CN106701976B - The cause of disease multiple PCR detection kit of one breeder common bacteria disease - Google Patents
The cause of disease multiple PCR detection kit of one breeder common bacteria disease Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- 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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- C—CHEMISTRY; METALLURGY
- 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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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses the cause of disease multiple PCR detection kits of a breeder common bacteria disease, the kit includes PCR reaction solution and pasteurella multocida, salmonella, Escherichia coli O 157: the specific primer of this 4 kinds of bacteriums of H7 and staphylococcus aureus, positive control (pasteurella multocida, salmonella, Escherichia coli O 157: H7 and staphylococcus aureus standard items or pasteurella multocidaKMT1Gene, salmonellainvAGene, Escherichia coliO157:H7 rfbEGene and staphylococcus aureusnucThe positive plasmid of gene), negative control (sterilizing without enzyme distilled water), DNA Marker.The present invention uses Quadruple- PCR, can quickly, it is accurate, synchronously detect 4 kinds of pathogens, and sensibility is good, high specificity, and Monitoring lower-cut reaches 0.01ng/ μ L, and accuracy rate is up to 92.6%.
Description
Technical field
The invention belongs to technical field of biological, and in particular to the cause of disease multiplex PCR of a breeder common bacteria disease detects
Kit further relates to the application method of the kit.
Background technique
Escherichia coli O 157: H7, salmonella, staphylococcus aureus and pasteurella multocida are in chicken growth course
The cause of disease of common bacteriosis.They not only seriously endanger the development of China's aviculture, but also Escherichia coli O 157: H7,
Salmonella, staphylococcus aureus can also infect the mankind by food pollution, threaten to human health.
Escherichia coli (E.Coli) O157:H7 is a main bacterial type of enterohemorrhagic escherichia coli, and people and animals can be caused total
Illness, potential outbreak of epidemic trend, strong pathogenic, lethal, there is it greatly to aquaculture and human security
Harm.Salmonella (Salmonella) is zoonosis pathogen, easily generation drug resistance, is endangered seriously aquaculture,
The account for the first in the pathogen for causing mankind's food origin disease.Pasteurella multocida (Pasteurella multocida) is
Cause the conditioned pathogen of humans and animals contact, strong bacterial infectious disease (also known as avian cholera, hueppe's disease), it is high-incidence
Sick rate and the death rate influence seriously, to be listed in one of the poultry disease of the anti-system of emphasis, had case gradually to increase in recent years on aquaculture
More trend.Staphylococcus aureus (Staphylococcus aureus) is that animal suppurative infection, cross-infection are most common
Pathogen has the nickname of " thermophilic meat bacterium ", is to endanger one of disease of most serious in current poultry production.China's law is to upper simultaneously
Stating quantity of the bacterium (except Pasteurella) in food has stringent restriction.
It is directed to the detection of farm's bacterial disease at present, depends on bacteriology culture, general time-consuming 4-7d, detection method
It is comparatively laborious, time-consuming, separation farm's various pathogens are detected simultaneously and mixed infection situation is cumbersome.Immunoblotting skill
Art, enzyme linked immunosorbent assay, nucleic acid hybridization technique, round pcr etc. are due to its high specificity, sensibility are high, easy to operate quick
It is widely used, but these methods can only usually detect a kind of pathogen every time.Multiplex PCR (Multiplex PCR) is normal
A kind of Novel DNA amplification technique for improving and growing up on the basis of rule PCR, is in same reaction system while to be added multipair
The a plurality of target DNA fragment of primer amplification is, it can be achieved that multiple pathogenic microorganisms detect simultaneously.The advantage of multiple PCR technique is can
To carry out quick, comprehensive, system, accurate detection and identification to cause of disease, and operation is simple as regular-PCR, so can
To greatly improve detection efficiency, shorten detection cycle, reduction testing cost, it is particularly suitable for the detection of pathogen in groups, has aobvious
The economic benefit and social benefit of work.
