CN107201400A - The five weight PCR detection methods and detection kit of avian escherichia coli, Salmonella gallinarum, Salmonella Pullorm etc. - Google Patents
The five weight PCR detection methods and detection kit of avian escherichia coli, Salmonella gallinarum, Salmonella Pullorm etc. Download PDFInfo
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Abstract
The invention provides five weight PCR detection kits of a kind of avian escherichia coli, Salmonella gallinarum, Salmonella Pullorm, Salmonella enteritidis, the multiple PCR primer group of Salmonella typhimurtum including the primer sets and five heavy PCR detection methods.Primer sets include：Primer pair ECphoA F and the ECphoA R of specific amplification avian escherichia coli phoA genes；Primer pair SGglgC F and the SGglgC R of specific amplification Salmonella gallinarum glgC genes；Primer pair SGPspeC F and the SGPspeC R of specific amplification Salmonella gallinarum and Salmonella Pullorm speC genes；The F of primer pair SEsdf I and SEsdf I R of the genes of specific amplification Salmonella enteritidis sdf I；Primer pair STstm4495 F and the STstm4495 R of specific amplification Salmonella typhimurtum stm4495 genes.
Description
Technical field
The invention belongs to biological technical field, and in particular to one kind is used to detect avian escherichia coli, Salmonella gallinarum, chicken
Dysentery characterized by white mucous stool detection of Salmonella, Salmonella enteritidis, the multiple PCR primer group of Salmonella typhimurtum include five weight PCR detection examinations of the primer sets
Agent box and the five heavy PCR detection methods using the primer sets.
Background technology
With the fast development of scale aviculture, the generation of epidemic disease and popular also corresponding increase cause aviculture economy
Decline in benefits.The important bacterial infectious disease such as significant bacterial such as avian colibacillosis, white diarrhea, fowl typhoid disease, which is seriously endangered, supports
Fowl industry.
At present, the country relies primarily on antibiotic and prevents and treats bacterial disease, but due to it is long-term, unreasonably caused using antibiotic：
On the one hand extensive drug resistance is produced, another aspect medicament residue directly affects the safety of animal product.Therefore, strengthen to this
The long term monitoring of a little bacteriosises and research, it is all significant to aviculture and public health.
Diagnosis and monitoring technology species both at home and abroad to bacteriosis is various, mainly includes：Bacteria distribution culture and life
Change identification, Protocols in Molecular Biology, immunological technique etc..Round pcr is because of its high specificity, sensitiveness is high, simple to operate, detect
It is widely used the advantages of quick.
At present, a variety of detection methods are established for avian escherichia coli and different serotypes detection of Salmonella, however, not being directed to
Avian escherichia coli, Salmonella gallinarum, Salmonella Pullorm, Salmonella enteritidis, the quick detection of Salmonella typhimurtum and discriminating
Diagnose PCR method.
The content of the invention
In order to solve the above problems, present invention employs following technical scheme：
It is an object of the present invention to provide it is a kind of can accurately, it is quick, stably, specificity and the high detection fowl of sensitivity
Escherichia coli, Salmonella gallinarum, Salmonella Pullorm, Salmonella enteritidis, the primer sets of Salmonella typhimurtum, its feature exist
In, including：The primer pair ECphoA-F and ECphoA-R of specific amplification avian escherichia coli phoA genes；Specific amplification chicken hinders
The primer pair SGglgC-F and SGglgC-R of cold detection of Salmonella glgC genes；Specific amplification Salmonella gallinarum and white diarrhea sramana
The primer pair SGPspeC-F and SGPspeC-R of bacterium speC genes；The primer pair of the genes of specific amplification Salmonella enteritidis sdf I
- the F of the SEsdf I and-R of SEsdf I；The primer pair STstm4495-F of specific amplification Salmonella typhimurtum stm4495 genes and
STstm4495-R,
The nucleotide sequence of each primer is respectively：
ECphoA-F, 5 '-GGCAATACACTCACTATGCGCTG-3 ',
ECphoA-R, 5 '-AGGATTCGCAGCATGATCCTG-3 '；
SGglgC-F, 5 '-CGTCGCTATAAAGCGGAATATGTC-3 ',
SGglgC-R, 5 '-CCTTTTCAAAACATACGCGAGTAG-3 '；
SGPspeC-F, 5 '-GTTGCCGTACCGGTCGTAACG-3 ',
SGPspeC-R, 5 '-TTCCTGACGGGCATCGACGGC-3 '；
SEsdf I-F, 5 '-GATGTGGTTGGTTCGTCACTG-3 ',
SEsdf I-R, 5 '-GCGAGACCTCAAACTTACTCAG-3 '；
STstm4495-F, 5 '-CAGCGGTATGATGCGGTAGT-3 ',
STstm4495-R, 5 '-TCACCGGTGGACATGCCTGC-3 '.
