CN107012239A - A kind of multiplex PCR classifying method of C.perfringens - Google Patents

A kind of multiplex PCR classifying method of C.perfringens Download PDF

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Publication number
CN107012239A
CN107012239A CN201710314609.XA CN201710314609A CN107012239A CN 107012239 A CN107012239 A CN 107012239A CN 201710314609 A CN201710314609 A CN 201710314609A CN 107012239 A CN107012239 A CN 107012239A
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perfringens
toxin
bacterium
pcr
detection
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关平原
雷宇
李平安
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Inner Mongolia Agricultural University
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雷宇
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • 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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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

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Abstract

The conserved sequence of α, β and ε toxin encoded according to common tetra- kinds of bacterial type bacterial strains of C.perfringens A, B, C, D, separately designs the specific primer synthesized for this 3 kinds of toxin, has invented the method that multiplex PCR detects different shaped C.perfringens.Show that this method is not only quick, accurate by the parting detection to reference strain, and with good specificity and sensitiveness.The C.perfringens of separation is detected, also produced a desired effect.

Description

A kind of multiplex PCR classifying method of C.perfringens
Technical field
The invention belongs to Bacteria Detection field, and in particular to the detection method of bacterial infectious disease, more particularly to aerogenesis The detection method and application field of capsular clostridium.
Background technology
C.perfringens (Clostridium perfringens), it is extremely wide in distributed in nature, under certain condition may be used Cause a variety of serious diseases.The bacterium can produce a variety of exotoxins, but play principal causative and have 4 kinds of toxin of α, β, ε, ι, root According to the difference of bacterium toxin producing, this bacterium is divided into 5 types of A, B, C, D, E.A type bacterium mainly produce alpha toxin, cause people and dynamic Thing emphysematous gangrene and food poisoning, can also cause the enterotoxemia of ox, lamb, goat, reinder, piglet and rabbit etc.;Type B bacterium It is main to produce α, β, ε toxin, cause lamb dysentery, can also cause coltfoal, calf, lamb, the enterotoxemia or bad of sheep and goat Dead property enteritis;C-type bacterium mainly produces α, β toxin, is the cause of disease of struck, can also cause lamb, calf, piglet and sheep Enterotoxemia, the necrotic enteritis of necrotic enteritis and people;D types bacterium mainly produces α, ε toxin, cause lamb, sheep, The enterotoxemia of goat, ox and squirrel;E types bacterium mainly produces α, ι toxin, can cause calf, lamb enterotoxemia, but seldom Occur.
This research is with perfringens alpha, β, ε toxincap、cpb、etxGene is target sequence, establishes detection aerogenesis The multiple PCR method of capsular clostridium.Detection and parting for C.perfringens provide effective method.
The content of the invention
According to the perfringens alpha, the gene order of β, ε toxin logged in GenBank, 3 pairs of specific primers are designed. Establish detection and parting C.perfringens multiple PCR method so that realize to C.perfringens it is sensitive, efficient, Quickly, accurate detection and parting.
It is an object of the invention to carry out Classification Identification to the C.perfringens in animal body by multiple PCR method.
It is an object of the invention to provide the primer that one group of multiplex PCR detects C.perfringens, the primer such as SEQ ID NO:1 and SEQ ID NO:Shown in 2.
It is an object of the invention to provide the kit of C.perfringens multiplex PCR parting, the kit includes inspection Survey detection perfringens alpha, the primer of β, ε toxin.
A kind of method that the present invention specifically provides multiplex PCR detection and parting C.perfringens, is comprised the following steps that：
1. the design and synthesis of primer：
According to the C.perfringens coding for alpha, the gene order of 3 kinds of toxin of β, ε delivered in GenBank（cap、cpb、etx）Protect Defending zone designs 3 pairs of primers, and is synthesized by Huada gene company.
2. the preparation of target template DNA
Bacterium solution 1.0mL is taken, is extracted according to the specification of Tiangeng bacterial genomes DNA extraction kit.
25.0 μ LMix, 11.0 μ LddH are added in 50 μ L systems2O, template, each 2 μ L of upstream and downstream primer.Loop parameter is： 94 DEG C of min of pre-degeneration 5；94 DEG C of 45s, 55 DEG C of 45s, 72 DEG C of 30s, totally 30 circulations, last 72 DEG C of extensions 2min；Purpose fragment Length is respectively 288bp, 151bp, 678bp.
In the present invention, reference gene storehouse（Genbank）Middle accession number is X17300 alpha toxin gene order, and accession number is L13198 β toxin gene sequences, accession number designs primer for X60694 ε toxin, and those of ordinary skill in the art can lead to The channel for crossing the public is known.
The present invention is further confirmed to the specificity and sensitiveness of C.perfringens detection method.
The amplification system provided with the present invention has expanded clostridium septicum, Pasteurella, ox, Brucella melitensis, large intestine respectively The nucleic acid samples such as bacillus, sample does not have positive amplification band, shows that this method has preferable specificity.
It is 1 × 10 to choose dilution factor5Copies/ μ L DNA profiling, is carried out sensitive in 4 10 times of doubling dilutions, foundation group Property experiment.As a result show sensitiveness 1 × 102Copies/ μ L, show that this method has good sensitiveness.
By implementing the specific content of the invention of the present invention, following beneficial effect can be reached.
