CN106811527A - A kind of kit of quick Direct PCR detection ox mycoplasma pneumoniae and its application - Google Patents
A kind of kit of quick Direct PCR detection ox mycoplasma pneumoniae and its application Download PDFInfo
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- 238000001514 detection method Methods 0.000 title claims abstract description 31
- 241000202934 Mycoplasma pneumoniae Species 0.000 title claims abstract description 22
- 239000013641 positive control Substances 0.000 claims abstract description 16
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 14
- 239000013642 negative control Substances 0.000 claims abstract description 13
- 201000010099 disease Diseases 0.000 claims description 16
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 16
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- 238000012408 PCR amplification Methods 0.000 claims description 7
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- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 claims description 3
- 229960005542 ethidium bromide Drugs 0.000 claims description 3
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- 238000004519 manufacturing process Methods 0.000 claims description 3
- 210000001989 nasopharynx Anatomy 0.000 claims description 3
- 238000000246 agarose gel electrophoresis Methods 0.000 claims 1
- 241001138504 Mycoplasma bovis Species 0.000 abstract description 17
- 206010035664 Pneumonia Diseases 0.000 abstract description 10
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Classifications
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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
-
- 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]
Abstract
Kit and its application of ox mycoplasma pneumoniae are detected the present invention relates to a kind of kit and its application, more particularly to a kind of quick Direct PCR, belongs to biological technical field.Kit of the invention includes following reagent：PCR amplifing reagents, positive control and negative control.The kit can fast and accurately detect ox mycoplasma pneumoniae.Using when, the single sample of Mycoplasma bovis pneumonia can be detected, the detection of mixed infection in clinical case can also be can be used for the generaI investigation of ox mycoplasma pneumoniae, Molecule Epidemiology Investigation and vaccine screening and be monitored.
Description
Technical field
The present invention relates to a kind of kit and its application, more particularly to a kind of quick Direct PCR detection ox mycoplasma pneumoniae
Kit and its application, belong to biological technical field.
Background technology
Mycoplasma bovis (Mycoplasma bovis) mainly cause the pneumonia of ox, arthritis, mammitis, keratoconjunctivitis,
Genital inflammation and miscarriage and the disease such as infertile.1961, American Hale etc. was first from suffering from the milk of mammitis milk cow
In isolate Mycoplasma bovis.Mycoplasma bovis in 1976 are described as causing the Etiological of breathing problem, afterwards, Mycoplasma bovis and
Its relevant disease diffuses to rapidly the cows in world's every country and area, and the sound development to world's cattle-raising is caused greatly
Threat and serious economic loss.Since 2008, many ground of China occur in succession and it is pandemic to generate heat, cough and
It is cardinal symptom to have a running nose, and the bovine respiratory infectious disease with necrotizing pneumonia as major lesions is also confirmed as Mycoplasma bovis infection
Caused Mycoplasma bovis pneumonia, due to the sick conventional medicine unsatisfactory curative effect, and is prevented without effective vaccine, therefore to China
Cattle-raising causes serious financial consequences.
Because Mycoplasma bovis pneumonia clinical symptoms are without particularity, clinically easily mutually obscure with other breathing problems, because
This, the laboratory diagnostic method of current Mycoplasma bovis pneumonia mainly has etiological diagnosis, immunology diagnosis, diagnosis of molecular biology
Deng.Wherein, etiological diagnosis are the most certain, but need to expend longer time.And it is conventional anti-in serological diagnostic method
Body detecting method Diagnosis of Cattle Eaton agent pneumonia sensitivity is low, non-specific high.Therefore, quick, sensitive and special Mycoplasma bovis
The foundation of detection method is to the timely of Mycoplasma bovis pneumonia, Accurate Diagnosis and effectively controls significant.
PCR method Sensitivity and Specificity is a kind of more universal method in clinical etiological diagnosis preferably.But pass
The PCR method of system is extracted from the cracking of sample, DNA, PCR amplifications need more than 5 hours to electrophoresis detection.And bacteria distribution culture
Extracting amplification to purifying and DNA at least needs 3~4 days can just make Preliminary Identification.Therefore common PCR method and separation is identified
Method not only wastes time and energy, as a result also inaccurate, and sensitiveness is poor, easily produces the result of false positive, is unfavorable for raiser
Cause of disease is quick and precisely diagnosed with scale enterprise, corresponding vaccine and medicine are taken immediately timely to be treated to reduce damage
The purpose of mistake.