The primer sets that the present invention is provided, also have the feature that：Primer pair ECphoA-F and ECphoA-R are used to expand
Purpose fragment size be 761bp；Primer pair SGglgC-F and SGglgC-R are for the size of the purpose fragment of amplification
83bp；The size that primer pair SGPspeC-F and SGPspeC-R are used for the purpose fragment expanded is 249bp；- the F of primer pair SEsdf I
It is 409bp with the-R of SEsdf I sizes for being used for the purpose fragment of amplification；Primer pair STstm4495-F and STstm4495-R are used for
The purpose fragment size of amplification is 569bp.
It is a further object to provide a kind of five heavy PCR detection kits, for detecting avian escherichia coli, chicken wound
Cold detection of Salmonella, Salmonella Pullorm, Salmonella enteritidis and Salmonella typhimurtum, it is characterised in that including：It is anti-for setting up PCR
2 × PCR the premixed liquids and primer sets of system are answered, wherein, each primer pair that PCR reaction systems are used in primer sets carries out special
Property amplification, the above-mentioned primer sets of primer sets.
The five heavy PCR detection kits that the present invention is provided, also have the feature that：Wherein, 2 × PCR PreMix body
Product is 12.5 μ L, in primer sets, and primer pair ECphoA-F and ECphoA-R volume are respectively 0.5 μ L, primer pair SGglgC-F
Volume with SGglgC-R is respectively 0.2 μ L, and primer pair SGPspeC-F and SGPspeC-R volume are respectively 0.3 μ L, primer pair
- the F of SEsdf I and the-R of SEsdf I volume are respectively that 0.4 μ L, primer pair STstm4495-F and STstm4495-R volume are respectively 0.5
μL。
It is also another object of the present invention to provide one kind detection avian escherichia coli, fowl typhoid door bacterium, Salmonella Pullorm, intestines
Scorching detection of Salmonella and the five heavy PCR detection methods of Salmonella typhimurtum, it is characterised in that comprise the following steps：Step 1, based on treating
Detection bacterium prepares pcr template；Step 2, specific primer group is prepared, is drawn using any one in claim 1 or 2
Thing group prepares specific primer group；Step 3, pcr amplification reaction, the pcr template obtained based on step 1, using step are carried out
Rapid 2 obtained specific primers set up the PCR reaction systems of the vertical primer sets included, then using the PCR reaction systems
Pcr amplification reaction is carried out under the conditions of predetermined PCR response parameters and obtains pcr amplification product；Step 4, electrophoresis detection is carried out, to expanding
Volume increase thing carries out electrophoresis detection and obtains electrophoresis result, determines whether avian escherichia coli, fowl typhoid sramana occur according to electrophoresis result
Bacterium, Salmonella Pullorm, Salmonella enteritidis or Salmonella typhimurtum.
The five heavy PCR detection methods that the present invention is provided, also have the feature that：The pcr template obtained in step 1 is to treat
The bacterium solution of detection bacterium, detailed process is：It it is 37 DEG C in temperature by the inoculation of bacterium to be detected to LB fluid nutrient mediums
Under the conditions of cultivate to OD600=1.0, obtained bacterium solution as pcr template.
The five heavy PCR detection methods that the present invention is provided, also have the feature that：The pcr template obtained in step 1 is thin
Bacterium slightly carries DNA, and detailed process is：By the inoculation of bacterium to be detected to LB fluid nutrient mediums, in the condition that temperature is 37 DEG C
Lower culture obtains bacterium solution, then the bacterium solution centrifuges 1-2min under 10000-12000r/min by 1mL, goes to OD600=1.0
Supernatant, plus 0.1-0.5mL sterilizing ultra-pure waters are resuspended, and boil 5-10min, then centrifuge 3- under 10000-12000r/min
5min, obtained supernatant is that bacterium slightly carries DNA.
The five heavy PCR detection methods that the present invention is provided, also have the feature that：The PCR reaction systems of step 3 include：
12.5 μ L 2 × PCR PreMix, 1.0 μ L template, 7.7 μ L ultra-pure water, and primer sets, in primer sets, primer pair
ECphoA-F and ECphoA-R volume is respectively 0.5 μ L, and primer pair SGglgC-F and SGglgC-R volume are respectively 0.2 μ L, are drawn
Thing to SGPspeC-F and SGPspeC-R volume respectively for 0.3 μ the L ,-F of primer pair SEsdf I and the-R of SEsdf I volume everybody 0.4
μ L, primer pair STstm4495-F and STstm4495-R volume are respectively 0.5 μ L.
The five heavy PCR detection methods that the present invention is provided, also have the feature that：When preparing specific primer group, primer
The concentration of each primer used in group is 10pM, is used again after the dilution of each primer used is placed in into -20 DEG C of preservations.