This method detection time is short, in traditional toxin and experimental method at least will be to parting time of C.perfringens More than 7 days, the invention detection time included sample pre-treatments to testing result is obtained within 3 days, is tested than traditional neutralization Method at least will also shorten 4 days or so.Detection sensitivity is high, this method detectable 1 × 102Copies/ μ L copies DNA,.Specificity is good, by being detected to DNA samples such as C.perfringens, clostridium septicum, Pasteurellas, only perfringens Clostridium test positive.This method flow is simple, strong operability, it is easy to grasp, as long as possessing molecular biology mechanism knowledge, nothing Need good special training just can complete very well.It can be achieved that process of the test is implemented to monitor by the use of this method, effective solution Determine false negative result present in traditional detection method, quality monitoring can have been carried out to laboratory, it is ensured that the standard of testing result True property.Parting is carried out to the C.perfringens of 11 plants of separation with the inventive method, expected results are obtained.
This method can differentiate 3 kinds of toxin genes of α, β, ε or progress single-gene mirror that detection C.perfringens is produced simultaneously It is fixed.The research for being established as Related Bacteria inspection and quarantine technology of this method provides preferable reference, and is specification detection Reagent has to the development of standardization instructs reference.This method can be widely applied for inspection and quarantining for import/export portion Door, animal and veterinary department and cultivation unit, are that effective prevention and control of epidemic disease are significant.
Brief description of the drawings
Fig. 1：C.perfringens Reference Strains multiplexed PCR amplification α, β, ε result：Wherein 1,2,3,4 be respectively perfringens Clostridium A types（Alpha toxin）Amplified production, C.perfringens Type B（α, β, ε toxin）Amplified production, C. perfringens Type C（α、β Toxin）Amplified production, C.perfringens D types（α, ε toxin）Amplified production, M is DL2000marker, and 5 be blank control.
Fig. 2：C.perfringens Type B refers to verticillium toxin gene PCR sensitiveness amplification：Wherein 1~6 swimming lane is respectively 1×105~1 × 100Copies/ μ L are the multiplexed PCR amplification of the progress of template, and 7 be blank control, and M is DL2000marker.
Fig. 3：C.perfringens refers to bacterium A, B, C, D type multiplex PCR specific amplification result：Wherein 1 is that A types refer to bacterium 2 be that Type B refers to bacterium, and 3 be that c-type refers to bacterium, and 4 be that D types refer to bacterium, and 5 be clostridium septicum, and 6 be Escherichia coli, and 7 be Pasteurella, M For DL2000marker.
Fig. 4：The C.perfringens result that multiplexed PCR amplification is clinically separated：Wherein M is DL2000 maker, and 1 is A Type refers to bacterium, and 2 be that Type B refers to bacterium, and 3 be that c-type refers to bacterium, and 4 be that D types refer to bacterium, and 5 ~ 15 be the C.perfringens being clinically separated, 16 be blank control, and M is DL2000marker.
Embodiment
The design of primers of embodiment one and synthesis
According to the C.perfringens coding for alpha, the gene order of 3 kinds of toxin of β, ε delivered in GenBank（cap、cpb、etx）Protect Defending zone designs 3 pairs of primers, and is synthesized by Huada gene company.
Primer code name sequence in the present invention is shown in Table 1
Embodiment two：The acquisition of To Template
Ox, sheep enteron aisle content and its hetero-organization are taken under aseptic condition, is dipped with oese, streak inoculation is in through tryptone-Asia Sulfate-seromycin agar（TSC）On, 37 DEG C of Anaerobic culturel 18h.Black colonies on picking TSC flat boards, are inoculated into lonely 18h is cultivated in liver bouillon, extracting DNA with Tiangeng bacterial genomes kit is used as masterplate.
Embodiment three：The foundation of multiplexed PCR amplification system
PCR reaction systems are 50 μ L, take PCR pipe to sequentially addPremix Ex Tap25 μ L, 3 pairs of primers (10 pmol/ μ L) Each 2 μ L, DNA masterplate 2 μ L, ddH2O 11µL.Reacted as follows in PCR instrument：95 DEG C of pre-degeneration 5min, 95 DEG C are denatured 30s, 54 DEG C annealing 30s, 72 DEG C extension 1min carry out 30 circulation, then again 72 DEG C extension 10 min.7 μ L PCR are taken to produce after the completion of PCR Thing, with the 110V min of electrophoresis 30, is observed and taken pictures with gel imager in 1% Ago-Gel.
Multiplexed PCR amplification is with reference to verticillium toxin gene map referring to accompanying drawing 1
Example IV：Sensitivity tests and specific test
The concentration of bacterium DNA masterplates is referred to 260nm spectrophotometric determinations Type B, and copy number is calculated with formula, 1 × 10 is taken5~1 ×100Copies/ μ L totally 6 dilution factors, each dilution factor takes 2 μ L as multi-PRC reaction template respectively, carries out multiplex PCR Expand to detect its sensitiveness.
Sensitiveness PCR figures are referring to accompanying drawing 2
Reaction system and response parameter are pressed with clostridium septicum DNA, Pasteurella DNA and e. coli dna, enters performing PCR specificity Experiment.Aerogenesis clostridium A, B, C, D type reference strain DNA is taken as positive control, while setting up negative control.
Specific PCR figure is referring to accompanying drawing 3
Embodiment five：Detection and parting to sample
The C.perfringens of 11 portions of oxen, small sheep intestines separation is detected.As seen from the figure, there is corresponding amplification bar in positive control Band, negative control does not occur amplified band.It is C. perfringens Type A wherein to have 3 parts, and 3 parts are C. perfringens Type C, 5 parts For C.perfringens D types.
Sample detection parting PCR figures are referring to accompanying drawing 4
Above example further illustrates present disclosure, but should not be construed as limiting the invention.Without departing substantially from this In the case of spirit and essence, the modifications or substitutions done to the inventive method, step or condition belong to the present invention's Category.

