CN106701976A - Pathogen multiplex PCR (polymerase chain reaction) detection kit for chicken common bacterial diseases - Google Patents
Pathogen multiplex PCR (polymerase chain reaction) detection kit for chicken common bacterial diseases Download PDFInfo
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Abstract
The invention discloses a pathogen multiplex PCR (polymerase chain reaction) detection kit for chicken common bacterial diseases. The kit comprises a PCR reaction solution, specific primers for Pasteurella multocida, Salmonella, Escherichia coli O157:H7 and Staphylococcus aureus, positive controls (standard substances of Pasteurella multocida, Salmonella, Escherichia coli O157:H7 and Staphylococcus aureus, or positive plasmids of Pasteurella multocida KMT1 gene, Salmonella invA gene, Escherichia coli O157:H7 rfbE gene and Staphylococcus aureus nuc gene), a negative control (sterile enzyme-free double distilled water), and a DNA marker. The quadruplex PCR is utilized to quickly, accurately and synchronously detect the four pathogenic bacteria; the kit has the advantages of high sensitivity and high specificity; the lower detection limit reaches 0.01ng/mu L; and the accuracy 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 detection of breeder common bacteria disease
Kit, further relates to the application method of the kit.
Background technology
Escherichia coli O 157:During H7, salmonella, staphylococcus aureus and pasteurella multocida are chicken growth course
The cause of disease of common bacteriosis.The development of their not only serious harm China aviculture, and Escherichia coli O 157:H7、
Salmonella, staphylococcus aureus can also infect the mankind by food pollution, be threatened to human health.
Escherichia coli (E.Coli) O157:H7 is a main bacterial type of EHEC, and people and animals can be caused to be total to
Illness, its potential outbreak of epidemic trend, strong pathogenic, lethal, makes it have greatly aquaculture and human security
Harm.Salmonella (Salmonella) is zoonosis pathogen, easily produces drug resistance, serious to aquaculture harm,
First place is accounted in the pathogen for causing mankind's food origin disease.Pasteurella multocida (Pasteurella multocida) is
Cause humans and animals contact, the conditioned pathogen of strong bacterial infectious disease (also known as cholera fowl, hueppe's disease), its is occurred frequently
Sick rate and the death rate, it is serious on aquaculture influence, one of poultry disease of the anti-system of emphasis is listed in, there is case gradually to increase in recent years
Many trend.Staphylococcus aureus (Staphylococcus aureus) is most common animal suppurative infection, cross-infection
Pathogen, the another name for having " thermophilic meat bacterium " is one of disease of harm most serious in poultry production at present.China's law is to upper simultaneously
Stating quantity of the bacterium (except Pasteurella) in food has strict restriction.
Currently for the detection of plant's bacterial disease, bacteriology culture is depended on, typically take 4-7d, detection method
It is comparatively laborious, time-consuming, to detect that separation plant's various pathogens and mixed infection situation are cumbersome simultaneously.Western blotting skill
Art, enzyme linked immunosorbent assay, nucleic acid hybridization technique, round pcr etc. are because its high specificity, sensitiveness are high, simple to operate quick
It is widely used, but these methods can only generally 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 adding multipair
Primer expands a plurality of target DNA fragment, is capable of achieving multiple pathogenic microorganisms and detects simultaneously.The advantage of multiple PCR technique is can
To carry out quick, comprehensive, system, accurately 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, with aobvious
The economic benefit and social benefit of work.
It is contemplated that setting up a kind of Quadruple- PCR method for quick, realize to colon bacillus 0157：H7, salmonella,
The simultaneously and rapidly detection of four kinds of infectious diseases common to human beings and animals that pasteurella multocida, staphylococcus aureus cause, pop disease
Investigating and effectively carrying out livestock and poultry pestilence monitoring prevention and control, raising public health security has definite meaning.
The content of the invention
The purpose of the present invention is the cause of disease multiple PCR detection kit that there are provided breeder common bacteria disease, the examination
Agent box is made up of multi-PRC reaction liquid, positive control and negative controls.Detected that pathogen passed through mostly in poultry in the past
The separation identification of bacterium is detected with the PCR for single bacterium, and this detection kit is thin for most common 4 kinds of chicken bacterial disease
Bacterium, designs and filters out non-interfering 4 pairs of detection primers, and 4 kinds of pathogens of detection complete in being reacted at one, highly efficient,
And sensitiveness 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 that there are provided breeder common bacteria disease
Application method, method is simple, and quickly, all detecting steps can be completed in 4 hours.
