CN105755130A - Primer, kit and method for detecting swine chlamydiosis - Google Patents
Primer, kit and method for detecting swine chlamydiosis Download PDFInfo
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- CN105755130A CN105755130A CN201610209856.9A CN201610209856A CN105755130A CN 105755130 A CN105755130 A CN 105755130A CN 201610209856 A CN201610209856 A CN 201610209856A CN 105755130 A CN105755130 A CN 105755130A
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- 238000000034 method Methods 0.000 title claims abstract description 18
- 241000282898 Sus scrofa Species 0.000 title abstract description 29
- 238000012408 PCR amplification Methods 0.000 claims abstract description 10
- 238000001514 detection method Methods 0.000 claims description 15
- 238000012360 testing method Methods 0.000 claims description 15
- 230000004087 circulation Effects 0.000 claims description 3
- 238000004925 denaturation Methods 0.000 claims description 3
- 230000036425 denaturation Effects 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 241001647378 Chlamydia psittaci Species 0.000 abstract description 16
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 238000001962 electrophoresis Methods 0.000 abstract description 3
- 244000052769 pathogen Species 0.000 abstract description 3
- 230000002265 prevention Effects 0.000 abstract description 2
- 230000009182 swimming Effects 0.000 description 7
- 239000000203 mixture Substances 0.000 description 5
- 230000003321 amplification Effects 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 241000606748 Actinobacillus pleuropneumoniae Species 0.000 description 3
- 241000204045 Mycoplasma hyopneumoniae Species 0.000 description 3
- 241000579205 Mycoplasma suis Species 0.000 description 3
- 241000606856 Pasteurella multocida Species 0.000 description 3
- 241000194021 Streptococcus suis Species 0.000 description 3
- 241000223996 Toxoplasma Species 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 229940051027 pasteurella multocida Drugs 0.000 description 3
- 230000001717 pathogenic effect Effects 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 206010000234 Abortion spontaneous Diseases 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- 208000004232 Enteritis Diseases 0.000 description 2
- 238000002738 Giemsa staining Methods 0.000 description 2
- 206010036030 Polyarthritis Diseases 0.000 description 2
- 206010003246 arthritis Diseases 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 238000000386 microscopy Methods 0.000 description 2
- 208000015994 miscarriage Diseases 0.000 description 2
- 230000010355 oscillation Effects 0.000 description 2
- 208000030428 polyarticular arthritis Diseases 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 208000000995 spontaneous abortion Diseases 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000606161 Chlamydia Species 0.000 description 1
- 208000000362 Chlamydial Pneumonia Diseases 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 206010010741 Conjunctivitis Diseases 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical class CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 1
- 101710164702 Major outer membrane protein Proteins 0.000 description 1
- 108010079246 OMPA outer membrane proteins Proteins 0.000 description 1
- 208000006735 Periostitis Diseases 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 206010035673 Pneumonia chlamydial Diseases 0.000 description 1
- 208000006399 Premature Obstetric Labor Diseases 0.000 description 1
- 206010036600 Premature labour Diseases 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 210000002718 aborted fetus Anatomy 0.000 description 1
- 229960000583 acetic acid Drugs 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 210000003756 cervix mucus Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 210000003837 chick embryo Anatomy 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
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- 238000002474 experimental method Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 210000000281 joint capsule Anatomy 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L magnesium chloride Substances [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 201000011475 meningoencephalitis Diseases 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 208000008494 pericarditis Diseases 0.000 description 1
- 210000003460 periosteum Anatomy 0.000 description 1
- 208000030773 pneumonia caused by chlamydia Diseases 0.000 description 1
- 208000026440 premature labor Diseases 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 210000001550 testis Anatomy 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 210000001325 yolk sac Anatomy 0.000 description 1
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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Abstract
The invention discloses a primer, a kit and a method for detecting swine chlamydiosis. The primer is shown in SEQ ID No:1 and SEQ ID No.2, and the method comprises the step of carrying out PCR amplification reaction and electrophoretic analysis by taking a to-be-detected pig sample genome DNA as a template. The primer for detecting the swine chlamydiosis can specifically amplify chlamydia psittaci and is unresponsive to other common pathogens; the method has high sensitivity and can conveniently and quickly detect the chlamydia psittaci of various clinical pig samples, specific prevention and control measures can be beneficially rapidly taken, and operation is simple and practical.