Claims (2)

1. a kind of multiplex PCR classifying method of C.perfringens, includes the primer of α, β, ε toxin of C.perfringens.
2. described in primer be used for C.perfringens produce α, β, ε toxin detection and C.perfringens A, B, C, D type point Type.
CN201710314609.XA 2017-05-06 2017-05-06 A kind of multiplex PCR classifying method of C.perfringens Pending CN107012239A (en)

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Citations (7)

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Publication number Priority date Publication date Assignee Title
CN101613746A (en) * 2009-08-05 2009-12-30 广州军区广州总医院 The fast quantitative measurement method for detecting of clostridium perfringens and detection kit
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
CN104131022A (en) * 2014-08-01 2014-11-05 中国农业科学院兰州兽医研究所 Clostridium perfringens alpha, beta 1, beta 2 and epsilon coexpression vector and construction method and expression method thereof
WO2015044472A1 (en) * 2013-09-27 2015-04-02 Universidad De Oviedo Method for the simultaneous detection of pathogenic micro-organisms
CN104531867A (en) * 2014-12-25 2015-04-22 湖北省农业科学院畜牧兽医研究所 Clostridium perfringens enterotoxin positive bacteria dual fluorescent quantitative PCR rapid detection kit
KR20160014960A (en) * 2014-07-30 2016-02-12 대한민국 (식품의약품안전처장) Multiplex real-time PCR detection primer set for pathogenic genes of food borne pathogenic microorganism, and uses thereof
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Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101613746A (en) * 2009-08-05 2009-12-30 广州军区广州总医院 The fast quantitative measurement method for detecting of clostridium perfringens and detection kit
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
WO2015044472A1 (en) * 2013-09-27 2015-04-02 Universidad De Oviedo Method for the simultaneous detection of pathogenic micro-organisms
KR20160014960A (en) * 2014-07-30 2016-02-12 대한민국 (식품의약품안전처장) Multiplex real-time PCR detection primer set for pathogenic genes of food borne pathogenic microorganism, and uses thereof
CN104131022A (en) * 2014-08-01 2014-11-05 中国农业科学院兰州兽医研究所 Clostridium perfringens alpha, beta 1, beta 2 and epsilon coexpression vector and construction method and expression method thereof
CN104531867A (en) * 2014-12-25 2015-04-22 湖北省农业科学院畜牧兽医研究所 Clostridium perfringens enterotoxin positive bacteria dual fluorescent quantitative PCR rapid detection kit
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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鲍长磊: "产气荚膜梭菌不同毒素型多重PCR和α毒素抗体ELISA检测方法的建立及初步应用", 《中国优秀硕士学位论文全文数据库 农业科技辑》 *

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