The five heavy PCR detection methods that the present invention is provided, also have the feature that：Predetermined PCR response parameters condition is 95
DEG C pre-degeneration 5min；30s at 95 DEG C, 30s at 57 DEG C, 50s at 72 DEG C, carry out 35 circulations；72 DEG C, 10min.
The five heavy PCR detection methods that the present invention is provided, also have the feature that：The detailed process of step 4 is to take 10 μ L
Pcr amplification product, point sample is in 2.0% agarose gel electrophoresis plate hole, under 80-100V voltages, electrophoresis 30-40min, Yu Ning
Taken pictures under glue imaging system and obtain electrophoresis result, the precondition judged electrophoresis result is negative control without any band
Occur, be determined as avian escherichia coli when 761bp bands occurs in electrophoresis result, sentence when 83bp and 249bp bands occurs in electrophoresis result
It is set to Salmonella gallinarum, Salmonella Pullorm is determined as when 249bp bands occurs in electrophoresis result, when electrophoresis result occurs
409bp bands are determined as Salmonella enteritidis, and Salmonella typhimurtum is determined as when 569bp bands occurs in electrophoresis result.
Prepared present invention also offers a kind of according to above-mentioned primer sets for detecting avian escherichia coli, fowl typhoid sramana
Application in bacterium, Salmonella Pullorm, Salmonella enteritidis, the five weight PCR detection kits of Salmonella typhimurtum.
Invention effect and effect
Provided by the present invention for detection avian escherichia coli, Salmonella gallinarum, Salmonella Pullorm, Salmonella enteritidis,
Five weight PCR detection kits of the multiple PCR primer group of Salmonella typhimurtum including the primer sets and utilize the primer sets
Five heavy PCR detection methods, due to the high specificity of primer sets therein, sensitivity is high, meanwhile, the primer sets have good weight
Renaturation, thus the present invention provide include using above-mentioned primer sets five weight PCR detection methods or containing above-mentioned primer sets five
Weight PCR detection kit is to avian escherichia coli, Salmonella gallinarum, Salmonella Pullorm, Salmonella enteritidis, Salmonella typhimurtum
Detection have the advantages that it is sensitive, special, quick and stable, be quick detection with identification avian escherichia coli, Salmonella gallinarum,
Salmonella Pullorm, Salmonella enteritidis, Salmonella typhimurtum provide effective means, while the primer sets can also prepared
For detect avian escherichia coli, Salmonella gallinarum, Salmonella Pullorm, Salmonella enteritidis, Salmonella typhimurtum five heavy PCR
Application in detection kit.
Brief description of the drawings
Fig. 1 is the electrophoresis result of the pcr amplification product of embodiment 1；
Fig. 2 is the result of the test for evaluating Evaluation on specificity in example 1；
Fig. 3 is to evaluate in example 2 using bacterial solution as the result of the test of the sensitivity assessment of template；
Fig. 4 is the result of the test for evaluating example 2 using bacterial genomes DNA as the sensitivity assessment of template.
Embodiment
Illustrate the embodiment of the present invention below in conjunction with accompanying drawing.For specific method used in embodiment or
Material, those skilled in the art can carry out conventional replacement according to existing technology on the basis of the technology of the present invention thinking
Selection, is not limited solely to the specific record of the embodiment of the present invention.
Experimental method used in implementation is conventional method unless otherwise specified；Used material, reagent etc.,
Unless otherwise specified, commercially obtain.
The heavy PCR detection method of embodiment 1 five
The present embodiment, which is used, is used for multiplex PCR detection avian escherichia coli, Salmonella gallinarum, Salmonella Pullorm, enteritis
Detection of Salmonella, the primer sets of Salmonella typhimurtum are to avian escherichia coli, Salmonella gallinarum, Salmonella Pullorm, Salmonella enteritidis
Detected with Salmonella typhimurtum, specifically include following steps：
Step 1, pcr template is prepared based on bacterium to be detected
Bacterium bacterial strain to be checked is seeded to LB fluid nutrient mediums, cultivated under conditions of temperature is 37 DEG C to OD600=1.0,
Obtain that the bacterium solution of pcr template can be used as.