The content of the invention
The purpose of the present invention is directed to the defect of prior art presence, a kind of quick Direct PCR detection ox pneumonia of proposition
The kit of mycoplasma and its application, sample simple process are directly expanded, fast reaction, and whole process is completed in 1 hour, inspection
Survey rapid, sensitivity is good.
The present invention solves technical problem by the following technical programs：A kind of quick Direct PCR detection ox mycoplasma pneumoniae
Kit, comprising following reagent：PCR amplifing reagents, positive control and negative control.
It is anti-that PCR amplifing reagents described above contain 2 × Direct PCR Mix, 10 μ l, 10pM forward primers, 1 μ l, 10pM
To the μ l of primer 1, the μ l of 5U/ μ l Hot Strart Taq enzymes 0.5, add sterilizing ddH2The μ of O to 19 l.The forward primer is PF：5’-
CCTTAGGCAGTTTCTTTATAA-3 ', the reverse primer is PR：5’-CATGCATGTCGAGCGATGAT-3’.
The kit that the present invention is provided can fast and accurately detect ox mycoplasma pneumoniae.Can be to ox mycoplasma pneumoniae sense
The single sample detection of dye, can also can be used for the generaI investigation of Mycoplasma bovis pneumonia cause of disease, divides to the detection of mixed infection in clinical case
Sub- epidemiology survey and vaccine screening and monitoring.
The present invention further provides a kind of application of the kit of quick Direct PCR detection ox mycoplasma pneumoniae, including with
Lower step：
The collection of the first step, sample, is divided into pathological material of disease and Nasopharyngeal swabs collection, and the pathological material of disease collection is collection under aseptic condition
Pathological material of disease 10mg~100mg, is fitted into standby in the centrifuge tube without RNase pollution, and the Nasopharyngeal swabs collection is collection nasopharynx liquid, dress
Enter without RNase pollution centrifuge tube in it is standby；
Second step, sample treatment, add 200 μ l sterilizings ddH in Nasopharyngeal swabs2O, discards swab, pathological material of disease grinding after extruding
Solid-like sample 2mg afterwards, adds 20 μ l sterilizings ddH2O, concussion is mixed；It is quick to be vortexed 5 seconds, 1 μ l supernatants are taken as template.
3rd step, Direct PCR reaction, reaction system is 20 μ l, takes the μ l of Sample supernatants 1 after treatment, adds PCR amplification examinations
The μ l of agent 19, fully mix, and positive control and negative control take 1 μ l, 19 μ l PCR amplifing reagents of addition respectively, and PCR amplification programs are
98℃30s；Then 35 circulations：98 DEG C of 5s, 55 DEG C of 5s, 72 DEG C of 5s；Last 72 DEG C of 1min；
4th step, the detection of PCR primer, take 5 μ l amplified productions and are applied directly in the Ago-Gel containing ethidium bromide, together
When add DNA Marker (2000bp) control, voltage is 100V, electrophoresis 20min, and result is then observed in gel imaging instrument,
If sample can amplify 1390bp bands, illustrate that sample is the positive, positive control also amplifies 1390bp bands, negative control
There is no band.
Reaction system in 3rd step of the above method is 20 μ l, containing the Sample supernatants 1 μ l after treatment, 2 ×
μ l, the 5U/ μ l Hot Strart Taq enzymes 0.5 of 10 μ l, 10pM forward primers of Direct PCR Mix, 1 μ l, 10pM reverse primer 1
μ l, plus sterilizing ddH2The μ of O to 20 l.It is sufficiently mixed uniform rear of short duration centrifugation.Amplification length is 1390bp.Agar in 4th step
Gel strength during sugared gel electrophoresis is 1.0%, wherein containing 1% gel stain.
The method of the present invention is built upon on molecular biology mechanism, negative and positive control is provided in detection, greatly
The big degree of accuracy that improve detection, reduces the occurrence probability of false positive, and specificity is relatively strong, time saving and energy saving, and the kit will be detected
Time foreshortens to 1h, substantially increases operating efficiency.The invention is particularly suited to a large amount of clinical sample of ox mycoplasma pneumoniae infection
This quick diagnosis, can provide scientific basis for the preventing and treating and treatment of Mycoplasma bovis pneumonia in clinical and scientific research, and to carrying
Detection, monitoring, the vaccine development of ox mycoplasma pneumoniae high etc. are significant.