To achieve these goals, the present invention takes following technical measures：
The cause of disease multiple PCR detection kit of one breeder common bacteria disease, including (the Taq DNA polymerizations of multi-PRC reaction liquid
Enzyme, without Mg2+PCR reaction buffers, Mg2+, dNTPs), pasteurella multocida, salmonella, Escherichia coli O 157:H7
With staphylococcus aureus this 4 kinds of specific primers of bacterium (as shown in table 1), positive control (pasteurella multocida, sramana
Salmonella, Escherichia coli O 157:H7 and staphylococcus aureus standard items or pasteurella multocida KMT1 genes, salmonella
InvA genes, Escherichia coli O 157:The positive plasmid of H7rfbE genes and staphylococcus aureus nuc genes), negative control
(sterilizing without enzyme distilled water), DNA Marker.
The multiplex PCR specific primer sequence table of table 1
The application method of the cause of disease multiple PCR detection kit of one breeder common bacteria disease, its step is as follows：
1st, the treatment of sample and the extraction of DNA profiling：
Infection early stage enteron aisle is the main parasitic position of 4 kinds of pathogenic bacteria of the above, and live-bird sampling is main by gathering anus cotton
Swab, dead fowl can use intestinal contents, and meat products can directly take fritter tissues.The genomic DNA of sample is extracted, 2 μ L are taken
(20ng) is used as testing sample.Detection every time all should set up positive control and negative control.
2nd, reaction system and reaction condition：
Reaction system is following (the μ L of cumulative volume 25)：
Reaction condition：94 DEG C of 4min of predegeneration；94 DEG C of 30s of denaturation, annealed 56 DEG C of 30s, extends 72 DEG C of 45S, and 30 are circulated altogether
It is secondary；Extend 72 DEG C of 10min eventually,.
PCR primer is identified：Take 8 μ L pcr amplification products plus 2 μ 6 × Loading of L Buffer are mixed, with DL2000DNA
Marker is indicated as relative molecular mass, with 1.8% Ago-Gel (the μ L/mL containing the EB 0.03) electrophoresis under 100V voltages
30min, to put and carry out interpretation of result in gel imaging system.
3rd, the judgement of result：
(1) quality control standard
Negative control：The corresponding swimming of loading wells is taken without band；
Positive control：The corresponding swimming of loading wells has taken 4 obvious positive bands：Escherichia coli O 157:H7 amplifications
Positive fragment 678bp (between 500-700bp), salmonella 284bp (slightly above 250bp), pasteurella multocida 460bp
(close to 500bp), staphylococcus aureus 229bp (are less than 250bp)；
This detection is invalid if negative control or positive control have one not to be inconsistent standardization.
(2) result of determination
Without band on the corresponding swimming lane of detection sample well, result judgement is feminine gender；
Occur≤4 swimming bands on the corresponding swimming lane of detection sample well, band corresponding with standard positive hole is compared, such as
Point-blank, corresponding Bacteria Detection result is judged as the positive to really bar band；If not point-blank or detection hole
Occur >=5 swimming bands, possible process of the test pollution, it is proposed that reform on swimming lane.
The present invention compared with prior art, with advantages below and beneficial effect：
1st, at present, the various cross infection of disease of aquaculture clinical case, mixed infection situation it is of common occurrence, or even have more
Carry out more complicated situation, but detection fowl bacterial clinical case and carry out epidemiology survey and mainly take bacteria distribution to identify
Method, needs multiple detecting steps, complex operation, time and effort consuming, and can only once detect a kind of pathogen.It is many that this research is set up
Weight PCR detection method overcomes the deficiency of the above, after Bacteria Culture, completes to detect Escherichia coli O 157 simultaneously：H7, Salmonella
Bacterium, pasteurella multocida, four kinds of tasks of pathogen of staphylococcus aureus, only need about 4h, have preferably specificity and
Sensitiveness (Monitoring lower-cut is up to 10pg/uL), it is easier than traditional bacteriological method economical, there is larger application value.
