CN104328177A - Clostridium perfringens isothermal PCR field rapid analysis kit - Google Patents
Clostridium perfringens isothermal PCR field rapid analysis kit Download PDFInfo
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- CN104328177A CN104328177A CN201410586237.2A CN201410586237A CN104328177A CN 104328177 A CN104328177 A CN 104328177A CN 201410586237 A CN201410586237 A CN 201410586237A CN 104328177 A CN104328177 A CN 104328177A
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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Abstract
The invention discloses a Clostridium perfringens isothermal PCR field rapid analysis kit. The kit is composed of a set of primers, 10fold loop-mediated isothermal amplification reaction liquid, DNA polymerase, positive control, negative control and a color developing agent, wherein the set of primers is composed of a pair of primers and a pair of inner primers. The kit can be used for qualitatively and quantitatively detecting the Clostridium perfringens, four primers are designed, six areas of a target sequence are recognized due to strand displacement characteristic of Bst DNA enzyme, a great number of repeated stem ring shaped structures are generated under the isothermal condition, any special equipment is not required during qualitative detection, the color change of a reaction pipe is only required to be watched with naked eyes, and thus, the judgment can be realized; and the kit has the characteristics of high specificity, high sensitivity, short detection time and the like compared with the common PCR, and is suitable for field rapid detection for culture farms.
Description
Technical field
The invention belongs to technical field of bioengineering, relate to a kind of clostridium perfringens isothermal PCR field quick detection test kit.
Background technology
Clostridium perfringens disease is a kind of infecting both domestic animals and human acute infectious disease caused by clostridium perfringens, and this bacterium is distributed widely in nature, is also one of the bacterium of being everlasting in animal intestinal.Clostridium perfringens can produce multiple extracellular toxin, has pathogenic, causes necrotic enteritis, enterotoxemia is the characteristic feature that this bacterium infects to many animals such as pig, ox, sheep, chicken, rabbit, dog, people.Clostridium perfringens infects the animal Sudden Death Syndrome caused, and general rapid onset, the course of disease are short, mortality ratio is high, often has little time treatment, causes serious threat to aquaculture development and human health.
Ox Sudden Death Syndrome is caused by C. perfringens Type A bacterium, also has C, D type bacterium to cause a disease.External report be with C, D type bacterium cause the necrotic enteritis of ox or enterotoxemia more.Sick ox main manifestations be disease just apocleisis, lassitude, be reluctant the stench ight soil of walking about, fervescence, asoscope are dry, expiratory dyspnea, palpitating speed show uneasiness, stomachache, row's scattered paste shape band blood subsequently, finally spit out white foams, the spasm that falls down to the ground and extremely.
Rabbit enterotoxemia is by A type or the microbial enterotoxemia of E type by clostridium perfringens, is mainly in after wean to the rabbit grown up.Clinical symptom is that disease rabbit spirit is depressed, and body temperature is not high, is off one's feed or goes on a hunger strike, and the distortion of sick just ight soil is thinning, very soon row's band blood, gel-shaped or black watery stools, and sharply suffer from diarrhoea, though single cases can stop watery diarrhea through pharmacological agent, but mortality ratio is up to 100%.
When the sporadic intestinal mucosa of the chicken that A type bacterium and C type clostridium perfringens cause is downright bad, the rapid decline and death of the most acute person's sudden onset, can't see manifest symptom; Acute infection chicken spirit is depressed, feather is fluffy and disorderly, breastbone lands, appetite weakens, arrange band bloody stool just.Most.Most sick cockscomb eyelid is livid purple, and minority is pale asphyxia, and lethality rate is high; Mild case takes a turn for the better after showing general toxicity symptom 2 ~ 3d gradually, increases and rehabilitation with appetite.
C type clostridium perfringens causes pig Sudden Death Syndrome to be more common in 3 age in days piglets, shows as red bloody stool, also referred to as piglet.Sick pig has a delicate constitution, discharge sorrel loose stool containing fragment of tissue, mortality ratio 12%, 10 age rear section piglet there is dysentery characterized by white mucous stool, after recover normal gradually.The sick pig of minority does not descend bloody flux just to faint and death.