Brief description of the drawings
Fig. 1 is the testing result of PCR primer, and Marker in figure is DL 2000bp, be followed successively by from top to bottom 2000bp,
1000bp, 750bp, 500bp, 250bp, 100bp, as shown in Figure 1, template PCR amplifications fragment is about 1390bp, it was demonstrated that amplification piece
Section is purpose fragment, meets desired design size, and 1 is positive control, and 2 is the fragment of mycoplasma amplification, and 3 is negative control.
Fig. 2 is kit specific detection result, and the Marker in figure is DL 2000bp, is followed successively by from top to bottom
2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp.1 is positive control, and 2 expand for mycoplasma, and 3~9 are followed successively by pig
Streptococcus, haemophilus parasuis, actinobacillus pleuropneumoniae, Escherichia coli, pasteurella multocida, bordetella bacilli,
Salmonella, does not amplify.As shown in Figure 2, kit specificity is good.
Fig. 3 is kit sensitivity Detection result, and the Marker in figure is DL 2000bp, is followed successively by from top to bottom
2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp, 1 is positive control, and 2~7 are followed successively by template 100~10-5Dilution
Degree.From the figure 3, it may be seen that template dilution factor is 100、10-1、10-2、10-3、10-4、10-5When, can amplify clear band, it was demonstrated that examination
The susceptibility of agent box detection is 10-4, kit sensitiveness is good.
Fig. 4 is the clinical practice testing result of kit, and the Marker in figure is DL 2000bp, is followed successively by from top to bottom
2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp, 1 is positive control, and 2~22 is clinical pathological material of disease, and 23 is negative right
According to.As shown in Figure 4, kit can be used for the detection of clinical pathological material of disease.
Specific embodiment
The present invention is further illustrated by the following examples, and the percentage composition in text is such as not particularly illustrated and refers both to weight
Percentage.
Embodiment 1
The foundation of the method for the Direct PCR detection ox mycoplasma pneumoniae of the present embodiment：
(1) PCR amplifing reagents, reversely draw containing 2 × Direct PCR Mix, 10 μ l, 10pM forward primers 1 μ l, 10pM
The μ l of thing 1, the μ l of 5U/ μ l Hot Strart Taq enzymes 0.5, plus sterilizing ddH2The μ of O to 19 l.Wherein 2 × Direct PCR Mix contain
dNTPs、MgCl2, the agent of PCR buffer, PCR increased responses, stabilizer, blue tracer dye.
(2) positive control and negative control.Positive control is mycoplasma bovis vaccine strain, and negative control is sterilizing ddH2O。
Reaction system is 20 μ l, the μ l of 1 μ l, PCR amplifing reagent of Sample supernatants 19 after treatment.
Comprise the following steps：
(1) collection of sample：
1. pathological material of disease：Aseptic collection pathological material of disease 10mg~100mg, is fitted into standby in the centrifuge tube without RNase pollution.
2. Nasopharyngeal swabs：Nasopharynx liquid is gathered with Nasopharyngeal swabs, is fitted into standby in the centrifuge tube without RNase pollution.
(2) sample treatment：
1. 200 μ l sterilizings ddH is added in Nasopharyngeal swabs2O, discards swab after extruding.
2. the animal tissue of solid-like is first ground, and takes the solid-like sample 2mg after grinding, adds 20 μ l sterilizings
ddH2O。
3. concussion is mixed, quick to be vortexed 5 seconds, takes 1 μ l supernatants as template.
(3) Direct PCR reaction：
1. PCR reaction systems are 20 μ l, take the μ l of Sample supernatants 1 after treatment, add the μ l of PCR amplifing reagents 19, are fully mixed
It is even, while setting positive and negative control.
2. PCR amplification programs are 98 DEG C of 30s；Then 35 circulations：98 DEG C of 5s, 55 DEG C of 5s, 72 DEG C of 5s；Last 72 DEG C
1min；
(4) detection of PCR primer：5 μ l amplified productions are taken to be applied directly in the Ago-Gel containing ethidium bromide, while plus
Enter DNA Marker (2000bp) controls, voltage is 100V, electrophoresis 20min, and result is then observed in gel imaging instrument, if
Sample can amplify 1390bp bands, illustrate that sample is the positive, otherwise be feminine gender.Positive control also amplifies 1390bp bands,
Negative control does not have band.Sequence table is shown in into the sequencing of positive pcr amplification product, sequence, by comparing, with Mycoplasma bovis
HB0801-P180 plants of (Genbank no.CP007591) homology 100%.