Step 2, specific primer group is prepared
Prepare for multiplex PCR detection avian escherichia coli, Salmonella gallinarum, Salmonella Pullorm, Salmonella enteritidis, mouse
The primer sets of Salmonella typhi：Using the concentration of each primer sets therein as 10pM, the dilution of the primer of the concentration is placed in-
20 DEG C save backup, it is to avoid multigelation,
Above-mentioned primer sets include following primer pair：
The primer pair ECphoA-F and ECphoA-R of specific amplification avian escherichia coli phoA genes；Specific amplification chicken hinders
The primer pair SGglgC-F and SGglgC-R of cold detection of Salmonella glgC genes；Specific amplification Salmonella gallinarum and white diarrhea sramana
The primer pair SGPspeC-F and SGPspeC-R of bacterium speC genes；The primer pair of the genes of specific amplification Salmonella enteritidis sdf I
- the F of the SEsdf I and-R of SEsdf I；The primer pair STstm4495-F of specific amplification Salmonella typhimurtum stm4495 genes and
STstm4495-R,
The nucleotide sequence of each primer is respectively：
ECphoA-F, 5 '-GGCAATACACTCACTATGCGCTG-3 ',
ECphoA-R, 5 '-AGGATTCGCAGCATGATCCTG-3 '；
SGglgC-F, 5 '-CGTCGCTATAAAGCGGAATATGTC-3 ',
SGglgC-R, 5 '-CCTTTTCAAAACATACGCGAGTAG-3 '；
SGPspeC-F, 5 '-GTTGCCGTACCGGTCGTAACG-3 ',
SGPspeC-R, 5 '-TTCCTGACGGGCATCGACGGC-3 '；
SEsdf I-F, 5 '-GATGTGGTTGGTTCGTCACTG-3 ',
SEsdf I-R, 5 '-GCGAGACCTCAAACTTACTCAG-3 '；
STstm4495-F, 5 '-CAGCGGTATGATGCGGTAGT-3 ',
STstm4495-R, 5 '-TCACCGGTGGACATGCCTGC-3 '.
Wherein, the size that primer pair ECphoA-F and ECphoA-R is used for the purpose fragment expanded is 761bp；Primer pair
The size that SGglgC-F and SGglgC-R is used for the purpose fragment expanded is 83bp；Primer pair SGPspeC-F and SGPspeC-R are used
In amplification purpose fragment size be 249bp；- the F of the primer pair SEsdf I and-R of SEsdf I are used for the big of the purpose fragment expanded
Small is 409bp；The purpose fragment size that primer pair STstm4495-F and STstm4495-R are used to expand is 569bp.
Step 3, pcr amplification reaction is carried out
Based on the pcr template obtained in step 1, the vertical primer included is set up using the specific primer in step 2
The PCR reaction systems of group：PCR pipe is taken, Jia 2 respectively × the μ L of PCRPreMix 12.5, each 0.5 μ of primer ECphoA-F/ECphoA-R
Each 0.2 μ L of L, primer SGglgC-F/SGglgC-R, each 0.3 μ L of primer the SGPspeC-F/SGPspeC-R ,-F/ of primer SEsdf I
Each 0.4 μ L of-R of SEsdf I, each 0.5 μ L of primer STstm4495-F/STstm4495-R, the μ L of template 1.0, the μ L of ultra-pure water 7.7, are mixed
It is even, obtain 25 μ L PCR reaction systems.
Then pcr amplification reaction is carried out under the conditions of predetermined PCR response parameters using above-mentioned PCR reaction systems and obtains PCR
Amplified production：Predetermined PCR response parameters condition is 95 DEG C of pre-degeneration 5min；95 DEG C of 30s, 57 DEG C of 30s, 72 DEG C of 50s, carry out 35
Circulation；72 DEG C, 10min.
Step 4, electrophoresis detection is carried out,
Electrophoresis detection is carried out to the amplified production obtained in step 3 and obtains electrophoresis result, is determined whether according to electrophoresis result
There is avian escherichia coli, Salmonella gallinarum, Salmonella Pullorm, Salmonella enteritidis or Salmonella typhimurtum：
10 μ L pcr amplification products are taken, point sample is in 2.0% agarose gel electrophoresis plate hole, under 80-100V voltages, electricity
Swim 30-40min, obtains electrophoresis result in being taken pictures under gel imaging system, the precondition judged electrophoresis result is the moon
Property control without any band occur：
It is determined as avian escherichia coli when 761bp bands occurs in electrophoresis result,
It is determined as Salmonella gallinarum when 83bp and 249bp bands occurs in electrophoresis result,
It is determined as Salmonella Pullorm when 249bp bands occurs in electrophoresis result,
It is determined as Salmonella enteritidis when 409bp bands occurs in electrophoresis result,
It is determined as Salmonella typhimurtum when 569bp bands occurs in electrophoresis result.
Fig. 1 is the electrophoresis result of the pcr amplification product of embodiment 1.
As shown in figure 1, swimming lane 1 is avian escherichia coli, Salmonella gallinarum, Salmonella Pullorm, Salmonella enteritidis, mouse wound
The heavy PCR primer of cold detection of Salmonella five；Swimming lane 2 is Salmonella gallinarum (83bp and 249bp)；Swimming lane 3 is Salmonella Pullorm
(249bp)；Swimming lane 4 is Salmonella enteritidis (409bp)；Swimming lane 5 is Salmonella typhimurtum (569bp)；Swimming lane 6 is avian escherichia coli
(761bp)；Swimming lane 7 is negative control.
The weight PCR detection kit of embodiment 2 five is detected
The present embodiment, it is consistent the step of with embodiment 1, unlike the present embodiment directly using including carrying in embodiment 1
To five weight PCR detection kits of primer sets set up above-mentioned PCR reaction systems.