The sudden subcutaneous ulcer of sheep, sheep enterotoxemia, lamb dysentery are microbial by clostridium perfringens C, D, Type B respectively.The sudden subcutaneous ulcer of sheep is with ulcerative enteritis and peritonitis for feature, and sick sheep falls group, planted agent, and performance is uneasy, weak, spasm, ophthalmoptosis, abdominal cavity, thoracic cavity, a large amount of ponding of pericardium, dead in a few hours; The sick sheep of sheep enterotoxemia is before dying, and strong paddling appears in four limbs, muscular tremor, and Rotation of eyeball is ground one's teeth in sleep, and saliva is more significantly twitches subsequently, and often die from death in 2-4 hour, upper lower jaw is chuckleed and made a sound, and then goes into a coma, and silently dies; Lamb dysentery main manifestations is strong diarrhoea, spirit difference ight soil stench, and head is to layback, and body temperature reduces, small intestine generation ulcer and blood, pericardial effusion, pulmonary congestion, extravasated blood;
The extracellular toxin of pathogenic and its secretion of clostridium perfringens is relevant, has found 12 kinds of extracellular toxins at present, and that wherein play principal causative effect is α, β, ε, ι 4 kinds, according to these 4 kinds of toxin, this bacterium is divided into serotype in A, B, C, D, E five.Clostridium perfringens A, B, C, D, E type all can produce alpha toxin, and alpha toxin is the major virulent factor of humans and animals gas gangrene and ox, horse, chicken necrotizing enterocolitis.β toxin produces primarily of B, C type bacterium, and ruminating animal (based on sheep) can be caused to suffer from enterotoxemia, and cardinal symptom is vomiting, and bloody diarrhea, even can cause intestinal obstruction and enteric necrosis.ε toxin produces primarily of B, D type bacterium, and it mainly causes sheep enterotoxemia.Only having E type bacterium can produce ι toxin, is a kind of cutaneous necrotizing and mortality toxin, is that generally acknowledged cause lamb, calf and rabbit suffers from the primary toxins of enterotoxemia.
Summary of the invention
The object of the invention is to overcome the defect existed in prior art, there is provided a kind of clostridium perfringens isothermal PCR field quick detection test kit, test kit of the present invention is made up of the primer of an Analysis of Nested Design, 10 × LAMP reaction solution (10 times of loop-mediated isothermal amplification liquid), archaeal dna polymerase, positive control, negative control and developer.The present invention is adopted to infect the clostridium perfringens of the many animals such as qualitative detection pig, ox, sheep, chicken, people.The present invention utilizes the strand displacement characteristic of Bst DNA enzymatic, design 4 primers, identify 6 regions of target sequence, generate a large amount of stem ring texture repeated under isothermal conditions, any specific installation is not needed when qualitative detection, only need observe reacting pipe colour-change can judge therefore have specificity, susceptibility is high and detection time is shorter than regular-PCR, be applicable to the features such as the rapid detection at plant scene.Object of the present invention is embodied in the following aspects:
1. Standard PCR detection technique compares this technical speed slowly, and from sampling out, result is minimum needs 48h, and this technology only needs 30-90 minute;
2. Standard PCR needs large-scale, expensive these technology of plant and instrument such as PCR instrument, electrophoresis system, gel imaging system only to need a thermostat container or thermos cup;
3. the result of Standard PCR must be observed under gel imaging instrument, and the test kit of this technology after completion of the reaction naked eyes gets final product result of determination;
4. the susceptibility, the specificity that detect of this test kit is higher than Standard PCR.
Its concrete technical scheme is:
A kind of clostridium perfringens isothermal PCR field quick detection test kit,
Be made up of a set of primer, 10 times of loop-mediated isothermal amplification liquid, archaeal dna polymerase, positive control, negative control and developers; Described a set of primer is made up of pair of primers and a pair inner primer, and its sequence is:
Primer 1:AGAGAACATGCATGAGCT
Primer 2: CTCTCCAAGATAGAATGTAGCT
Inner primer 1:TCTGTATCAGGATCCCAGAAATGATACTTATCCAGATTATGATAAGAACG
Inner primer 2:ATACCTGACACAGGGGAATCACTTTGCCATTCATATCTAGCT
Wherein, described pair of primers is 1: 6 ~ 9 with the ratio of the mole number of a pair inner primer; Described archaeal dna polymerase is the Bst archaeal dna polymerase with strand displacement effect, and vigor is 8 activity unit/microlitres; Described positive control is the positive plasmid containing inserting clostridium perfringens alpha toxin gene one section of distinguished sequence, and loop-mediated isothermal amplification amplification region is arranged in this distinguished sequence, and its concentration is 10
7copy/μ L; Described negative control is sterilizing distilled water; Described developer is 20 times of SYBR Green I.
Preferably, 10 times of described loop-mediated isothermal amplification liquid are made up of the dNTP of 200mmol/L Tri(Hydroxymethyl) Amino Methane Hydrochloride, 100mmol/L Repone K, 100mmol/L ammonium sulfate, 40 ~ 100mmol/L magnesium sulfate, 4 ~ 10mol/L trimethyl-glycine, 5 ~ 18mmol/L and the Triton X-100 of mass percentage concentration 0.1%.
Preferably, described dNTPs is the equal amount of mixture containing four kinds of dNTP.