Description
Technical field
The invention belongs to technical field of molecular biology, more particularly it relates to an the detection primer of porcine chlamydiosis, test kit and method.
Background technology
Chlamydia is the quasi-microorganism between antibacterial and virus, diameter 0.2 μm～1.5 μm, and it can only breed with a kind of developmental history inside host cell protoplasm.This developmental history is to become bigger reticulate body for feature with little substance, and reticulate body is bred with divisional mode, and reticulate body breaks after reaching maturity, and discharges substantial amounts of substance.Substance has infectivity, and new host cell is had no infectious by reticulate body.Chlamydia psittaci Gram’s staining is negative.The structure of cell wall is similar to other gram negative bacterias with composition, but only has trace or without 3-O-.alpha.-carboxyethyl-D-glucosamine..
The how symptomatic infectious disease of one that porcine chlamydiosis is mainly caused by chlamydia psittaci (Chlamydiapsittaci) infected pigs, clinical manifestation is the piglet that farrowing sow miscarriage, premature labor and output vitality are weak；Piglet pneumonia, enteritis, pericarditis, meningoencephalitis, polyarthritis and conjunctivitis；The symptoms such as Testis of Boar Pig is scorching.Wherein swine C.psittaci is miscarried and causes the chlamydial pneumonia-enteritis of piglet large quantities of death intensive industrialized piggery to be endangered serious.
The diagnostic method of porcine chlamydiosis mainly have Giemsa staining microscopy and chick embryo yolk sac inoculation carry out the methods such as pathogen separation at present.Giemsa staining microscopy method is big by subjective impact, and result error is big.And pathogen separation takes time and effort, separation rate is low.
Summary of the invention
Based on this, in order to overcome the defect of above-mentioned prior art, the invention provides the detection method of a kind of porcine chlamydiosis and the primer that this detection method uses, this primer can specific amplification chlamydia psittaci, quickly clinical sample is detected.
In order to realize foregoing invention purpose, this invention takes techniques below scheme:
A kind of primer detecting porcine chlamydiosis, described primer is such as shown in SEQIDNo:1 and SEQIDNo:2.
The application in the test kit of preparation detection porcine chlamydiosis of the above-mentioned primer.
A kind of test kit detecting porcine chlamydiosis, described test kit includes the primer of above-mentioned detection porcine chlamydiosis.
A kind of method detecting porcine chlamydiosis, adopts above-mentioned primer, comprises the following steps:
With pig sample genomic dna to be checked for template, carry out pcr amplification reaction, electroresis appraisal；The reaction system of described pcr amplification reaction is:
The response procedures of described pcr amplification reaction is: 94 DEG C of 5min carry out denaturation；Then 94 DEG C of 30s, 52 DEG C of 30s, 72 DEG C of 50s, carry out 35 circulations altogether；Last 72 DEG C extend 10min.
Compared with prior art, the present invention has following remarkable result:
1, the primer of the detection porcine chlamydiosis of the present invention can expand pig chlamydia psittaci specifically, and other encountered pathogenics such as pig toxoplasma, eperythrozoon suis, mycoplasma hyopneumoniae, pig pasteurella multocida, Streptococcus suis, actinobacillus pleuropneumoniae etc. is reactionless；
2, the method for the detection porcine chlamydiosis of the present invention has significantly high susceptiveness, sensitivity is up to about 25ng, easily and efficiently the pig chlamydia psittaci of various clinical pig samples can be detected, be conducive to rapid development prevention and control measure targetedly, simple to operate, practical.
Accompanying drawing explanation
Fig. 1 is porcine chlamydiosis clinical sample detection collection of illustrative plates in the embodiment of the present invention 1, and wherein swimming lane 1 is pig chlamydia psittaci positive control, and swimming lane 2 is pig chlamydia psittaci negative control, and swimming lane 3,4,5,6 is clinical sample；
Fig. 2 is the specific electrophoresis pattern of the PCR detection method of pig chlamydia psittaci in test example 1 of the present invention, wherein: swimming lane 1 is pig chlamydia psittaci positive, swimming lane 2 is pig chlamydia psittaci negative sample, swimming lane 3-8 respectively pig toxoplasma, eperythrozoon suis, mycoplasma hyopneumoniae, pig pasteurella multocida, Streptococcus suis, actinobacillus pleuropneumoniae sample；
Fig. 3 is the electrophoresis pattern of the sensitivity of the PCR detection method of porcine chlamydiosis in test example 2 of the present invention, wherein the DNA concentration of swimming lane 1,2,3,4,5,6,7 respectively 2.48 μ g, 0.248 μ g, 24.8ng, 2.48ng, 0.248ng, 0.0248ng, 2.48pg.