The composition of five weight PCR detection kits now includes 2 × PCR PreMix and primer sets, wherein 2 × PCR
PreMix volume is that the volume of primer pair ECphoA-F and ECphoA-R in 12.5 μ L, primer sets are respectively 0.5 μ L, primer pair
SGglgC-F and SGglgC-R volume is respectively 0.2 μ L, and primer pair SGPspeC-F and SGPspeC-R volume are respectively 0.3 μ L,
- the F of primer pair SEsdf I and the-R of SEsdf I volume are respectively 0.4 μ L, primer pair STstm4495-F and STstm4495-R volume
It is respectively 0.5 μ L.
Evaluate the Evaluation on specificity of example 1
The present embodiment is by taking five weight PCR detection kits of embodiment 2 as an example to the primer sets in embodiment 1 and embodiment 2
Specific detection is carried out：
Choose riemerella anatipestifer, staphylococcus aureus, pseudomonas aeruginosa PA14, Streptococcus suis, mouse typhus husky
Door bacterium, Salmonella Pullorm, Listeria monocytogenes, avian pasteurella multocida, fowl enteropathogenic E. Coli, Salmonella gallinarum, fowl branch
Substance, Salmonella typhimurtum, fowl Podbielniak bacterium, staphylococcus aureus, Salmonella enteritidis (CVCC 1805), chicken virus mycoplasma etc.
Bacterium is operated into performing PCR according to five heavy PCR detection kit operation instructions, and product is identified in 2% agarose gel electrophoresis.
Fig. 2 is the experimental result for evaluating Evaluation on specificity in example 1.
As shown in Fig. 2 swimming lane M：DNA Maker；Swimming lane 1：Five heavy PCR；Swimming lane 2：Serum 1 type riemerella anatipestifer
CH3；Swimming lane 3：Staphylococcus aureus (CVCC 543)；Swimming lane 4：Pseudomonas aeruginosa PA14；Swimming lane 5：Streptococcus suis；Swimming lane
6：The type riemerella anatipestifer Yb2 of serum 2；Swimming lane 7：Salmonella typhimurtum SAT52；Swimming lane 8：Mo Shi bars in the type pest of duck of serum 10
Bacterium HXb2；Swimming lane 9：Salmonella gallinarum；Swimming lane 10：Listeria monocytogenes 10403s；Swimming lane 11：Avian pasteurella multocida (CVCC
493)；Swimming lane 12：Fowl enteropathogenic E. Coli；Swimming lane 13, riemerella anatipestifer (wg4)；Swimming lane 14：Salmonella Pullorm
(CVCC 519)；Swimming lane 15：Avian mycoplasmas (CVCC 274)；Swimming lane 16：Salmonella typhimurtum SL1344；Swimming lane 17：Fowl Podbielniak
Bacterium (IPDH 591-77)；Swimming lane 18：Staphylococcus aureus (CVCC543)；Swimming lane 19：Salmonella enteritidis (CVCC 1805)；
Swimming lane 20：Chicken virus mycoplasma (CVCC 1651)；Swimming lane 21：Negative control.
As a result show, hindered with fowl enteropathogenic E. Coli, Salmonella gallinarum, Salmonella Pullorm, Salmonella enteritidis, mouse
Cold detection of Salmonella is amplifiable to arrive specific band respectively as template；And the control samples such as other bacteriums are expanded, PCR results are equal
For feminine gender.Illustrate that the specificity of embodiment 1 and the primer sets in embodiment 2 is stronger, using five heavy PCR detection methods of its foundation
Or detection kit can accurately, quickly, stably, specifically by avian escherichia coli, Salmonella gallinarum, white diarrhea sramana
Bacterium, Salmonella enteritidis, Salmonella typhimurtum are distinguished with other bacterium.
Evaluate the sensitivity assessment of example 2
The present embodiment is by taking five weight PCR detection kits of embodiment 2 as an example to the primer sets in embodiment 1 and embodiment 2
Sensitiveness tested and assessed：
The present embodiment enters performing PCR amplification by template of the bacterial cultures and DNA of bacteria of doubling dilution respectively.
(1) using bacterial cultures as the sensitivity tests of template
Avian escherichia coli, Salmonella gallinarum, Salmonella Pullorm, Salmonella enteritidis, Salmonella typhimurtum are inoculated with respectively
Into LB fluid nutrient mediums, 37 DEG C are shaken bacterium to OD600=1.0, respectively take 1mL bacterium solutions 12000r/min to centrifuge 1min, remove supernatant, go out
The ultrapure washing of bacterium 3 times, 1mL sterilizing ultra-pure waters are resuspended, and bacterium solution are prepared respectively, concentration is 106CFU/μL、105CFU/μL、104CFU/
μL、103CFU/μL、102CFU/μL、10CFU/μL.Each 1 μ L that draw add PCR reaction systems, according to five heavy PCR detection kits
Operating instruction enters performing PCR, and it is husky to avian escherichia coli, Salmonella gallinarum, white diarrhea to determine above-mentioned primer sets according to result of the test
Door bacterium, Salmonella enteritidis, the sensitiveness of Salmonella typhimurtum.