2nd, there is multipair primer and multiple template in multi-PRC reaction system, any both of which is in the presence of interacting
Situation, cause the appearance of false positive, thus primer the key factor for being designed to multi-PCR detection method foundation, this hair
The detection primer of bright offer ensures between different primers pair, does not have affinity, four of amplification between primer and non-target sequences
Genes of interest fragment length also has certain gap, and makes Ago-Gel in certain concentration range, it is ensured that purpose during electrophoresis
Band can be distinguished substantially.
3rd, the kit that the present invention is provided can be used for the detection of the live-bird anus swab such as chicken, be conducive to the popularization of clinic.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
Four kinds of detections of bacterium provide effective instrument and method with prevention and control, and accuracy rate is up to 92.6%.
Brief description of the drawings
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
Really；C is the specific detection result of pasteurella multocida primer；D is the specific detection knot of staphylococcus aureus primer
Really.
Fig. 2 is four kinds of bacterial primers mixing specific detection results in multiple PCR detection kit of the present invention
Note：A is the specific amplification result for detecting a kind of bacterium；B is two kinds of specific amplification results of bacterium of detection；C
It is three kinds of specific amplification results of bacterium of detection；D is four kinds of testing results of bacterium solution mixed liquor of detection.
Fig. 3 is the result figure of multiple PCR detection kit sensitivity test of the present invention
Note：M：DL2000Marker；1：10ng/μL；2：1ng/μL；3：0.1ng/μL；4：0.01ng/μL；5：
0.001ng/μL；6：0.1pg/μL.
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：Killing property more
Pasteurella；7、11、12：Staphylococcus aureus；13-16：Four plants of positive controls of bacterium；17-20：Negative control.
Specific embodiment
The present invention is further described with specific embodiment below in conjunction with the accompanying drawings, these embodiments are merely to illustrate this
Invention rather than limitation the scope of the present 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, the kit includes：Multi-PRC reaction liquid, sun
Property control, negative control, DNAMarker2000 (100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp；TaKaRa
Ltd), four pairs of specific primers of bacterium (10uM)：RfbE-F, rfbE-R, invA-F, invA-R, KMT1-F, KMT1-R,
Nuc-F, nuc-R；
Described multi-PRC reaction liquid is：Taq archaeal dna polymerases (2-5U/ μ L), without Mg2+10 × PCR reaction buffering
Liquid, 25mM Mg2+With 10mM dNTPs；
Described positive control is：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 masculines plasmid, salmonella invA gene masculines plasmid,
Pasteurella multocida KMT1 gene masculines plasmid, the positive plasmid of staphylococcus aureus nuc genes；
Described negative control is sterilizing without enzyme distilled water；
Described specific primer is：
rfbE-F 5′-AACGGTTGCTCTTCATTTAG-3′
rfbE-R 5′-GAGACCATCCAATAAGTGTG-3′
invA-F 5′-TCATCGCACCGTCAAAGGAACC-3′
invA-R 5′-GTGAAATTATCGCCACGTTCGGGCAA-3′
KMT1-F 5′-ATCCGCTATTTACCCAGTGG-3′
KMT1-R 5′-GCTGTAAACGAACTCGCCAC-3′
nuc-F 5′-AGATAACGGCGTAAATAGAAGTGG-3′
nuc-R 5′-TGCACTTGCTTCAGGACCATA-3′。
The application method of the cause of disease multiple PCR detection kit of one breeder common bacteria disease, its step is as follows：
1st, the treatment of sample and the extraction of DNA profiling：
Infection early stage enteron aisle is the main parasitic position of 4 kinds of pathogenic bacteria of the above, and live-bird sampling is main by gathering anus cotton
Swab, dead fowl can use intestinal contents, and meat products can directly take fritter tissues.Samples taken uses QIAGEN companies of Germany
QIAxtractor high-throughput nucleic acids Purification Station extracts DNA profiling, it is possible to use other DNA extraction kits are extracted, specifically
Step takes 2 μ L (20ng) as testing sample with reference to specification.Detection every time all should set up positive control and negative control.
