CN107164557A - It is a kind of while detecting primer and the application of pasteurella multocida and its capsular serotype A type dual real-time fluorescence quantifying PCR method - Google Patents
It is a kind of while detecting primer and the application of pasteurella multocida and its capsular serotype A type dual real-time fluorescence quantifying PCR method Download PDFInfo
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- CN107164557A CN107164557A CN201710612652.4A CN201710612652A CN107164557A CN 107164557 A CN107164557 A CN 107164557A CN 201710612652 A CN201710612652 A CN 201710612652A CN 107164557 A CN107164557 A CN 107164557A
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Abstract
It is a kind of while detecting primer and the application of pasteurella multocida and its capsular serotype A type dual real-time fluorescence quantifying PCR method, it is related to biological technical field.The present invention primer be：Kmt‑F：5'‑GCTYGTTGTGAGTGGGCTTGT‑3'；Kmt‑R：5'‑GCCGTTGTCAAGGAAGCAGAT‑3'；Kmt‑P：5'FAM‑TTTGTTGGGCRGAGTTTGGTG‑BHQ13'；CapA‑F：5'‑GGGCTGGCTATGTTGGTTTAT‑3'；CapA‑R：5'‑CTTTATCTGTGATGGGCGATT‑3'；CapA‑P：5'HEX‑TAGCTCAACACCACAATGTGATCT3'.Kmt1 and hyaD hyaC genes are detected simultaneously.
Description
Technical field
It is more particularly to a kind of to detect pasteurella multocida and its capsular serotype A type simultaneously the invention belongs to biological technical field
Dual Real-time PCR methods primer and application.
Background technology
Pasteurella multocida (Pasteurella multocida, Pm) can cause a variety of diseases of humans and animals, including
Cholera fowl, ox hueppe's disease, atrophic rhinitis, rabbit hemorrhagic septicemia and subcutaneous infection of people etc., are caused to animal
Higher pathogenicity rate and the death rate.Pasteurella multocida is divided into according to the difference of Buccal mucosa flap：A, B, D, E and F type.Conventional detection
The method of pasteurella multocida antigen is to be detected using after bacteria distribution identification, or increasing bacterium by regular-PCR.Wherein, carefully
Bacterium separation identification method is very complicated in practical operation, and easily by miscellaneous bacteria interference effect, this method time and effort consuming；And it is common
PCR method identifies detection method compared to traditional separation, although more sensitive quick, but the content to object bacteria in bacterium solution
And require more strict in terms of miscellaneous bacteria quantity, and it is possible to the results such as false negative and false positive occur.Point of bacterial capsule
Type mainly uses serological method and capsular PCR typing：Serological method needs Buccal mucosa flap standard serum, it is difficult to buy, takes
When it is laborious.
The content of the invention
It is an object of the invention to：Two pairs of high specific primers of design are crossed, using Taqman real time fluorescence quantifying PCR methods
Pasteurella multocida and its capsular serotype A type bacterium are identified simultaneously, so as to substitute the method for regular-PCR, make detection more accurate
With it is efficient.The present invention two pairs of high specific fluorescence quantitative PCR detection primers of design, are easy to pasteurella multocida and its pod membrane
The detection of A type bacterium, so as to improve detection efficiency.
The a kind of of the present invention detects pasteurella multocida and its capsular serotype A type dual real-time fluorescence quantifying PCR method simultaneously
Primer and application, described primer includes upstream and downstream primer and Taqman probe primers, specific as follows：
Sense primer Kmt-F：5'-GCTYGTTGTGAGTGGGCTTGT-3'；
Anti-sense primer Kmt-R：5'-GCCGTTGTCAAGGAAGCAGAT-3'；
Probe primer Kmt-P：5'FAM-TTTGTTGGGCRGAGTTTGGTG-BHQ1 3'；
Sense primer CapA-F：5'-GGGCTGGCTATGTTGGTTTAT-3'；
Anti-sense primer CapA-R：5'-CTTTATCTGTGATGGGCGATT-3'；
Probe primer CapA-P：5'HEX-TAGCTCAACACCACAATGTGATCT3'.
The present invention includes following beneficial effect：
The present invention utilizes the capsular serotype A type of the CapA primer detections bacterium by Kmt primer detection pasteurella multocida.