Preferably, the making method of described positive plasmid is: adopt pcr amplification one section of clostridium perfringens alpha toxin gene specific sequence, then PMD18-T carrier is loaded, be converted into DH5 α competence bacterium, extract plasmid DNA after sequence is determined, adopt ultraviolet spectrophotometer measure the concentration of plasmid DNA and use sterilizing distilled water adjustment copy number to 10
7copy/μ L.
Preferably, described SYBR Green I is highly sensitive DNA fluorescence dye.
Compared with prior art, beneficial effect of the present invention is:
1, the present invention utilizes the strand displacement characteristic of Bst DNA enzymatic, designs 4 primers, identifies 6 regions of target sequence, generates a large amount of stem ring texture repeated under isothermal conditions, does not need any specific installation when qualitative detection.
2, specificity of the present invention, susceptibility are all higher than regular-PCR technology.
3, the present invention is not owing to needing the recirculation of 3 temperature through round pcr, and detection time is shorter than PCR.
4, result of the present invention judges not need through electrophoresis, simpler than PCR, very easily promotes.For detection by quantitative, although by means of quantitative real time PCR Instrument, do not need to synthesize fluorescent probe, only need by fluorescence dye SYBR Green I, cost lowers greatly.
5, temperature of reaction of the present invention is at 60 DEG C ~ about 65 DEG C, and identifies target gene 6 specific regions, and primer polymer and non-specific amplification probability reduce greatly, and dosing accuracy improves greatly.
Embodiment
Below in conjunction with specific embodiment, technical scheme of the present invention is described in more detail.
A kind of clostridium perfringens isothermal PCR field quick detection test kit, comprises the following steps:
Be made up of a set of primer, 10 times of loop-mediated isothermal amplification liquid, archaeal dna polymerase, positive control, negative control and developers; Described a set of primer is made up of pair of primers and a pair inner primer, and its sequence is:
Primer 1:AGAGAACATGCATGAGCT
Primer 2: CTCTCCAAGATAGAATGTAGCT
Inner primer 1:TCTGTATCAGGATCCCAGAAATGATACTTATCCAGATTATGATAAGAACG
Inner primer 2:ATACCTGACACAGGGGAATCACTTTGCCATTCATATCTAGCT
Wherein, described pair of primers is 1: 6 ~ 9 with the ratio of the mole number of a pair inner primer; Described archaeal dna polymerase is the Bst archaeal dna polymerase with strand displacement effect, and vigor is 8 activity unit/microlitres; Described positive control is the positive plasmid containing inserting clostridium perfringens alpha toxin gene one section of distinguished sequence, and loop-mediated isothermal amplification amplification region is arranged in this distinguished sequence, and its concentration is 10
7copy/μ L; Described negative control is sterilizing distilled water; Described developer is 20 times of SYBR Green I.
Described 10 times of loop-mediated isothermal amplification liquid (usually representing by the form of " 10 × LAMP reaction solution ", the multiple diluted when what numeral " 10 " represented is and uses) are made up of the dNTPs of 200mmol/L Tri(Hydroxymethyl) Amino Methane Hydrochloride, 100mmol/L Repone K, 100mmol/L ammonium sulfate, 40 ~ 100mmol/L magnesium sulfate, 4 ~ 10mol/L trimethyl-glycine, 5 ~ 18mmol/L and the Triton X-100 of mass percentage concentration 0.1%.Described mM is the unit of volumetric molar concentration, refers to the mole number of contained solute in often liter of solution.
Described dNTPs is the equal amount of mixture containing four kinds of dNTP.The making method of described positive plasmid is: adopt pcr amplification one section of clostridium perfringens alpha toxin gene specific sequence, then PMD18-T carrier is loaded, be converted into DH5 α competence bacterium, extract plasmid DNA after sequence is determined, adopt ultraviolet spectrophotometer measure the concentration of plasmid DNA and use sterilizing distilled water adjustment copy number to 10
7copy/μ L.Described SYBR Green I is highly sensitive DNA fluorescence dye.The avidity of SYBR Green I and double-stranded DNA is very high.Restraining effect is not had to enzyme conventional in molecular biology.
Principle of work of the present invention is: loop-mediated isothermal amplification process be first by primer amplification go out inner primer increase required for template and the synthesis of initial reactant template, and then synthesis target gene DNA fragmentation is guided by inner primer, because the DNA fragmentation of inner primer amplification contains this primer 5, the reverse complementary sequence of end DNA fragmentation, thus loop-stem structure is formed by hybridization between these reverse complementary sequences, guiding chain replacement synthesis after an other inner primer and its complementary strand anneal, new loop-stem structure is created in other one end of the DNA fragmentation of amplification, form dumbbell structure, in one hour, this circulating reaction can make target DNA be accumulated to 109 copies, after electrophoresis, visible amplification end product is made up of the DNA fragmentation differed in size, in scalariform band, also amplification is observed by fluorescence dye.The whole reaction of LAMP divides three steps to complete, i.e. the originally synthesis of reactant template, cyclic amplification stage, extension and recirculation.