Fig. 3 is the experimental result for evaluating the sensitive stock comment valency in example 2 by template of bacterial solution.
As shown in figure 3, interpretation of result such as table 1, showing that the PCR reaction systems set up using above-mentioned primer sets are minimum can examine
Measure 1000CFU bacteriums.
Sensitiveness evaluating result of the table 1 using bacterial solution as template
(+：There is specific band；-:Do not occur specific band)
(2) using bacterial genomes DNA as the sensitivity tests of template
Avian escherichia coli, Salmonella gallinarum, Salmonella Pullorm, Salmonella enteritidis, Salmonella typhimurtum are inoculated with respectively
Into LB fluid nutrient mediums, 37 DEG C are shaken bacterium to OD600=1.0, wherein 1mL enrichment liquids are taken respectively, and according to kit, (Tiangeng is biological
Technology Co., Ltd.) operated, extract DNA of bacteria.Diluted concentration is 100ng/ μ L, 50ng/ μ L, 10ng/ μ L, 1ng/ respectively
μL、500pg/μL、100pg/μL、10pg/μL.Each 1 μ L that draw add PCR reaction systems, are said according to five weight PCR kit operations
It is bright enter performing PCR, above-mentioned primer sets are determined to avian escherichia coli, Salmonella gallinarum, Salmonella Pullorm, intestines according to result of the test
Scorching detection of Salmonella, the sensitiveness of Salmonella typhimurtum.
Fig. 4 is the experimental result for evaluating example 2 using bacterial genomes DNA as the sensitivity assessment of template.
As shown in figure 4, interpretation of result such as table 2, avian escherichia coli that the surface present invention is set up, Salmonella gallinarum, chicken are white
Dysentery detection of Salmonella, Salmonella enteritidis, Salmonella typhimurtum five weight PCR detection kit minimum fowl that can detect 100pg it is big
Enterobacteria, Salmonella gallinarum, Salmonella enteritidis, Salmonella typhimurtum, and it is only capable of detecting 500pg Salmonella Pullorm.
Sensitiveness evaluating result of the table 2 using bacterial genomes DNA as template
(+：There is specific band；-:Do not occur specific band)
Illustrate that the sensitiveness of embodiment 1 and the primer sets in embodiment 2 is stronger, using five weight PCR detection sides of its foundation
Method or five weight PCR detection kits can be delicately husky by avian escherichia coli, Salmonella gallinarum, Salmonella Pullorm, enteritis
Door bacterium, Salmonella typhimurtum are distinguished with other bacterium.
Evaluate the reproducibility of example 3
Avian escherichia coli of the present embodiment by taking the five of embodiment 2 weight PCR detection kits as an example to preparation, fowl typhoid sramana
Bacterium, Salmonella Pullorm, Salmonella enteritidis, the repeatability of the five weight PCR detection kits of Salmonella typhimurtum are tested and assessed：
The present embodiment enters under different time, different operating condition (including PCR instrument, operating personnel) to positive template respectively
Row detection, testing result is shown in Table 3.
The repeated testing result of table 3
(+：There is specific band；-:Do not occur specific band)
As shown in table 3, according to PCR testing results, illustrate that the repeatability of embodiment 1 and the primer sets in embodiment 2 is stronger,
Can be repeated preferably by avian escherichia coli, chicken using five weight PCR detection methods of its foundation or five weight PCR detection kits
Salmonella typhi, Salmonella Pullorm, Salmonella enteritidis, Salmonella typhimurtum are distinguished with other bacterium.
The effect of embodiment 1 and embodiment 2 and effect
It is can be seen that by evaluating example 1 to example 3 is evaluated due to the positive strain that the primer sets of use have been preserved to laboratory
Expanded, it is amplifiable to arrive specific band, and the control sample such as other bacterium bacterial strains is not expanded to band, illustrates that this draws
The high specificity of thing group, and with the positive that different dilution factors are detected using the primer sets, its lowest detection sensitiveness is reachable
To 1000CFU bacterial solutions and 500pg bacterial genomes DNA, illustrate that the sensitivity of the primer sets is high, meanwhile, the primer sets
With good repeatability, illustrate that its stability is stronger.So embodiment 1 and embodiment 2 use above-mentioned primer at including of providing
Five weight PCR detection methods of group or five containing above-mentioned primer sets heavy PCR detection kits are to avian escherichia coli, fowl typhoid sramana
Bacterium, Salmonella Pullorm, Salmonella enteritidis, the detection of Salmonella typhimurtum have the advantages that sensitive, special, quick and stabilization,
For quick detection and identification avian escherichia coli, Salmonella gallinarum, Salmonella Pullorm, Salmonella enteritidis, Salmonella typhimurtum
There is provided effective means, while the primer sets can also be prepared for detecting avian escherichia coli, Salmonella gallinarum, white diarrhea
Detection of Salmonella, Salmonella enteritidis, the five heavy PCR detection kits of Salmonella typhimurtum.