The present invention is directed to establish a kind of Quadruple- PCR rapid detection method, realize to colon bacillus 0157: H7, salmonella,
The simultaneously and rapidly detection of four kinds of infectious diseases common to human beings and animals caused by pasteurella multocida, staphylococcus aureus, right pop disease
Investigation and effectively progress livestock and poultry pestilence monitor prevention and control, raising public health security has definite meaning.
Summary of the invention
The purpose of the invention is to provide the cause of disease multiple PCR detection kit of a breeder common bacteria disease, the examinations
Agent box is made of multi-PRC reaction liquid, positive control and negative controls.Pathogen is detected in poultry in the past to pass through mostly
The separation identification of bacterium is detected with the PCR for single bacterium, this detection kit is thin for most common 4 kinds of chicken bacterial disease
Non-interfering 4 pairs of detection primers are designed and filtered out to bacterium, detects 4 kinds of pathogens and completes in a reaction, highly efficient,
And sensibility is good, high specificity, Monitoring lower-cut reaches 0.01ng/ μ L.
A further object of the invention is the cause of disease multiple PCR detection kit for being the provision of a breeder common bacteria disease
Application method, method is simple, and quickly, all detecting steps can be completed in 4 hours.
To achieve the goals above, the present invention takes following technical measures:
The cause of disease multiple PCR detection kit of one breeder common bacteria disease, including (the TaqDNA polymerization of multi-PRC reaction liquid
Enzyme is free of Mg2+PCR reaction buffer, Mg2+, dNTPs), pasteurella multocida, salmonella, Escherichia coli O 157: H7
With the specific primer (as shown in table 1) of staphylococcus aureus this 4 kinds of bacteriums, positive control (pasteurella multocida, sramana
Salmonella, Escherichia coli O 157: H7 and staphylococcus aureus standard items or pasteurella multocida KMT1 gene, salmonella
InvA gene, Escherichia coli O 157: the positive plasmid of H7rfbE gene and staphylococcus aureus nuc gene), negative control
(sterilizing without enzyme distilled water), DNA Marker.
1 multiplex PCR specific primer sequence table of table
The application method of the cause of disease multiple PCR detection kit of one breeder common bacteria disease, its step are as follows:
1, the extraction of the processing of sample and DNA profiling:
Infection early stage enteron aisle is the main parasitic position of above 4 kinds of pathogenic bacteria, and live-bird sampling mainly passes through acquisition anus cotton
Swab, dead fowl can use intestinal contents, and meat products can directly take fritter tissues.The genomic DNA for extracting sample, takes 2 μ L
(20ng) is used as sample to be tested.Detection should all set up positive control and negative control every time.
2, reaction system and reaction condition:
Reaction system is following (25 μ L of total volume):
Reaction condition: 94 DEG C of 4min of initial denaturation；94 DEG C of 30s are denaturalized, anneal 56 DEG C of 30s, extends 72 DEG C of 45S, recycles 30 altogether
It is secondary；Extend 72 DEG C of 10min eventually,.
PCR product identification: take 8 μ L pcr amplification products that 2 μ 6 × Loading of L Buffer is added to mix, with DL2000 DNA
Marker is indicated as relative molecular mass, with 1.8% Ago-Gel (the 0.03 μ L/mL containing the EB) electrophoresis under 100V voltage
30min sets and carries out interpretation of result in gel imaging system.
3, the judgement of result:
(1) quality control standard
Negative control: the corresponding swimming of loading wells takes no band；
Positive control: the corresponding swimming of loading wells takes 4 apparent positive bands: Escherichia coli O 157: H7 amplification
Positive fragment 678bp (between 500-700bp), salmonella 284bp (slightly above 250bp), pasteurella multocida 460bp
(close to 500bp), staphylococcus aureus 229bp (are lower than 250bp)；
This detection is invalid if negative control or positive control have one not to be inconsistent standardization.
(2) determine result
Without band on the corresponding swimming lane in test sample hole, result judgement is feminine gender；
Occur≤4 swimming bands on the corresponding swimming lane in test sample hole, band corresponding with standard positive hole is compared, such as
Point-blank, corresponding Bacteria Detection result is judged as positive to really bar band；If not point-blank or detection hole
Occur >=5 swimming bands on swimming lane, process contamination may be tested, it is proposed that reform.