The specificity of the kit of quick Direct PCR detection ox mycoplasma pneumoniae
By the Streptococcus suis of separated identification, haemophilus parasuis, actinobacillus pleuropneumoniae, Escherichia coli,
Pasteurella multocida, bordetella bacilli, salmonella culture, take single bacterium colony and are dissolved in 20 μ l ddH2O, takes 1 μ l and adds Direct PCR
Reaction system is expanded, and as a result as Fig. 2 does not have amplified band, the positive pathological material of disease only containing Mycoplasma bovis amplifies specific bar
Band, it was demonstrated that the present embodiment has preferable specificity.
The sensitivity of the kit of Direct PCR detection ox mycoplasma pneumoniae
1 μ l supernatants are taken after the sample treatment of collection as template, 10 times are diluted to 10 successively-5, 1 μ l are respectively taken as direct
PCR is expanded, and determines its sensitiveness.Such as Fig. 3, template dilution factor is 100、10-1、10-2、10-3、10-4When, it is clear to amplify
Band, it was demonstrated that the susceptibility of kit detection is 10-4。
The clinical practice of the kit of quick Direct PCR detection ox mycoplasma pneumoniae
The infected cattle that classical symptom occurs from clinic gathers 57 parts of nose swab, is processed according to the method for this kit, carries out straight
PCR is met, electrophoresis detection is as a result positive 32 parts, meanwhile, sample DNA is extracted, Standard PCR is carried out, it is positive 32 parts, with Direct PCR knot
Really one, such as Fig. 4, it was demonstrated that this kit and Standard PCR coincidence rate 100%, are adapted to the quick detection of clinical sample.
In addition to above-mentioned implementation, the present invention can also have other embodiment.All use equivalents or equivalent transformation are formed
Technical scheme, all fall within the protection domain of application claims.
Sequence table
Sample amplification sequence：CCTTAGGCAGTTTCTTTATAAACCGACTTCGGGCATTACCAGCTCCCATGGTTTGACGGG
CGGTGTGTACAAGACCCGAGAACGTATTCACCGTAGCGTAGCTGATCTACGATTACTAGCGATTCCGACTTCATGAA
GTCGAGTTGCAGACTTCAATCCGAACTGAGAACGGTTTTTTGAGGTTTGCTCCATGTCACCACTTCGCTTCTCTTTG
TACCGTCCATTGTAGCACGTGTGTAGCCCCACTCGTAAGAGGCATGATGATTTGACGTCGTCCCCACCTTCCTCCCG
ATTACTCGGGCAGTCTCCTTAGAGTGCTCAACTAAATGGTAGTAACTAAGGATAAGGGTTGCGCTCGTTGCAGGACT
TAACCGAACATCTCACGACACGAGCTGACGACAACCATGCACCATCTGTCATTCTGTTAACCTCCACTATGTCTCCA
TAGCTTTGCAGAAGATGTCAAGAGTGGGTAAGGTTCTACGCGTAACATCAAATTAAACCACATGCTCCACCGCTTGT
GCGGATCCCCGTCAATTCCTTTAAGTTTTATTCTTGCGAACGTACTACTCAGGCGGATCATTTAATGCGTTAGCTGC
GTCGATGAGTTCCCCATCAACTAATGATCATCGTTTAGGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCC
ACGCTTTCGTCTCTCAGTGTCAGTGTATGCCCAGTTAGCTGCCTTCGCCATATTGATGTTCTTCCTTATATCTACGC
ATTTCACCGCTTCACAAGGAATTCCGCTAACCTCTACATAACTCTAGTCTGCCAGTATCCAACGCGTTTTGGGGTTG
AGCCCCAAAATTTAACGCCAGACTTAACAAACAACCTACAGACGCTTTACGCCCAATAATTTCGGATAACGCTTGCA
ACCTATGTATTACCGCGGCTGCTGGCACATAGTTAGCCGTTGCTTTCTAATCAGGTACCGTCAAGGTAGCATCATTT
CCTATGCTACTTTTTCTTCCCTAACCACAGCAGTTTACAACCCATAGGGCCTTCATCCTGCACGCTGTGTCGCTCCA
TCAGACTTTCGTCCATTGTGGAATATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTATCTCAGTCCCAGTGT
GGCGGATCAATCTCTCAACCCCGCTAAACATCATCGCCTTGGTGGGCCGTTACCTCACCAACTAGCTAATGTTGCGC
ACTCCGATCTTTTAGCGAAGCAAACGCTTCCTTTTATATTACTTTCATGCAAAAATAATAAGTATTCGGTATTATCG
GATGTTTCCATCCGCTATCCCAATCTAAAAGGTACGTTGAGTACGTGTTACTCACCCATTCGCCGCTATGATATTGC
TATCATCGCTCGACATGCATG
Primer sequence：PF:5’-CCTTAGGCAGTTTCTTTATAA-3’
PR: 5’-CATGCATGTCGAGCGATGAT-3’
Claims (7)
1. a kind of quick Direct PCR detects the kit of ox mycoplasma pneumoniae, comprising following reagent：
PCR amplifing reagents, positive control and negative control.