The high special primer that the present invention is used, using fluorescence quantitative PCR method, in bacterial content 3 × 10-12During g/ μ L still
Pasteurella multocida and its capsular serotype A type can be accurately detected, and not by miscellaneous bacteria interference effect；The real-time fluorescence of use is determined
PCR methods detection pasteurella multocida antigen is measured, the characteristics of with convenient, accurate, high sensitivity.
Brief description of the drawings
Fig. 1 is FAM probes mark (kmt1 genes) and HEX probes mark (CapA genes) dual real-time fluorescence quantitative PCR
The standard curve of detection；Wherein A is kmt1 gene curves, and B is CapA gene curves；
Fig. 2 is dual real-time fluorescence quantitative PCR sensitivity Detection figure；Wherein, N is negative control；
Fig. 3 is conventional dual PCR electrophoretograms；Wherein, N is negative control；
Fig. 4 is real-time fluorescence quantitative PCR specific detection figure；Wherein, A is P.multocida C48-1 capsular serotype A types (C
apA)HEX；B is P.multocida C48-1FAM；C is P.multocida C44-1FAM；D is P.multoci da
901FAM；E is other bacterial strains.
Embodiment
Embodiment one：The a kind of of present embodiment detects that pasteurella multocida and its capsular serotype A type are dual simultaneously
The primer of real time fluorescence quantifying PCR method and application, described primer include upstream and downstream primer and Taqman probe primers, tool
Body is as follows：
Sense primer Kmt-F：5'-GCTYGTTGTGAGTGGGCTTGT-3'；
Anti-sense primer Kmt-R：5'-GCCGTTGTCAAGGAAGCAGAT-3'；
Probe primer Kmt-P：5'FAM-TTTGTTGGGCRGAGTTTGGTG-BHQ1 3'；
Sense primer CapA-F：5'-GGGCTGGCTATGTTGGTTTAT-3'；
Anti-sense primer CapA-R：5'-CTTTATCTGTGATGGGCGATT-3'；
Probe primer CapA-P：5'HEX-TAGCTCAACACCACAATGTGATCT3'.
Embodiment two：
PCR reaction systems are 20 μ L；
PCR reaction conditions：95℃5min；95 DEG C of 15sec, 53 DEG C of 20sec, 72 DEG C of 30sec, 35 are circulated；In prolonging for circulation
Fluorescence signal detection is carried out when stretching.
Present embodiment has unlike one from embodiment：Described primer is applied to detection killing property Pasteur's bars more
Bacterium and and its capsular serotype A type in.It is other identical with embodiment one.
Present invention is not limited only to the content of the respective embodiments described above, the group of one of them or several embodiments
Contract sample can also realize the purpose of invention.
Beneficial effects of the present invention are verified by following examples：
Embodiment 1
Pasteurella multocida and its design of capsular serotype A type dual real-time fluorescence quantification PCR primer
With reference to the kmt1 sequences (No. GenBank of GenBank Pm70 plants of the pasteurella multocida included：AE004339),
Sequence alignment is carried out to reference strain by bioinformatics software, analyzes and obtains pasteurella multocida kmt1 and hyaD-
The conservative region of hyaC genes.Using the real-time fluorescence quantitative PCR primer-design softwares of ABI PrimerExpress 3.0, design
Synthesize TaqMan probe and primer.Design 2 pairs of primer pairs and probe.
Primer and the detection of the dual Real-time PCR methods of pasteurella multocida and its capsular serotype A type are detected simultaneously
Method, described primer includes upstream and downstream primer and probe primer, specific as follows：
Sense primer Kmt-F：5'GCTYGTTGTGAGTGGGCTTGT 3', as shown in SEQ ID NO.1；
Anti-sense primer Kmt-R：5'GCCGTTGTCAAGGAAGCAGAT3', as shown in SEQ ID NO.2；
Probe primer Kmt-P：Shown in 5'FAM-TTTGTTGGGCRGAGTTTGGTG-BHQ1 3', SEQ ID NO.3.