Compared with prior art, the present invention having the following advantages and beneficial effects:
1, currently, the case where a variety of cross infection of disease of aquaculture clinical case, mixed infection is commonplace, or even have more
Carry out more complicated situation, but detects fowl bacterial clinical case and carry out epidemiological survey and mainly take bacterium separation identification
Method needs multiple detecting steps, complicated for operation, time and effort consuming, and can only once detect a kind of pathogen.This research is established more
Weight PCR detection method overcomes above deficiency, after Bacteria Culture, completes while detecting Escherichia coli O 157: H7, Salmonella
The task of bacterium, four kinds of pasteurella multocida, staphylococcus aureus pathogens only needs about 4h, have preferable specificity and
Sensibility (Monitoring lower-cut is up to 10pg/uL), it is easier than traditional bacteriological method economical, there is biggish application value.
2, there are multipair primer and multiple templates in multi-PRC reaction system, arbitrarily both exist and interact
The case where, cause the appearance of false positive, therefore the key factor for being designed to multi-PCR detection method foundation of primer, this hair
The detection primer of bright offer ensures between different primers pair, does not have affinity between primer and non-target sequences, four of amplification
Target gene fragment length also has certain gap, and makes Ago-Gel in certain concentration range, guarantees purpose when electrophoresis
Band can be distinguished obviously.
3, kit provided by the invention can be used for the detection of the live-birds anus swab such as chicken, be conducive to clinical popularization.This examination
Agent box can also be used for the germ contamination detection during meat products processing.The kit be in aquaculture and food hygiene more than
The detection and prevention and control of four kinds of bacteriums provide effective tool and method, and accuracy rate is up to 92.6%.
Detailed description of the invention
Fig. 1 is the specific detection result of single primer in multiple PCR detection kit of the present invention
Note: A is the specific detection result of Escherichia coli O 157 primer；B is the specific detection knot of salmonella primer
Fruit；C is the specific detection result of pasteurella multocida primer；D is the specific detection knot of staphylococcus aureus primer
Fruit.
Fig. 2 is that four kinds of bacterial primers mix specific detection result in multiple PCR detection kit of the present invention
Note: A is the specific amplification result for detecting a kind of bacterium；B is the specific amplification result for detecting two kinds of bacteriums；C
For the specific amplification result for detecting three kinds of bacteriums；D is the testing result for detecting four kinds of bacterium solution mixed liquors.
Fig. 3 is the result figure of multiple PCR detection kit sensitivity test of the present invention
Note: M:DL2000Marker；1:10ng/ μ L；2:1ng/ μ L；3:0.1ng/ μ L；4:0.01ng/ μ L；5:
0.001ng/μL；6:0.1pg/ μ L.
Fig. 4 is the result figure that multiple PCR detection kit of the present invention detects analog sample
Note: M:DL2000Marker；1,4,9: Escherichia coli O 157: H7；2,5,8: salmonella；3,6,10: more killing property
Pasteurella；7,11,12: staphylococcus aureus；The positive control of 13-16: four plants of bacterium；17-20: negative control.
Specific embodiment
Invention is further explained in the following with reference to the drawings and specific embodiments, these embodiments are merely to illustrate this
It invents rather than limits the scope of the invention.