The above; be only the present invention's preferably embodiment; protection scope of the present invention is not limited thereto; anyly be familiar with those skilled in the art in the technical scope that the present invention discloses, the simple change of the technical scheme that can obtain apparently or equivalence are replaced and are all fallen within the scope of protection of the present invention.
Claims (5)
1. a clostridium perfringens isothermal PCR field quick detection test kit, is characterized in that,
Be made up of a set of primer, 10 times of loop-mediated isothermal amplification liquid, archaeal dna polymerase, positive control, negative control and developers; Described a set of primer is made up of pair of primers and a pair inner primer, and its sequence is:
Primer 1:AGAGAACATGCATGAGCT
Primer 2: CTCTCCAAGATAGAATGTAGCT
Inner primer 1:TCTGTATCAGGATCCCAGAAATGATACTTATCCAGATTATGATAAGAACG
Inner primer 2:ATACCTGACACAGGGGAATCACTTTGCCATTCATATCTAGCT
Wherein, described pair of primers is 1: 6 ~ 9 with the ratio of the mole number of a pair inner primer; Described archaeal dna polymerase is the Bst archaeal dna polymerase with strand displacement effect, and vigor is 8 activity unit/microlitres; Described positive control is the positive plasmid containing inserting clostridium perfringens alpha toxin gene one section of distinguished sequence, and loop-mediated isothermal amplification amplification region is arranged in this distinguished sequence, and its concentration is 10
7copy/μ L; Described negative control is sterilizing distilled water; Described developer is 20 times of SYBR Green I.
2. clostridium perfringens isothermal PCR field quick detection test kit according to claim 1, it is characterized in that, 10 times of described loop-mediated isothermal amplification liquid are made up of the dNTPs of 200mmol/L Tri(Hydroxymethyl) Amino Methane Hydrochloride, 100mmol/L Repone K, 100mmol/L ammonium sulfate, 40 ~ 100mmol/L magnesium sulfate, 4 ~ 10mol/L trimethyl-glycine, 5 ~ 18mmol/L and the Triton X-100 of mass percentage concentration 0.1%.
3. clostridium perfringens isothermal PCR field quick detection test kit according to claim 2, is characterized in that, described dNTPs is the equal amount of mixture containing four kinds of dNTP.
4. clostridium perfringens isothermal PCR field quick detection test kit according to claim 1, it is characterized in that, the making method of described positive plasmid is: adopt pcr amplification one section of clostridium perfringens alpha toxin gene specific sequence, then PMD18-T carrier is loaded, be converted into DH5 α competence bacterium, extract plasmid DNA after sequence is determined, adopt ultraviolet spectrophotometer measure the concentration of plasmid DNA and use sterilizing distilled water adjustment copy number to 10
7copy/μ L.
5. clostridium perfringens isothermal PCR field quick detection test kit according to claim 1, is characterized in that, described SYBR Green I is highly sensitive DNA fluorescence dye.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104830698A (en) * | 2015-04-30 | 2015-08-12 | 广西壮族自治区农业科学院甘蔗研究所 | Pokkah boeng disease pathogen separating and identifying method |
| CN106148548A (en) * | 2016-08-31 | 2016-11-23 | 青海省畜牧兽医科学院 | A kind of can detect bacillus perfringens, clostridium hemolyticum and the multiple PCR detection kit of Type B Nuo Weishi clostridium and application thereof simultaneously |
Citations (2)
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| CN102134590A (en) * | 2010-12-07 | 2011-07-27 | 浙江省质量技术监督检测研究院 | Fast detection method for clostridium perfringens, detection primer group and detection kit |
| CN104087685A (en) * | 2014-07-04 | 2014-10-08 | 陕西溯源农业发展有限公司 | Pseudorabies isothermal PCR (polymerase chain reaction) field rapid detection kit |
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2014
- 2014-10-20 CN CN201410586237.2A patent/CN104328177A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102134590A (en) * | 2010-12-07 | 2011-07-27 | 浙江省质量技术监督检测研究院 | Fast detection method for clostridium perfringens, detection primer group and detection kit |
| CN104087685A (en) * | 2014-07-04 | 2014-10-08 | 陕西溯源农业发展有限公司 | Pseudorabies isothermal PCR (polymerase chain reaction) field rapid detection kit |
Non-Patent Citations (1)
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104830698A (en) * | 2015-04-30 | 2015-08-12 | 广西壮族自治区农业科学院甘蔗研究所 | Pokkah boeng disease pathogen separating and identifying method |
| CN106148548A (en) * | 2016-08-31 | 2016-11-23 | 青海省畜牧兽医科学院 | A kind of can detect bacillus perfringens, clostridium hemolyticum and the multiple PCR detection kit of Type B Nuo Weishi clostridium and application thereof simultaneously |
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