Sense primer CapA-F：5'-GGGCTGGCTATGTTGGTTTAT-3', as shown in SEQ ID NO.4；
Anti-sense primer CapA-R：5'-CTTTATCTGTGATGGGCGATT-3', as shown in SEQ ID NO.5；
Probe primer CapA-P：5'HEX-TAGCTCAACACCACAATGTGATCT3', as shown in SEQ ID NO.6；
2nd, the preparation of sample
1 antigen is detected
1.1 sampled acquisitions are died of illness liver, the spleen of fowl；Pig and the nose swab of ox, take out nose swab and are put into what is dispensed in advance
In BHI culture mediums containing 30% glycerine；
1.2 bacterium solutions prepare liver, the spleen for gathering the fowl that dies of illness；The nose swab of pig and ox carries out bacterium Zengjing Granule；37℃
Shaking table, under the conditions of 200rpm, incubated overnight 16h；Another go bail for deposits strain Pm, and gradient dilution is after recovery, colony counting
Various concentrations are used as positive control.
The extraction of 1.3 pasteurella multocida genomic DNAs
Pasteurella multocida 48h blood agar cultures are taken to be suspended in 1.5mL equipped with the 200 sterile PBS of μ L under aseptic condition
In, concentration is adjusted to 1 Maxwell turbidity.According to genome DNA extracting reagent kit operation instruction, DNA is extracted.Use uv-spectrophotometric
Instrument determines gained pasteurella multocida genomic DNA concentration, is detected or be placed in immediately -20 DEG C of preservations.
Embodiment 2
Pasteurella multocida and its drafting of capsular serotype A type dual real-time fluorescence quantitative PCR standard curve
The PCR amplifications of pasteurella multocida kmt1 and hyaD-hyaC gene, are expanded purpose fragment and are connected to pM
D-18T vector construction plasmid standards.
The μ L of kmt1 reaction systems 50 include：
25 μ 2 × Taq of L premixed liquids (purchased from precious bioengineering Dalian Co., Ltd), 1 μ L forward primers KMT1-SP6：5’-
GCTGTAAACGAACTCGCCAC-3 ' (10pmol), 1 μ L reverse primer KMT1-T7：5’-ATCCGCTATTTACCCAGTGG-
3 ' (10pmol), 1 μ L DNA, 22 μ L ultra-pure waters；
HyaD-hyaC reaction systems 50uL：25 μ L 2 × Taq premixed liquids, 1 μ L forward primers CapA-F：5’-
GGGCTGGCTATGTTGGTTTAT-3 ' (10pmol), 1 μ L reverse primer CapA-R：5’-
CTTTATCTGTGATGGGCGATT-3 ' (10pmol), 1 μ L DNA, 22 μ L ultra-pure waters.
PCR reaction conditions are：94 DEG C of pre-degenerations 5min, 94 DEG C of 30sec, 53 DEG C of 30sec, 72 DEG C of 1min, 30 circulations；72
DEG C extension 10min.
Take the μ L agarose gel electrophoresis of pcr amplification product 5 to detect, 457bp and 120bp purpose band agar will be obtained
Sugared gel reclaims kit is reclaimed and purified.Purifying pasteurella multocida kmt1 and CapA genetic fragment is carried with pMD18-T
Body is attached, by connection product thermal transition into bacillus coli DH 5 alpha competent cell, is coated with the LB containing ampicillin
Flat board, 37 DEG C of incubated overnights.The selected single bacterium colony containing positive colony is cultivated, DNA is extracted, is identified simultaneously
Sequencing.
Sequencing result shows to obtain correct sequence, by the correctly recombinant plasmid containing target gene, is named as pMD-
Kmt and pMD-CapA.Determined by ultraviolet spectrophotometry OD260Nm values calculate copy number, as a result copied to determine plasmid DNA concentration
Shellfish number is 1 × 109Copy/μ L, -20 DEG C of preservations.
2nd, standard items are detected
PMD-kmt and pMD-CapA DNAs are carried out into pasteurella multocida sonde method real-time fluorescence as template to determine
PCR detections are measured, standard curve is set up respectively.
Concrete operations are as follows：DNA progress is serially diluted into 1 × 10 for 10 times9Copy/μ L, 1 × 108Copy/μ L, 1
×107Copy/μ L, 1 × 106Copy/μ L, 1 × 105Copy/μ L, 1 × 104Copy/μ L, 1 × 103Copy/μ L, 1 × 102Copy
Shellfish/μ L totally 8 dilution factors.Each dilution factor repeats parallel test 2 times.3rd, the drafting of standard curve.