Embodiment 1:
The cause of disease multiple PCR detection kit and application method of chicken common bacteria disease
The cause of disease multiple PCR detection kit of one breeder common bacteria disease, which includes: multi-PRC reaction liquid, sun
Property control, negative control, DNAMarker2000 (100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp；TaKaRa
Ltd), the specific primer (10uM) of four pairs of bacteriums: rfbE-F (SEQ ID NO.1), rfbE-R (SEQ ID NO.2), invA-
F (SEQ ID NO.3), invA-R (SEQ ID NO.4), KMT1-F (SEQ ID NO.5), KMT1-R (SEQ ID NO.6),
Nuc-F (SEQ ID NO.7), nuc-R (SEQ ID NO.8)；
The multi-PRC reaction liquid are as follows: Taq archaeal dna polymerase (2-5U/ μ L) is free of Mg2+10 × PCR react buffering
Liquid, 25mM Mg2+With 10mM dNTPs；
The positive control are as follows: Escherichia coli O 157: H7 (CICC21530) standard items, salmonella (CVCC3375)
Standard items, pasteurella multocida (CVCC44801) standard items, staphylococcus aureus (SY1G7) standard items are (micro- purchased from China
Biological inoculum collection) or Escherichia coli O 157: H7rfbE gene masculine plasmid, salmonella invA gene masculine plasmid,
The positive plasmid of pasteurella multocida KMT1 gene masculine plasmid, staphylococcus aureus nuc gene；
The negative control is sterilizing without enzyme distilled water；
The specific primer are as follows:
rfbE-F 5′-AACGGTTGCTCTTCATTTAG-3′(SEQ ID NO.1)
rfbE-R 5′-GAGACCATCCAATAAGTGTG-3′(SEQ ID NO.2)
invA-F 5′-TCATCGCACCGTCAAAGGAACC-3′(SEQ ID NO.3)
invA-R 5′-GTGAAATTATCGCCACGTTCGGGCAA-3′(SEQ ID NO.4)
KMT1-F 5′-ATCCGCTATTTACCCAGTGG-3′(SEQ ID NO.5)
KMT1-R 5′-GCTGTAAACGAACTCGCCAC-3′(SEQ ID NO.6)
nuc-F 5′-AGATAACGGCGTAAATAGAAGTGG-3′(SEQ ID NO.7)
nuc-R 5′-TGCACTTGCTTCAGGACCATA-3′(SEQ ID NO.8)
The application method of the cause of disease multiple PCR detection kit of one breeder common bacteria disease, its step are as follows:
1, the extraction of the processing of sample and DNA profiling:
Infection early stage enteron aisle is the main parasitic position of above 4 kinds of pathogenic bacteria, and live-bird sampling mainly passes through acquisition anus cotton
Swab, dead fowl can use intestinal contents, and meat products can directly take fritter tissues.Samples taken uses QIAGEN company, Germany
QIAxtractor high-throughput nucleic acid Purification Station extracts DNA profiling, it is possible to use other DNA extraction kits are extracted, specifically
Step takes 2 μ L (20ng) as sample to be tested referring to specification.Detection should all set up positive control and negative control every time.
2, reaction system and reaction condition:
Reaction system is following (25 μ L of total volume), and the concentration of each ingredient is with embodiment 1:
Reaction condition: 94 DEG C of 4min of initial denaturation are denaturalized 94 DEG C of 30s, and anneal 56 DEG C of 30s, extend 72 DEG C of 45S, recycle 30 altogether
It is secondary, extend 72 DEG C of 10min eventually.
PCR product identification: take 8 μ L pcr amplification products that 2 μ 6 × Loading of L Buffer is added to mix, with DL2000 DNA
Marker is indicated as relative molecular mass, with 1.8% Ago-Gel (the 0.03 μ L/mL containing the EB) electrophoresis under 100V voltage
30min sets and carries out interpretation of result in gel imaging system.
3, the judgement of result:
(1) quality control standard:
Negative control: the corresponding swimming of loading wells takes no band；
Positive control: the corresponding swimming of loading wells takes 4 apparent positive bands: Escherichia coli O 157: H7 amplification
Positive fragment 678bp (between 500-700bp), salmonella 284bp (slightly above 250bp), pasteurella multocida 460bp
(close to 500bp), staphylococcus aureus 229bp (are lower than 250bp)；
This detection is invalid if negative control or positive control have one not to be inconsistent standardization.
(2) determine result:
Without band on the corresponding swimming lane in test sample hole, result judgement is feminine gender；
Occur≤4 swimming bands on the corresponding swimming lane in test sample hole, band corresponding with standard positive hole is compared, such as
Point-blank, corresponding Bacteria Detection result is judged as positive to really bar band；If not point-blank or detection hole
Occur >=5 swimming bands on swimming lane, process contamination may be tested, it is proposed that reform.