2nd, reaction system and reaction condition：
Reaction system is following (the μ L of cumulative volume 25), and the concentration of each composition is with embodiment 1：
Reaction condition：94 DEG C of 4min of predegeneration, are denatured 94 DEG C of 30s, and annealed 56 DEG C of 30s, extends 72 DEG C of 45S, and 30 are circulated altogether
It is secondary, 72 DEG C of 10min are extended eventually.
PCR primer is identified：Take 8 μ L pcr amplification products plus 2 μ 6 × Loading of L Buffer are mixed, with DL2000DNA
Marker is indicated as relative molecular mass, with 1.8% Ago-Gel (the μ L/mL containing the EB 0.03) electrophoresis under 100V voltages
30min, to put and carry out interpretation of result in gel imaging system.
3rd, the judgement of result：
(1) quality control standard:
Negative control：The corresponding swimming of loading wells is taken without band；
Positive control：The corresponding swimming of loading wells has taken 4 obvious positive bands：Escherichia coli O 157:H7 amplifications
Positive fragment 678bp (between 500-700bp), salmonella 284bp (slightly above 250bp), pasteurella multocida 460bp
(close to 500bp), staphylococcus aureus 229bp (are less than 250bp)；
This detection is invalid if negative control or positive control have one not to be inconsistent standardization.
(2) result of determination：
Without band on the corresponding swimming lane of detection sample well, result judgement is feminine gender；
Occur≤4 swimming bands on the corresponding swimming lane of detection sample well, band corresponding with standard positive hole is compared, such as
Point-blank, corresponding Bacteria Detection result is judged as the positive to really bar band；If not point-blank or detection hole
Occur >=5 swimming bands, possible process of the test pollution, it is proposed that reform on swimming lane.
Embodiment 2：
The design and screening of the cause of disease multiple PCR detection kit specific primer of chicken common bacteria disease
First, materials and methods
1st, bacterium bacterial strain
Reference culture：Pasteurella multocida (Pasteurella multocida CVCC44801), salmonella
(Salmonella enteritidis CVCC 3375), Escherichia coli O 157:H7(Escherichia coli
) and staphylococcus aureus (Staphylococcus aureus SY1G7) reference culture is purchased from Chinese micro- life CICC21530
Thing DSMZ.16 plants of laboratory clinicals separate bacterium and the numbering of identification：Fowl derived bacterium salmonella HZBL08121,
It is salmonella CMCC50051, salmonella HZBL08122, salmonella HZBL08123, pasteurella multocida HBNKM, many
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.
2nd, the extraction of DNA of bacteria template is with embodiment 1.
3rd, the design of primer and synthesis
According to announced gene order in GenBank, using Primer 5.0, Oligo 6.0 and BLAS T softwares point
4 pairs of specific primers of analysis design (are synthesized) by Shanghai life work, and primer sequence and genes of interest fragment are shown in Table 2.
The multiple PCR primer sequence of table 2
4th, the specificity of 4 kinds of bacteriums of detection compares when single primer PCR reacts
To determine the optimum reaction condition of the specific and single PCR of primer, by every pair of primers respectively to above-mentioned 20
Pathogen strain bacterium enters performing PCR specific amplification.Initial reaction system is 25 μ L：10 × EasyTaq Buffer2.5 μ L, dNTPs
2.0 μ L, upstream and downstream primer each μ L of 0.5 μ L, Taq DNA Polymerase 0.25, a kind of μ l of template 2.0 of bacterium, distilled water
Mend to 25 μ L.Initial reaction program is 94 DEG C of 4min of predegeneration；94 DEG C of 30s of denaturation, anneal 56 DEG C of 30s, extends 72 DEG C of 45s, altogether
Circulation 30 times；Extend 72 DEG C of 10min eventually, in 4 DEG C of preservations.PCR results are identified with embodiment 1.
5th, the results contrast of a kind of bacterium and various bacteria is detected after the mixing of four species-specific primers
In order to reduce the influence after primer mixing to genes of interest amplification, 4 experiments below ad hoc meter are drawn evaluating design
The specificity of thing.
Distinguish a kind of bacterium of PCR reaction detections after (1) four kind of primer mixing
Reaction condition adds any one bacterial template with above-mentioned 4.
Distinguish two kinds of bacteriums of PCR reaction detections after (2) four kinds of primer mixing
Reaction condition adds the bacterial template of any two kinds of combinations with above-mentioned 4.
