-GTTCAGCGGGTATTCCTA-3'.
Described foam pad 7 comprises and is positioned at inner the first top foam pad 7a of inner box 3, be positioned at the second foam pad 7b of inner box 3 interior central and be positioned at inner box 3 inside the 3rd foam pad 7c on the lower, and described first foam pad 7a, second foam pad 7b and the 3rd foam pad 7c be arranged in parallel up and down, described first foam pad 7a has 6 and place positive control standard substance 8 respectively, negative control standard substance 9, upstream primer 10, downstream primer 11, the groove of the circle of dNTPs Mixture 12 and Bst 2.0DNA Polymerase 13 reagent bottle, described first foam pad 7a is also provided with the through hole 20 of two circular put in fingers for taking out the first foam pad 7a, described second foam pad 7b has 5 and place two 10 × PCR Buffer 14a respectively, 14b and three ddH
2o 15a, the groove of the circle of 15b, 15c reagent bottle, described second foam pad 7b is also provided with the through hole 20 of two circular put in fingers, for taking out the second foam pad 7b, described 3rd foam pad 7c having 5 and placing five ddH respectively
2the groove of the circle of O15d, 15e, 15f, 15g, 15h reagent bottle.
Described positive criteria product 8 are the white PE plastics tubing of white cap, described negative controls 9 is the red PE plastics tubing of red cap, described upstream primer 10 is the white PE Plastic Bottle of black caps, described downstream primer 11 is the white PE plastics tubing of black caps, described dNTPs Mixture 12 is the blue PE plastics tubing of blue cap, described Bst2.0DNA Polymerase13 is the white PE Plastic Bottle of purple cap, and described 10 × PCR Buffer 14 is the white PE plastics tubing of white cap, described ddH
2o 15 is the white PE Plastic Bottle of yellow cap.
Described positive criteria product 8 are 1 pipe, 20 μ L, and described negative controls 9 is 1 pipe, 20 μ L, described upstream primer 10 is 1 pipe, 30 μ L, described downstream primer 11 is 1 pipe, and 30 μ L, described dNTPsMixture12 are 1 pipe, 30 μ L, described Bst 2.0DNA Polymerase 13 is 1 pipe, 10 μ L, and described 10 × PCR Buffer 14 is 2 pipes, often pipe 125 μ L, described ddH
2o 15 is 8 pipes, often pipe 125 μ L.
A PCR detection method for Pokkah boeng, it utilizes test kit mentioned above, carries out PCR method detection, comprise the steps: sample DNA
(1) extraction of template DNA: the template of DNA as PCR extracting sample;
(2) isothermal PCR reaction system is built: reaction system cumulative volume is 25 μ L, wherein:
Isothermal PCR amplified reaction: testing sample, positive control and negative control are carried out pcr amplification respectively, the parameter of described isothermal PCR amplified reaction is: 65 DEG C of isothermal 45min, 80 DEG C of inactivation 20min ,-20 DEG C of maintenances;
(3) pcr amplification product of testing sample, positive control and negative control is obtained respectively, and in-20 DEG C of preservations;
(4) isothermal PCR product carries out electrophoresis detection: each 5 μ L of pcr amplification product getting step (3) gained testing sample, positive control and negative control respectively, amplification is detected with 1% agarose gel electrophoresis, single amplified band is there is in described positive control at 550bp place, then there is not single amplified band at 550bp in described negative control, if testing sample is consistent with the electrophoresis result of positive control, it is then Pokkah boeng, if testing sample is consistent with the electrophoresis result of negative control, then it not Pokkah boeng.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (5)
1. a Pokkah boeng PCR detection kit, comprise box body (1) and be arranged on the lid (2) of described box body (1) opening part, it is characterized in that, described box body (1) comprises inner box (3), outer box (4), and the cavity (5) be positioned between inner box (3) and outer box (4), the outer bottom surrounding of described box body (1) is provided with vibration reducing rubber plate (6), described inner box (3) inside is provided with foam pad (7) for loaded reagent bottle and 1 part of working instructions, described foam pad (7) is provided with 2 reference standards and 14 bottles of reagent, 2 reference standards are respectively positive criteria product (8) and negative standards's product (9), 14 bottles of reagent are respectively 1 bottle of upstream primer (10), 1 bottle of downstream primer (11), 1 bottle of dNTPs Mixture (12), 1 bottle of Bst2.0DNA Polymerase (13), 2 bottles of 1 × Isothermal AmplificationBuffer (14) and 8 bottles of ddH
2o (15), described cavity is placed with multiple ice bag in (5), the side of described lid (2) and the opening part of box body (1) are rotationally connected, opposite side is bent to form connection section (16) downwards, described connection section (16) is provided with iron plate (17) towards the sidewall of box body (1), the sidewall of described box body (1) is provided with the magnet (18) corresponding with iron plate (17), and the internal surface of described lid (2) is provided with electronic thermometer (19).