Embodiment 2:
The design and screening of the cause of disease multiple PCR detection kit specific primer of chicken common bacteria disease
One, materials and methods
1, bacterium bacterial strain
Reference culture: pasteurella multocida (Pasteurella multocida CVCC44801), salmonella
(Salmonella enteritidis CVCC 3375), Escherichia coli O 157: H7 (Escherichia coli
CICC21530) and staphylococcus aureus (Staphylococcus aureus SY1G7) reference culture is purchased from the micro- life of China
Object Culture Collection Center.The bacterium and number that 16 plants of laboratory clinical separation are identified: fowl derived bacterium salmonella HZBL08121,
It is salmonella CMCC50051, salmonella HZBL08122, salmonella HZBL08123, pasteurella multocida HBNKM, more
Killing property Pasteurella HBNK1G1, staphylococcus aureus GX12G54, comma bacillus HS1201, morganella morganii HZBL08123, sieve
This Salmonella GX1202, enterococcus faecalis HS12E6, riemerella anatipestifer ATCC11845, Escherichia coli HZBL08121, large intestine bar
Bacterium HZBL08122, pseudo- osculant staphylococcus GX12G6, Staphylococcus sciuri GX12G5.
2, the extraction of DNA of bacteria template is the same as embodiment 1.
3, the design and synthesis of primer
According to gene order announced in GenBank, analyzed using Primer 5.0, Oligo 6.0 and BLAST software
4 pairs of specific primers (being synthesized by the raw work in Shanghai) is designed, primer sequence and target gene fragment are shown in Table 2.
2 multiple PCR primer sequence of table
4, the specificity of 4 kinds of bacteriums of detection compares when single primer PCR reacts
For determine primer specific and single PCR optimum reaction condition, by every pair of primers respectively to above-mentioned 20
Pathogen strain bacterium carries out PCR specific amplification.Initial reaction system is 25 μ L:10 × EasyTaq Buffer 2.5 μ L, dNTPs
2.0 μ L, each 0.5 μ L, Taq DNA Polymerase of upstream and downstream primer 0.25 μ L, a kind of 2.0 μ l of template of bacterium, distilled water
It mends to 25 μ L.Initial reaction program is 94 DEG C of 4min of initial denaturation；94 DEG C of 30s are denaturalized, anneal 56 DEG C of 30s, extends 72 DEG C of 45s, altogether
Circulation 30 times；Extend 72 DEG C of 10min eventually, is saved in 4 DEG C.PCR result is identified with embodiment 1.
5, a kind of bacterium is detected after the mixing of four species-specific primers and the result of various bacteria compares
In order to reduce the influence expanded after primer mixing to target gene, 4 tests below ad hoc meter are drawn to evaluate design
The specificity of object.
A kind of bacterium of PCR reaction detection is distinguished after (1) four kind of primer mixing
Any one bacterial template is added with above-mentioned 4 in reaction condition.
Two kinds of bacteriums of PCR reaction detection are distinguished after (2) four kinds of primer mixing
The bacterial template of any two kinds of combinations is added with above-mentioned 4 in reaction condition.
Three kinds of bacteriums of PCR reaction detection are distinguished after (3) four kinds of primer mixing
The bacterial template of three kinds of any combination is added with above-mentioned 4 in reaction condition.
Four kinds of bacteriums of PCR reaction detection are distinguished after (4) four kinds of primer mixing
Four kinds of bacterial templates are added with above-mentioned 4 in reaction condition.
Two, test result
1, single primer PCR result
Each pair of primer is distinguished into 20 bacterial strains of specific amplification, the result for adding a kind of primer amplification is single, and band is clear,
In the same size with expected segment, specific testing result is shown in Fig. 1, illustrates that primer specificity is good.
2, the specific detection result of four kinds of primer mixing PCR reaction
After the mixing of four kinds of primers, a kind of bacterium or various bacteria are either expanded, the amplification of target gene is all had no
It influences.After the target fragment electrophoresis of amplification, band is clear；Multiple target fragments are also easy to distinguish (see Fig. 2).Illustrate design primer
Quality is higher, can be completely used for the detection of clinical sample.