Distinguish three kinds of bacteriums of PCR reaction detections after (3) four kinds of primer mixing
Reaction condition adds three kinds of bacterial templates of any combination with above-mentioned 4.
Distinguish four kinds of bacteriums of PCR reaction detections after (4) four kinds of primer mixing
Reaction condition adds four kinds of bacterial templates with above-mentioned 4.
2nd, result of the test
1st, single primer PCR result
Each pair primer is distinguished into 20 bacterial strains of specific amplification, a kind of result of primer amplification of addition is single, and band is clear,
In the same size with expected fragment, specific testing result is shown in Fig. 1, illustrates that primer specificity is good.
2nd, four kinds of specific detection results of primer mixing PCR reactions
After four kinds of primer mixing, a kind of bacterium, or various bacteria are either expanded, the amplification to genes of interest all has no
Influence.After the purpose fragment electrophoresis of amplification, band is clear；Multiple purpose fragments are also easy to distinguish (see Fig. 2).Illustrate to design primer
Quality is higher, can be completely used for the detection of clinical sample.
Embodiment 3：
The specificity and sensitiveness of the common four kinds of bacterial diseases cause of disease multi-PCR detection method of chicken：
First, materials and methods
1st, reference culture is with embodiment 2.
2nd, according to the application method of multiple PCR detection kit described in embodiment 1, respectively extract pasteurella multocida,
Salmonella, Escherichia coli O 157:The DNA of H7 and staphylococcus aureus standard items sample simultaneously carries out multi-PRC reaction.
3rd, the specific detection of Quadruple- PCR reaction
16 plants of fowl derived bacterium (with embodiment 2) the extraction DNA that laboratory clinical is separate is chosen, multiplex PCR detection is carried out.
4th, the sensitivity Detection of Quadruple- PCR reaction
Determine four kinds of bacterial strains (pasteurella multocida, salmonella, Escherichia coli O 157s: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 be 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, each bacterial strain each
Gradient respectively takes 200 μ L and is well mixed, and makes in reaction system simultaneously containing four kinds of DNA, sterilizing without enzyme distilled water as negative control,
Expanded according to the multi-PRC reaction condition described in embodiment 1.Its sensitiveness and LDL are detected, each dilution ladder
Degree does 3 detections and repeats.
5th, analog sample detection：The Escherichia coli O 157 for being clinically separated and preserving is taken respectively:H7, salmonella, killing property more
Pasteurella and each 3 plants of staphylococcus aureus, numbering is 1-12, is positive control with the reference culture that this experiment is used, is gone out
Bacterium is negative control without enzyme distilled water, the multiple PCR method detection Clinical isolation after optimizing application, identifies that this is multiple
The feasibility of PCR method；Then by this 12 plants of bacteriums again random number, and with original numbering corresponding record, it is made manual simulation
Sample.
The preparation of manual simulation's sample：Cut open inspection will be carried out under healthy chick's aseptic condition, taken liver is divided into 12
Part, the 12 pathogen strain bacterium that will be renumberd are inoculated in taken liver respectively, the PBS200 μ L of each addition 10mM/L, and tissue is homogenized
After be separately added into 2mL TSB fluid nutrient mediums, be placed in 300r/min in constant-temperature table, 37 DEG C culture 5h.Above culture is pressed
DNA of bacteria is extracted as template according to boiling method.With the multi-PRC reaction system and condition after optimized to manual simulation's sample
Product are detected.
2nd, result of the test
1st, the testing result of standard items Quadruple- PCR
Add pasteurella multocida, salmonella, Escherichia coli O 157 simultaneously in a reaction system:H7 and golden yellow
The DNA of color staphylococcus standard items sample, as shown in Figure 2 D, amplifies obvious 4 swimming band, and large intestine bar is followed successively by from top to bottom
Bacterium O157:H7, pasteurella multocida, salmonella and staphylococcus aureus, one reaction system of display can be detected simultaneously
Containing 4 kinds of samples of bacterial strain, and testing result is not interfere with each other it.