2. a kind of Pokkah boeng PCR detection kit according to claim 1, it is characterized in that, the DNA that described positive criteria product (8) are extracted for Pokkah boeng, described negative controls (9) is the DNA of the sample extraction not containing Pokkah boeng, the primer sequence of described upstream primer (10) is: 5'-CTCTTGGTTCTGGCATCG-3', and the primer sequence of described downstream primer (11) is: 5'-GTTCAGCGGGTATTCCTA-3'.
3. a kind of Pokkah boeng PCR detection kit according to claim 1, it is characterized in that, described foam pad (7) comprises and is positioned at inner top the first foam pad (7a) of inner box (3), be positioned at second foam pad (7b) of inner box (3) interior central and be positioned at inner the 3rd foam pad (7c) on the lower of inner box (3), and described first foam pad (7a), second foam pad (7b) and the 3rd foam pad (7c) be arranged in parallel up and down, described first foam pad (7a) has 6 and place positive control standard substance (8) respectively, negative control standard substance (9), upstream primer (10), downstream primer (11), the groove of the circle of dNTPs Mixture (12) and Bst 2.0DNA Polymerase (13) reagent bottle, described first foam pad (7a) is also provided with the through hole (20) of two circular put in fingers for taking out the first foam pad (7a), described second foam pad (7b) has 5 and place two 1 × Isothermal Amplification Buffer (14a respectively, 14b) and three ddH
2o (15a, 15b, 15c) the groove of the circle of reagent bottle, described second foam pad (7b) is also provided with the through hole (20) of two circular put in fingers, for taking out the second foam pad (7b), described 3rd foam pad (7c) having 5 and placing five ddH respectively
2the groove of the circle of O (15d, 15e, 15f, 15g, 15h) reagent bottle.
4. a kind of Pokkah boeng PCR detection kit according to claim 1, it is characterized in that, the white PE plastics tubing that described positive criteria product (8) are white cap, the red PE plastics tubing that described negative controls (9) is red cap, the white PE Plastic Bottle that described upstream primer (10) is black caps, the white PE plastics tubing that described downstream primer (11) is black caps, the blue PE plastics tubing that described dNTPs Mixture (12) is blue cap, the white PE Plastic Bottle that described Bst 2.0DNA Polymerase (13) is purple cap, the white PE plastics tubing that described 10 × PCR Buffer (14) is white cap, described ddH
2the white PE Plastic Bottle that O (15) is yellow cap.
5. a kind of Pokkah boeng PCR detection kit according to claim 1, it is characterized in that, described positive criteria product (8) are 1 pipe, 20 μ L, described negative controls (9) is 1 pipe, 20 μ L, described upstream primer (10) is 1 pipe, 30 μ L, described downstream primer (11) is 1 pipe, 30 μ L, described dNTPs Mixture (12) is 1 pipe, 30 μ L, described Bst 2.0DNAPolymerase (13) is 1 pipe, 10 μ L, described 1 × Isothermal Amplification Buffer (14) is 2 pipes, often pipe 125 μ L, described ddH
2o (15) is 8 pipes, often pipe 125 μ L.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201510219774.8A CN104805211A (en) | 2015-04-30 | 2015-04-30 | PCR (polymerase chain reaction) detection kit for pathogenic bacteria of pokkah boeng disease |
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| CN201510219774.8A CN104805211A (en) | 2015-04-30 | 2015-04-30 | PCR (polymerase chain reaction) detection kit for pathogenic bacteria of pokkah boeng disease |
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| CN203768362U (en) * | 2014-02-14 | 2014-08-13 | 滨州职业学院 | Haemophilus paragallinarum pyrG gene PCR (Polymerase Chain Reaction) detection kit |
| CN203955244U (en) * | 2014-06-25 | 2014-11-26 | 王爱芹 | A kind of new medical kit |
| CN204185476U (en) * | 2014-10-31 | 2015-03-04 | 江苏所罗门兄弟医学科技有限公司 | A kind of ARMS-qPCR detection kit of NOSLAP gene type |
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| Title |
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Application publication date: 20150729 |