Painted according to gained CT values (cycle threshold) as the logarithm value of the corresponding standard items of ordinate as abscissa
The standard curve of the real-time fluorescence quantitative PCR detection of the pasteurella multocida of system is shown in Fig. 1, wherein, A slope is y=-
3.3607x+43.051；R2=0.9909；B slope is y=-3.2439x+41.612；R2=0.9873.Standard curve is shown：
The pasteurella multocida sonde method real-time fluorescence quantitative PCR detection method of foundation has the linear detection range of 7 orders of magnitude, enters
One step illustrates that the detection method has very high sensitivity.
Embodiment 3
The sensitiveness of dual real-time fluorescence quantitative PCR detecting method
Pasteurella multocida C48-1 pnca gene group DNA are serially diluted into 3 × 10 again as casting formwork 10-7g/μL、
3×10-8g/μL、3×10-9g/μL、3×10-10g/μL、3×10-11g/μL、3×10-12g/μL、3×10-13g/μL、3×10-14G/ μ L totally 8 dilution factors, using sterilized water as negative control, carry out sonde method real-time fluorescence quantitative PCR detection, evaluate the present invention
The sensitiveness of reaction system.
Dual real-time fluorescence quantitative PCR detection sensitiveness amplification curve result is shown：It is 3 × 10 in template concentrations-12g/μL
When detectable identification bacterium kind and capsular serotype A type fluorescence signal, as shown in Figure 2.The present invention sets up pasteurella multocida
And its capsular serotype A type dual real-time fluorescence quantitative PCR has good sensitiveness.
According to the diluted concentration of template in dual real-time fluorescence quantitative PCR, expanded with regular-PCR, carry out contrast and test
Card.Regular-PCR result is shown in accompanying drawing 3,3 × 10-10The amplifiable purpose band of genomic DNA of g/ μ L concentration, as shown in Figure 3.This hair
The dual real-time fluorescence quantifying PCR method of bright foundation is more sensitive 100 times than common dual-PCR method.
Embodiment 4
The specificity of dual real-time fluorescence quantitative PCR detecting method
Set water blank control, with staphylococcus aureus, salmonella, C.perfringens, riemerella anatipestifer,
Campylobacter jejuni, proteus mirabilis, Escherichia coli, John's acinetobacter calcoaceticus, nose tracheae bird bacillus, bronchus sepsis Podbielniak bar
The DNA of 17 plants of bacterial strains such as bacterium, Mannheimia haemolytica, pasteurella multocida capsular serotype A, B, D type carries out glimmering in real time as template
Fluorescent Quantitative PCR detection, the specificity of evaluation response system.Reaction system and reaction condition are same as Example 2.
As a result it is as shown in Figure 4.As a result show：Dual real-time fluorescence quantitative PCR only detects pasteurella multocida and pod
The bacterium of film A types, and to Type B and its fubaritic serotype of D types bacterium；Other bacterial strains can not be expanded.As a result illustrate：This
Inventing the pasteurella multocida set up and its capsular serotype A type dual real-time fluorescence quantitative PCR has good specificity.
Embodiment 5
Detection of the dual real-time fluorescence quantitative PCR detecting method to sample
With the pasteurella multocida and its capsular serotype A type dual real-time fluorescence quantifying PCR method of foundation to 30 parts C48-1 plants
Bacteria artificial is inoculated with the samples such as animal infected liver, spleen and detected, can detect bacterium kind and capsular serotype A type is special
Different fluorescence signal.The result for carrying out detecting after bacterium directly detection and Zengjing Granule to 46 clinical samples shows：It is dual real-time
Quantitative fluorescent PCR is respectively 18/46 and 30/46 to both recall rates, and common double PCR is respectively to both recall rates
10/46 and 13/46；Detect sensitiveer, accurate than regular-PCR.