2nd, the specific detection result of Quadruple- PCR reaction
By foregoing 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 the Quadruple- PCR specific primer of table 2
3rd, the sensitiveness of Quadruple- PCR reaction
Pasteurella multocida, salmonella, Escherichia coli O 157:H7 and staphylococcus aureus reference culture sample are passed through
Multiplexed PCR amplification is carried out after crossing doubling dilution, the testing result for various bacteriums is reproducible (see Fig. 3), four kinds of bacterial strain energy
Detect simultaneously to about 0.1ng/ μ L, wherein Escherichia coli O 157：H7, the minimum detectability of staphylococcus aureus are 10pg/ μ L.
4th, the testing result of clinical sample is simulated
Analog sample is carried out into testing result (see Fig. 4) and the complete phase of Clinical isolation identified after random number
Symbol, illustrates that the method can be used for analysis of clinic pathogenic microorganism detection.
Embodiment 4：
Multiple PCR method detects the contrast experiment of clinical sample with Bacteria Detection National Standard Method
90 parts of clinical samples of raiser's censorship are detected with the Quadruple- PCR method set up (can be in 4 hours
Complete).Enter with Bacteria Detection National Standard Method (respectively GB/T4789-2008 and GB4789-2010, GB4789.10-2010) simultaneously
Row contrasting detection.
Multiple PCR method after optimization is detected into clinical sample, and with International Standards Method carries out contrast experiment simultaneously,
In the random 513 parts of samples selected, 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%.Bacteria distribution qualification result also demonstrate that the correctness of multiplex PCR testing result.
The comparative test result of table 3
Claims (2)
1. the cause of disease multiple PCR detection kit of breeder common bacteria disease, including PCR reaction solutions, positive control, feminine gender are right
According to, specific primer and DNA Marker, it is characterised in that：Described specific primer is
rfbE-F 5′-AACGGTTGCTCTTCATTTAG-3′
rfbE-R 5′-GAGACCATCCAATAAGTGTG-3′
invA-F 5′-TCATCGCACCGTCAAAGGAACC-3′
invA-R 5′-GTGAAATTATCGCCACGTTCGGGCAA-3′
KMT1-F 5′-ATCCGCTATTTACCCAGTGG-3′
KMT1-R 5′-GCTGTAAACGAACTCGCCAC-3′
nuc-F 5′-AGATAACGGCGTAAATAGAAGTGG-3′
nuc-R 5′-TGCACTTGCTTCAGGACCATA-3′
Described positive control is：Escherichia coli O 157:H7, salmonella, pasteurella multocida, staphylococcus aureus
Standard items or Escherichia coli O 157:H7rfbE gene masculines plasmid, salmonella invA gene masculines plasmid, killing property Pasteur bar more
Bacterium KMT1 gene masculines plasmid, staphylococcus aureus nuc gene masculine plasmids；Described negative control is the double without enzyme of sterilizing
Steam water.
2. the cause of disease multiple PCR detection kit of chicken common bacteria according to claim 1 disease, it is characterised in that PCR is anti-
The system answered is as follows：
Reaction condition：94 DEG C of 4min of predegeneration；94 DEG C of 30s of denaturation, anneal 56 DEG C of 30s, extends 72 DEG C of 45s, circulates 30 times altogether；Eventually
Extend 72 DEG C of 10min.
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Cited By (2)
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| CN108251548A (en) * | 2018-04-10 | 2018-07-06 | 中国农业科学院上海兽医研究所 | Multiple PCR detection primer group, method and the kit of fowl enteropathogenic E. Coli etc. |
| CN111534619A (en) * | 2020-05-11 | 2020-08-14 | 贵州省畜牧兽医研究所 | Multiple PCR detection method and detection kit for chicken-derived pathogenic bacteria |
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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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| 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 |
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| CN108251548A (en) * | 2018-04-10 | 2018-07-06 | 中国农业科学院上海兽医研究所 | Multiple PCR detection primer group, method and the kit of fowl enteropathogenic E. Coli etc. |
| CN108251548B (en) * | 2018-04-10 | 2021-07-16 | 中国农业科学院上海兽医研究所 | Multiplex PCR detection primer group, method and kit for avian pathogenic escherichia coli and the like |
| CN111534619A (en) * | 2020-05-11 | 2020-08-14 | 贵州省畜牧兽医研究所 | Multiple PCR detection method and detection kit for chicken-derived pathogenic bacteria |
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