Detailed description of the invention
The present invention is described in detail below in conjunction with the drawings and specific embodiments.
The experiment material related in following example if no special instructions, derives from commercially available, the routine operation that the operation adopted in following example is well known to the skilled person.
Embodiment 1 detects primer and the method for porcine chlamydiosis
1、Design of primers
According to NCBI pig chlamydia psittaci MOMP (majoroutermembraneprotein) gene order (accession number: the AB468956 logged in, KM609426, CP003790, EU856033, CP002807, CP002805, CP002586, FQ482149) compare, and devise pig chlamydia psittaci PCR primer, respectively F1, R1.
F1:TCCTTACAAGCCTTGCCTGTAGG(SEQIDNO:1)
R1:AGCGTATTGGAAYTCRGCTC(SEQIDNO:2)
Wherein, base code is annexed: Y=C/T, R=A/G
2、Detection method
Comprise the following steps:
(1), pig sample genomic dna to be checked extracts
Love is adopted to pursue progress biotechnology (Hangzhou) company limited DNA/RNA small volume of reagent box.The fresh aborted fetus lungs of 100mg (numbering 3), lymph node (numbering 4), miscarriage sow vaginal secretions (numbering 5), suffer from the joint capsule liquid (numbering 6) of polyarthritis piglet.
Adding 1mLPBS buffer to be fully ground ,-20 DEG C of multigelations 3 times, 8000rpm is centrifuged 10min, takes supernatant 200 μ L, proceeds in 1.5mL centrifuge tube.Add 200 μ LBufferV-L, vortex oscillation mix homogeneously, stand 5min.Adding 75 μ LBufferV-N, vortex oscillation mix homogeneously, 12000g is centrifuged 5min.Supernatant is transferred in new 2mL centrifuge tube, add 300 μ L isopropanols (containing 1% glacial acetic acid), 6-8 mixing of turned upside down.Being placed in 2ml centrifuge tube by preparing pipe, prepare in pipe by mixed liquor immigration, 6000g is centrifuged 1min.Abandoning filtrate, put back into preparing pipe in 2mL centrifuge tube, add 500 μ LBufferW1A, room temperature stands 1min.12000g is centrifuged 1min.Abandon filtrate, put back into preparing pipe in 2mL centrifuge tube, add the centrifugal 1min of 800 μ LBufferW2,12000g.Putting back in 2mL centrifuge tube by preparing pipe, 12000g is centrifuged 1min.Being placed in preparing pipe in the 1.5mL centrifuge tube of cleaning, add 40 μ LBufferTE preparing periosteum central authorities, room temperature stands 1min.12000g is centrifuged 1min, is centrifuged the solution got off and is genomic DNA template.
(2), pcr amplification
After PCR reactant liquor is added PCR reaction tube mix homogeneously, add genomic DNA template, PCR pipe is placed in PCR instrument and carries out cyclic amplification reaction；
PCR reaction system is as follows, wherein PremixEXTaq is the mixture of 2 times of concentration of DNAPolymerase, Buffer, dNTPMixture of PCR reaction, comprises DNAPolymerase1.25U/25 μ L, Buffer (Tris-HCl, pH8.320mM, KCl100mM, MgCl23mM), each 0.4mM of dNTPMixture:
The response procedures of described pcr amplification reaction is: 94 DEG C of 5min carry out denaturation；Then 94 DEG C of 30s, 52 DEG C of 30s, 72 DEG C of 50s, carry out 35 circulations altogether；Last 72 DEG C extend 10min.
(3), electroresis appraisal
5 μ L product are taken after pcr amplification, the agarose gel of 1% (mass ratio) carries out electroresis appraisal, there is 609bp band in PCR primer, it is pig chlamydia psittaci positive, without band appearance is negative, the pig pattern detection result of the present embodiment is shown in Fig. 1, pig sample 6 test positive；Pig sample 3,4,5 detection is feminine gender.