In addition, in embodiment 1 and embodiment 2, the bacterium solution of the bacterium to be detected in step 1 with culture is prepared
Pcr template, as the present invention, can also continue to the bacterium solution in step 1 extract and obtains bacterium and slightly carry DNA for pcr template,
Now specific process is：By the inoculation of bacterium to be detected to LB fluid nutrient mediums, trained under conditions of temperature is 37 DEG C
Support to OD600=1.0, obtain bacterium solution, then the bacterium solution centrifuges 1-2min under 10000-12000r/min by 1ml, removes supernatant,
Plus 0.1-0.5mL sterilizing ultra-pure waters are resuspended, and boil 5-10min, then centrifuge 3-5min under 10000-12000r/min, obtain
Supernatant be that bacterium slightly carries DNA.
The scope of the present invention is not limited by specific embodiment, and embodiment is only used as illustrating various aspects of the present invention
Also include the method and component of functional equivalent in single example, the scope of the invention.In fact, in addition to this paper content, ability
Field technique personnel can easily grasp a variety of improvement to the present invention with reference to described above and accompanying drawing.Improvement also falls into appended
Within the scope of claims.
Claims (12)
1. one kind is used to detect avian escherichia coli, Salmonella gallinarum, Salmonella Pullorm, Salmonella enteritidis, mouse typhus sramana
The primer sets of the multiplex PCR of bacterium, it is characterised in that including：
The primer pair ECphoA-F and ECphoA-R of specific amplification avian escherichia coli phoA genes；Specific amplification fowl typhoid is husky
The primer pair SGglgC-F and SGglgC-R of door bacterium glgC genes；Specific amplification Salmonella gallinarum and Salmonella Pullorm
The primer pair SGPspeC-F and SGPspeC-R of speC genes；The primer pair of the genes of specific amplification Salmonella enteritidis sdf I
- the F of the SEsdf I and-R of SEsdf I；The primer pair STstm4495-F of specific amplification Salmonella typhimurtum stm4495 genes and
STstm4495-R,
The nucleotide sequence of each primer is respectively：
ECphoA-F, 5 '-GGCAATACACTCACTATGCGCTG-3 ',
ECphoA-R, 5 '-AGGATTCGCAGCATGATCCTG-3 '；
SGglgC-F, 5 '-CGTCGCTATAAAGCGGAATATGTC-3 ',
SGglgC-R, 5 '-CCTTTTCAAAACATACGCGAGTAG-3 '；
SGPspeC-F, 5 '-GTTGCCGTACCGGTCGTAACG-3 ',
SGPspeC-R, 5 '-TTCCTGACGGGCATCGACGGC-3 '；
SEsdf I-F, 5 '-GATGTGGTTGGTTCGTCACTG-3 ',
SEsdf I-R, 5 '-GCGAGACCTCAAACTTACTCAG-3 '；
STstm4495-F, 5 '-CAGCGGTATGATGCGGTAGT-3 ',
STstm4495-R, 5 '-TCACCGGTGGACATGCCTGC-3 '.
2. primer sets according to claim 1, it is characterised in that：
The size that primer pair ECphoA-F and ECphoA-R are used for the purpose fragment expanded is 761bp；
The size that primer pair SGglgC-F and SGglgC-R are used for the purpose fragment expanded is 83bp；
The size that primer pair SGPspeC-F and SGPspeC-R are used for the purpose fragment expanded is 249bp；
The size that-the F of the primer pair SEsdf I and-R of SEsdf I are used for the purpose fragment expanded is 409bp；
The purpose fragment size that primer pair STstm4495-F and STstm4495-R are used to expand is 569bp.
3. the primer sets according to any one right in claim 1 or 2 are being prepared for detecting avian escherichia coli, chicken wound
Application in cold detection of Salmonella, Salmonella Pullorm, Salmonella enteritidis, the five weight PCR detection kits of Salmonella typhimurtum.
4. a kind of five heavy PCR detection kits, for detecting avian escherichia coli, Salmonella gallinarum, Salmonella Pullorm, enteritis
Detection of Salmonella and Salmonella typhimurtum, it is characterised in that including：
2 × PCR premixed liquids and primer sets for setting up PCR reaction systems,
Wherein, each primer pair that the PCR reaction systems are used in the primer sets carries out specific amplification,
The primer sets are the primer sets described in any one in claim 1 or 2.