2. quick Direct PCR detects the kit of ox mycoplasma pneumoniae according to claim 1, it is characterised in that：The PCR
Amplifing reagent contains 2 × Direct PCR Mix, 10pM forward primer, 10pM reverse primers, 5U/ μ l Hot Strart Taq
Enzyme, sterilizing ddH2O。
3. quick Direct PCR detects the kit of ox mycoplasma pneumoniae according to claim 2, it is characterised in that：It is described just
It is PF to primer：5 '-CCTTAGGCAGTTTCTTTATAA-3 ', the reverse primer is PR：5’-
CATGCATGTCGAGCGATGAT-3’。
4. a kind of application of the kit of quick Direct PCR detection ox mycoplasma pneumoniae, comprises the following steps：
The collection of the first step, sample, is divided into pathological material of disease and Nasopharyngeal swabs collection, and the pathological material of disease is gathered to gather pathological material of disease under aseptic condition
10mg~100mg, is fitted into standby in the centrifuge tube without RNase pollution, and the Nasopharyngeal swabs collection is collection nasopharynx liquid, loads nothing
It is standby in the centrifuge tube of RNase pollution；
Second step, sample treatment, add 200 μ l sterilizings ddH in Nasopharyngeal swabs2O, discards swab after extruding, the solid-like after grinding
Sample 2mg, adds 20 μ l sterilizings ddH2O, concussion is mixed；It is quick to be vortexed 5 seconds, 1 μ l supernatants are taken as template；
3rd step, Direct PCR reaction, reaction system is 20 μ l, takes the μ l of Sample supernatants 1 after treatment, adds PCR amplifing reagents 19
μ l, fully mix, and positive control and negative control take 1 μ l and add 20 μ l PCR amplifing reagents respectively, and PCR amplification programs are 98 DEG C
30s；Then 35 circulations：98 DEG C of 5s, 55 DEG C of 5s, 72 DEG C of 5s；Last 72 DEG C of 1min；
4th step, the detection of PCR primer, take 5 μ l amplified productions and are applied directly in the Ago-Gel containing ethidium bromide, while plus
Enter DNA Marker (2000bp) controls, voltage is 100V, electrophoresis 20min, and result is then observed in gel imaging instrument, if
Sample can amplify 1389bp bands, illustrate that sample is the positive, and positive control also amplifies 1390bp bands, and negative control does not have
Band.
5. the application of the kit of quick Direct PCR detection ox mycoplasma pneumoniae according to claim 4, it is characterised in that：
Reaction system in 3rd step is 20 μ l, μ l, the 10pmol/ μ l of forward primer 1 of μ l, the 10pmol/ μ l of sample 1 after treatment
The μ l of 1 μ l, 2 × Direct PCR Mix of reverse primer 10, the μ l of 5U/ μ l Hot Strart Taq enzymes 0.5, plus sterilizing ddH2O
To 20 μ l, fully mix.
6. the application of the kit of ox mycoplasma pneumoniae Direct PCR detection is used for according to claim 5, it is characterised in that：
Amplification length is 1390bp.
7. the application of the kit of quick Direct PCR detection ox mycoplasma pneumoniae according to claim 4, it is characterised in that：
Gel strength in 4th step during agarose gel electrophoresis is 1.0%, wherein containing 1% gel stain.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710089520.8A CN106811527A (en) | 2017-02-20 | 2017-02-20 | A kind of kit of quick Direct PCR detection ox mycoplasma pneumoniae and its application |
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| CN201710089520.8A CN106811527A (en) | 2017-02-20 | 2017-02-20 | A kind of kit of quick Direct PCR detection ox mycoplasma pneumoniae and its application |
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Citations (3)
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