Test example 1 specific test
Specific detection is carried out with pig toxoplasma, eperythrozoon suis, mycoplasma hyopneumoniae, pig pasteurella multocida, Streptococcus suis, actinobacillus pleuropneumoniae sample.Using the method provided in embodiment 1 and primer to detect, result shows, does not all have amplification curve except positive controls, and amplification is shown in Fig. 2.
Test example 2 sensitivity test
It is the gradient dilution of 10 times, Genome DNA content respectively 2.48 μ g, 0.248 μ g, 24.8ng, 2.48ng, 0.248ng, 0.0248ng, 2.48pg with the genomic templates sterile purified water of preparation in embodiment 1.
Each dilution factor respectively takes 1 μ L as template, detects by embodiment 1 method, observes positive band, and the most high dilution to there is the positive expection template used amount of band calculates its sensitivity, and result shows that minimum detectable activity is 24.8ng, sees Fig. 3.
Embodiment described above only have expressed the several embodiments of the present invention, and it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that, for the person of ordinary skill of the art, without departing from the inventive concept of the premise, it is also possible to making some deformation and improvement, these broadly fall into protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (4)
1. the primer detecting porcine chlamydiosis, it is characterised in that described primer is such as shown in SEQIDNo:1 and SEQIDNo:2.
2. the application in the test kit of preparation detection porcine chlamydiosis of the primer described in claim 1.
3. the test kit detecting porcine chlamydiosis, it is characterised in that described test kit includes the primer of the detection porcine chlamydiosis described in claim 1.
4. the method detecting porcine chlamydiosis, it is characterised in that adopt the primer as shown in SEQIDNo:1 and SEQIDNo:2, comprise the following steps:
With pig sample genomic dna to be checked for template, carry out pcr amplification reaction, electroresis appraisal；The reaction system of described pcr amplification reaction is:
The response procedures of described pcr amplification reaction is: 94 DEG C of 5min carry out denaturation；Then 94 DEG C of 30s, 52 DEG C of 30s, 72 DEG C of 50s, carry out 35 circulations altogether；Last 72 DEG C extend 10min.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107937576A (en) * | 2017-11-27 | 2018-04-20 | 咸阳职业技术学院 | One breeding pigeon Chlamydia PCR diagnostic kits and its detection method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101608239A (en) * | 2009-03-26 | 2009-12-23 | 重庆大学 | Animal chlamydia multiple sleeve type PCR detection kit and detection method thereof |
| UA110546C2 (en) * | 2014-04-22 | 2016-01-12 | Ігор Миколайович Ксьонз | Method for the detection of dna of bacteria chlamydia abortus, chlamydia pecorum, chlamydia psittaci in polymerase chain reaction by amplification of gene fragment encoding endoribonuclease p (rnase p rna) |
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- 2016-04-06 CN CN201610209856.9A patent/CN105755130A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101608239A (en) * | 2009-03-26 | 2009-12-23 | 重庆大学 | Animal chlamydia multiple sleeve type PCR detection kit and detection method thereof |
| UA110546C2 (en) * | 2014-04-22 | 2016-01-12 | Ігор Миколайович Ксьонз | Method for the detection of dna of bacteria chlamydia abortus, chlamydia pecorum, chlamydia psittaci in polymerase chain reaction by amplification of gene fragment encoding endoribonuclease p (rnase p rna) |
Non-Patent Citations (1)
| Title |
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| 王建忠: "鹦鹉热衣原体Taqman实时荧光PCR检测方法的建立", 《中国优秀硕士学位论文全文数据库农业科技辑》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107937576A (en) * | 2017-11-27 | 2018-04-20 | 咸阳职业技术学院 | One breeding pigeon Chlamydia PCR diagnostic kits and its detection method |
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Address after: 527400 9 East Causeway North Road, Xinxing County new town, Yunfu, Guangdong Applicant after: Winson food group Limited by Share Ltd Address before: 527400 9 East Causeway North Road, Xinxing County new town, Yunfu, Guangdong Applicant before: Guangdong Wens Foodstuff Group Co., Ltd. |
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