Embodiment 3:
The specificity and sensibility of the common four kinds of bacterial diseases cause of disease multi-PCR detection method of chicken:
One, materials and methods
1, reference culture is the same as embodiment 2.
2, according to the application method of multiple PCR detection kit described in embodiment 1, extract respectively pasteurella multocida,
Salmonella, Escherichia coli O 157: the DNA of H7 and staphylococcus aureus standard items sample simultaneously carries out multi-PRC reaction.
3, the specific detection of Quadruple- PCR reaction
16 plants of the fowl derived bacterium (with embodiment 2) for choosing laboratory clinical separation extracts DNA, carries out multiplex PCR detection.
4, the sensitivity Detection of Quadruple- PCR reaction
Measure four kinds of bacterial strains (pasteurella multocida, salmonella, Escherichia coli O 157: H7 and staphylococcus aureus)
DNA concentration and be diluted, make initial concentration be about 100ng/ μ L, carry out 10 times of gradient dilutions, the bacterial strain after dilution
Concentration is 10ng/ μ L, 1ng/ μ L, 0.1ng/ μ L, 0.01ng/ μ L (i.e. 10pg/ μ L), 1pg/ μ L, 0.1pg/ μ L, and each bacterial strain is each
Gradient respectively takes 200 μ L to be uniformly mixed, and makes in reaction system to contain four kinds of DNA simultaneously, sterilizing without enzyme distilled water as negative control,
It is expanded according to multi-PRC reaction condition described in embodiment 1.Detect its sensibility and minimum detection limit, each dilution ladder
Degree does 3 detections and repeats.
5, analog sample detects: taking the Escherichia coli O 157 for being clinically separated and saving: H7, salmonella, more killing property respectively
Pasteurella and each 3 plants of staphylococcus aureus, number 1-12 go out using this experiment reference culture used as positive control
Bacterium is negative control without enzyme distilled water, and the multiple PCR method after optimizing application detects Clinical isolation, identifies that this is multiple
The feasibility of PCR method；Then by this 12 plants of bacteriums again random number, and with former number corresponding record, manual simulation is made
Sample.
The preparation of manual simulation's sample: will carry out dissect under healthy chick's aseptic condition, taken liver is divided into 12
Part, the 12 pathogen strain bacterium renumberd are inoculated in taken liver respectively, tissue is homogenized by each PBS200 μ L that 10mM/L is added
After be separately added into 2mL TSB fluid nutrient medium, be placed in 300r/min in constant-temperature table, 37 DEG C of culture 5h.The above culture is pressed
DNA of bacteria is extracted as template according to boiling method.With after optimized multi-PRC reaction system and condition to manual simulation's sample
Product are detected.
Two, test result
1, the testing result of standard items Quadruple- PCR
Pasteurella multocida, salmonella, Escherichia coli O 157 are added simultaneously in a reaction system: H7 and golden yellow
The DNA of color staphylococcus standard items sample amplifies apparent 4 swimming band, is followed successively by large intestine bar from top to bottom as shown in Figure 2 D
Bacterium O157:H7, pasteurella multocida, salmonella and staphylococcus aureus show that a reaction system can detect simultaneously
Sample containing 4 kinds of bacterial strains, and it does not interfere with each other testing result.
2, the specific detection result of Quadruple- PCR reaction
By aforementioned 16 kinds of bacterial strains and reference culture with present invention detection after, only pasteurella multocida, Salmonella
Bacterium, Escherichia coli O 157: H7 and staphylococcus aureus reaction result are positive, and testing result is shown in Table 2.
The identification of 2 Quadruple- PCR specific primer of table
3, the sensibility of Quadruple- PCR reaction
Pasteurella multocida, salmonella, Escherichia coli O 157: H7 and staphylococcus aureus reference culture sample warp
Multiplexed PCR amplification is carried out after crossing doubling dilution, four kind bacterial strain energy reproducible (see Fig. 3) for the testing result of various bacteriums
Detection simultaneously is to about 0.1ng/ μ L, wherein Escherichia coli O 157: H7, staphylococcus aureus minimum detectability be 10pg/ μ L.