Sequence table
<110>Harbin Veterinary Medicine Inst., China Academy of Agriculture
<120>It is a kind of at the same detect pasteurella multocida and its capsular serotype A type dual real-time fluorescence quantifying PCR method primer and
Using
<160>6
<210>1
<211>21
<212> DNA
<213>Artificial sequence
<220>
<223> Kmt-F。
<400> 1
GCTYGTTGTGAGTGGGCTTGT 21
<210>2
<211>21
<212> DNA
<213>Artificial sequence
<220>
<223>Kmt-R。
<400> 2
GCCGTTGTCAAGGAAGCAGAT 21
<210>3
<211>21
<212> DNA
<213>Artificial sequence
<220>
<223>Kmt-P。
<400> 3
TTTGTTGGGCRGAGTTTGGTG 21
<210>4
<211>21
<212> DNA
<213>Artificial sequence
<220>
<223>CapA-F。
<400> 4
GGGCTGGCTATGTTGGTTTAT 21
<210>5
<211>21
<212> DNA
<213>Artificial sequence
<220>
<223>CapA-R。
<400> 5
CTTTATCTGTGATGGGCGATT 21
<210>6
<211>24
<212> DNA
<213>Artificial sequence
<220>
<223>CapA-R。
<400> 6
TAGCTCAACACCACAATGTGATCT 24
Claims (3)
1. it is a kind of while detecting the primer of pasteurella multocida and its capsular serotype A type dual real-time fluorescence quantifying PCR method and answering
With, it is characterised in that described primer includes upstream and downstream primer and Taqman probe primers, specific as follows：
It is as follows for the primer and probe sequence of pasteurella multocida：
Sense primer Kmt-F：5'-GCTYGTTGTGAGTGGGCTTGT-3'；
Anti-sense primer Kmt-R：5'-GCCGTTGTCAAGGAAGCAGAT-3'；
Probe primer Kmt-P：5'FAM-TTTGTTGGGCRGAGTTTGGTG-BHQ1 3'；
It is as follows for the primer and probe sequence of capsular serotype A type：
Sense primer CapA-F：5'-GGGCTGGCTATGTTGGTTTAT-3'；
Anti-sense primer CapA-R：5'-CTTTATCTGTGATGGGCGATT-3'；
Probe primer CapA-P：5'HEX-TAGCTCAACACCACAATGTGATCT3'.
2. a kind of detection pasteurella multocida simultaneously according to claim 1 and its capsular serotype A type dual real-time fluorescence are fixed
Measure primer and the application of PCR method, it is characterised in that：PCR reaction systems are 20 μ l；
PCR reaction conditions：95℃5min；95 DEG C of 15sec, 53 DEG C of 20sec, 72 DEG C of 30sec, 35 are circulated：Last 72 DEG C of 10min.
3. it is a kind of while detecting the primer of pasteurella multocida and its capsular serotype A type dual real-time fluorescence quantifying PCR method and answering
With, it is characterised in that by primer as claimed in claim 1 be applied to detection pasteurella multocida and and its capsular serotype A type in.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108277288A (en) * | 2017-12-19 | 2018-07-13 | 吉林农业大学 | The detection kit of three kinds of pathogens of ox respiratory disease |
| CN108411014A (en) * | 2018-04-28 | 2018-08-17 | 金宇保灵生物药品有限公司 | Differentiate the primer and probe and detection method of A types and the dual real-time fluorescence quantitative PCR of Type B ox pasteurella multocida |
| CN109628622A (en) * | 2019-02-02 | 2019-04-16 | 广西壮族自治区兽医研究所 | A kind of real-time quantitative LAMP primer group and kit detecting pasteurella multocida |
| CN110885892A (en) * | 2019-11-01 | 2020-03-17 | 拱北海关技术中心 | Method for detecting pasteurella multocida, primers and probe thereof |
| CN112760394A (en) * | 2021-02-23 | 2021-05-07 | 福建省农业科学院畜牧兽医研究所 | Multiplex PCR primer for identifying avian pasteurella multocida and serotypes thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108277288A (en) * | 2017-12-19 | 2018-07-13 | 吉林农业大学 | The detection kit of three kinds of pathogens of ox respiratory disease |
| CN108411014A (en) * | 2018-04-28 | 2018-08-17 | 金宇保灵生物药品有限公司 | Differentiate the primer and probe and detection method of A types and the dual real-time fluorescence quantitative PCR of Type B ox pasteurella multocida |
| CN109628622A (en) * | 2019-02-02 | 2019-04-16 | 广西壮族自治区兽医研究所 | A kind of real-time quantitative LAMP primer group and kit detecting pasteurella multocida |
| CN110885892A (en) * | 2019-11-01 | 2020-03-17 | 拱北海关技术中心 | Method for detecting pasteurella multocida, primers and probe thereof |
| CN112760394A (en) * | 2021-02-23 | 2021-05-07 | 福建省农业科学院畜牧兽医研究所 | Multiplex PCR primer for identifying avian pasteurella multocida and serotypes thereof |
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