5. detection kit according to claim 4, it is characterised in that：
Wherein, the volume of 2 × PCR PreMix is 12.5 μ L,
In the primer sets, primer pair ECphoA-F and ECphoA-R volume is respectively 0.5 μ L, primer pair SGglgC-F and
SGglgC-R volume is respectively 0.2 μ L, everybody 0.3 μ L, primer pair SEsdf of primer pair SGPspeC-F and SGPspeC-R volume
I-F and the-R of SEsdf I volume everybody 0.4 μ L, primer pair STstm4495-F and STstm4495-R volume are respectively 0.5 μ L.
6. a kind of detect the five of avian escherichia coli, fowl typhoid door bacterium, Salmonella Pullorm, Salmonella enteritidis and Salmonella typhimurtum
Weight PCR detection method, it is characterised in that comprise the following steps：
Step 1, pcr template is prepared based on bacterium to be detected；
Step 2, specific primer group is prepared, is prepared specifically using the primer sets described in any one in claim 1 or 2
Property primer sets；
Step 3, pcr amplification reaction is carried out, the pcr template obtained based on step 1, the specificity obtained using step 2 is drawn
Thing sets up the vertical PCR reaction systems for obtaining including described primer sets, is then reacted using the PCR reaction systems in predetermined PCR
Pcr amplification reaction is carried out under Parameter Conditions and obtains pcr amplification product；
Step 4, electrophoresis detection is carried out, carrying out electrophoresis detection to the amplified production obtains electrophoresis result, according to the electrophoresis knot
Fruit determines whether the avian escherichia coli, Salmonella gallinarum, the Salmonella Pullorm, the Salmonella enteritidis or institute occur
State Salmonella typhimurtum.
7. five heavy PCR detection method according to claim 6, it is characterised in that：
The pcr template obtained in step 1 is the bacterium solution of the bacterium to be detected, and detailed process is：
By the inoculation of the bacterium to be detected to LB fluid nutrient mediums, cultivated under conditions of temperature is 37 DEG C to OD600=
1.0, that is, obtain the bacterium solution.
8. five heavy PCR detection method according to claim 6, it is characterised in that：
The pcr template obtained in step 1 is that bacterium slightly carries DNA, and detailed process is：
By the inoculation of the bacterium to be detected to LB fluid nutrient mediums, cultivated under conditions of temperature is 37 DEG C to OD600
=1.0, bacterium solution is obtained, then the bacterium solution centrifuges 1-2min under 10000-12000r/min by 1ml, removes supernatant, plus 0.1-
0.5mL sterilizing ultra-pure waters are resuspended, and boil 5-10min, then centrifuge 3-5min under 10000-12000r/min, obtained supernatant
As bacterium slightly carries DNA.
9. the five heavy PCR detection methods according to claim 6 to 8 any one, it is characterised in that：
The PCR reaction systems of step 3 include：
12.5 μ L 2 × PCR PreMix, the 1.0 μ L template, 7.7 μ L ultra-pure water, and the primer sets,
In the primer sets, primer pair ECphoA-F and ECphoA-R volume is respectively 0.5 μ L, primer pair SGglgC-F and
SGglgC-R volume is respectively 0.2 μ L, everybody 0.3 μ L, primer pair SEsdf of primer pair SGPspeC-F and SGPspeC-R volume
I-F and the-R of SEsdf I volume everybody 0.4 μ L, primer pair STstm4495-F and STstm4495-R volume are respectively 0.5 μ L.
10. the five heavy PCR detection methods according to claim 6 to 8 any one, it is characterised in that：
When preparing specific primer group, the concentration of each primer used in the primer sets is 10pM, by each institute used
State primer dilution be placed in -20 DEG C preserve after use again.
11. the five heavy PCR detection methods according to claim 6 to 8 any one,
It is characterized in that：
The predetermined PCR response parameters condition is 95 DEG C of pre-degeneration 5min；30s at 95 DEG C, 30s at 57 DEG C, 50s at 72 DEG C, enter
35 circulations of row；72 DEG C, 10min.
12. the five heavy PCR detection methods according to claim 6 to 8 any one, it is characterised in that：
The detailed process of step 4 is,
10 μ L pcr amplification products are taken, point sample is in 2.0% agarose gel electrophoresis plate hole, under 80-100V voltages, electrophoresis
30-40min, electrophoresis result is obtained in being taken pictures under gel imaging system, and the premise bar of the judgement is carried out to the electrophoresis result
Part is that negative control occurs without any band,
It is determined as avian escherichia coli when 761bp bands occurs in the electrophoresis result,
It is determined as Salmonella gallinarum when 83bp and 249bp bands occurs in the electrophoresis result,
It is determined as Salmonella Pullorm when 249bp bands occurs in the electrophoresis result,
It is determined as Salmonella enteritidis when 409bp bands occurs in the electrophoresis result,
It is determined as Salmonella typhimurtum when 569bp bands occurs in the electrophoresis result.
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