4, the testing result of clinical sample is simulated
By testing result (see Fig. 4) and the complete phase of identified Clinical isolation after analog sample progress random number
Symbol illustrates that the method can be used for analysis of clinic pathogenic microorganism detection.
Embodiment 4:
The comparative experiments of multiple PCR method and Bacteria Detection National Standard Method detection clinical sample
Being detected with the Quadruple- PCR method of foundation to 90 parts of clinical samples of raiser's inspection (can be in 4 hour
It completes).Simultaneously with Bacteria Detection National Standard Method (respectively GB/T4789-2008 and GB4789-2010, GB4789.10-2010) into
Row contrasting detection.
Multiple PCR method after optimization is detected into clinical sample, and compares experiment with International Standards Method simultaneously,
In the 513 parts of samples selected at random, the positive quantity of two methods detection is respectively 94 parts and 87 parts and is shown in Table 3, and accuracy is
92.6%.Bacterium separation qualification result also demonstrates the correctness of multiplex PCR testing result.
3 comparative test result of table
Sequence table
<110>Hua Zhong Agriculture University
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Claims (2)
1. the cause of disease multiple PCR detection kit of a breeder common bacteria disease, including PCR reaction solution, positive control, feminine gender are right
According to, specific primer and DNA Marker, it is characterised in that: the specific primer is
rfbE-F 5′-AACGGTTGCTCTTCATTTAG-3′(SEQ ID NO.1)
rfbE-R 5′-GAGACCATCCAATAAGTGTG-3′(SEQ ID NO.2)
invA-F 5′-TCATCGCACCGTCAAAGGAACC-3′(SEQ ID NO.3)
invA-R 5′-GTGAAATTATCGCCACGTTCGGGCAA-3′(SEQ ID NO.4)
KMT1-F 5′-ATCCGCTATTTACCCAGTGG-3′(SEQ ID NO.5)
KMT1-R 5′-GCTGTAAACGAACTCGCCAC-3′(SEQ ID NO.6)
nuc-F 5′-AGATAACGGCGTAAATAGAAGTGG-3′(SEQ ID NO.7)
nuc-R 5′-TGCACTTGCTTCAGGACCATA-3′(SEQ ID NO.8)
The positive control are as follows: Escherichia coli O 157: H7, salmonella, pasteurella multocida, staphylococcus aureus
Standard items or Escherichia coli O 157: H7 rfbE gene masculine plasmid, salmonella invA gene masculine plasmid, killing property Pasteur more
Bacillus KMT1 gene masculine plasmid, staphylococcus aureus nuc gene masculine plasmid；
The negative control is sterilizing without enzyme distilled water.
2. the cause of disease multiple PCR detection kit of chicken common bacteria disease according to claim 1, which is characterized in that PCR is anti-
The system answered is as follows:
Reaction condition: 94 DEG C of 4min of initial denaturation；94 DEG C of 30s are denaturalized, anneal 56 DEG C of 30s, extends 72 DEG C of 45s, recycles 30 times altogether；
Extend 72 DEG C of 10min eventually.
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| WO2006090945A1 (en) * | 2005-02-28 | 2006-08-31 | Samsung Everland Inc. | Primer for detecting food poisoning and method for rapid detection of food born pathogene |
| CN103436626A (en) * | 2013-09-13 | 2013-12-11 | 湖北省农业科学院畜牧兽医研究所 | Quadruple fluorescent PCR (Polymerase Chain Reaction) detection kit for common chicken food-borne bacteria and application method thereof |
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| CN103436626A (en) * | 2013-09-13 | 2013-12-11 | 湖北省农业科学院畜牧兽医研究所 | Quadruple fluorescent PCR (Polymerase Chain Reaction) detection kit for common chicken food-borne bacteria and application method thereof |
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|---|
| 水产品中4种食源性致病菌多重PCR检测方法的建立;童桂香，等;《南方农业学报》;20151231;第46卷(第12期);第2217-2222页 * |
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