CN107012214A - A kind of method of mycoplasma in quick detection milk frozen cattle semens - Google Patents
A kind of method of mycoplasma in quick detection milk frozen cattle semens Download PDFInfo
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- CN107012214A CN107012214A CN201710191035.1A CN201710191035A CN107012214A CN 107012214 A CN107012214 A CN 107012214A CN 201710191035 A CN201710191035 A CN 201710191035A CN 107012214 A CN107012214 A CN 107012214A
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- mycoplasma
- quick detection
- frozen cattle
- cattle semens
- milk frozen
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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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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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
Abstract
The invention provides a kind of method of mycoplasma in quick detection milk frozen cattle semens, it is related to mycoplasma infection detection method technical field in livestock body, it is by the milk frozen cattle semens centrifugal treating after defrosting, DNA is sequentially added into supernatant and extracts lysate and chloroform, extraction obtains DNA, then enter performing PCR amplification, finally detect using agarose gel electrophoresis method and observe result；The detection method sets up the rapid technology based on milk Mycoplasma bovis detection of nucleic acids, can obtain result in 3 hours, greatly improve detection efficiency by using PCR method, is mycoplasma infection diseases treatment and prevents the further diffusion of mycoplasma from winning the time.
Description
Technical field
The present invention relates to mycoplasma infection detection method technical field in livestock body, and in particular to a kind of quick detection milk cow
Freeze the method for mycoplasma in essence.
Background technology
Frozen semen semipermanent preserve (- 196 DEG C) under the conditions of, often can also extend bacterium, virus, mycoplasma,
The time-to-live of the organisms such as worm's ovum.Many pathogenic microorganisms can be propagated by seminal fluid, and the energy that seminal fluid disseminates infection
Amount is very big, and some infectious diseases may be disseminated to global several continents by the frozen semen of l headbands disease bull within the several years.
Available data shows:Some cattle diseases propagated by frozen semen mainly have:Aftosa, perlsucht johne's disease, ox Bu Shi
Bacillosis, trichomoniasis, ox reproductive organs vibriosis, various leptospirosis, mycoplasmosis, ox infectious rhinotracheitis
Inflammation, infectiousness fester balanoposthitis, bovine viral diarrhoea and parainfluenza, fibropapilloma disease, blue tongue disease, bovine leucosis
Deng.
Mycoplasma is the minimum microorganism that a class can be bred in without living cells culture medium, and acellular wall, reproductive forms thereof are more
Sample, with height polymorphy.Most of mycoplasma is all that the mucous membrane of host specificity is normal in bacterium, and main parasitic is in nasal cavity, secondly
In mammary gland.1976 first report Mycoplasma bovis it is relevant with ox respiratory disease, nineteen eighty-three China Li Ji Shen report first from
Suffer from ox in mastitis cow's milk and be separated to Mycoplasma bovis.
At present, it is main logical although being separately cultured for Mycoplasma bovis identifies and can made a definite diagnosis that complex operation, required condition is high
Culture Mycoplasma and physio-biochemical characteristics identification are crossed, these methods consuming time is long, generally requires 3 days or so, is unfavorable for branch former
The quick diagnosis of body infectious disease.
The content of the invention
(1) technical problem solved
In view of the shortcomings of the prior art, the invention provides a kind of method of mycoplasma in quick detection milk frozen cattle semens, make
Obtain detection time to greatly shorten, greatly improve detection efficiency.
(2) technical scheme
To realize object above, the present invention is achieved by the following technical programs：
A kind of method of mycoplasma in quick detection milk frozen cattle semens, step is as follows：(1) milk frozen cattle semens are thawed at normal temperatures
Take 50uL to be placed in 1.5mL centrifuge tubes after 2-3min, defrosting, 5min is centrifuged under 12000r/min rotating speeds；(2) by after centrifugation
Supernatant is abandoned, and is added 0.5mL DNA and is extracted lysate, stands 5min；(3) 100uL chloroforms are continuously added after standing, mixing is equal
It is even, 3min is centrifuged under 12000r/min rotating speeds, supernatant is moved on in silicagel column, 1min is centrifuged under 12000r/min rotating speeds；(4)
After centrifugation, 50uL distilled water eluted dnas are added；(5) take the DNA after 2uL elutions to be added in 18uL PCR mixed liquors, carry out
PCR reacts；(6) agarose gel electrophoresis detection method is selected, electrophoresis 20min observes result.
It is preferred that, the compound method that step (2) DNA extracts lysate is：Take 57.6g guanidine hydrochlorides, 50mL water saturations
Phenol, adds the solution that distilled water is configured to 100mL.
It is preferred that, step (5) PCR mixed liquors are formulated as：Take 2.5uM dNTP, 160uL primer 1,40uL primers
2nd, 40uL Taq archaeal dna polymerases, 40uL buffer solutions, add 640uL distilled water and are configured to 0.92mL mixed liquors.
It is preferred that, the primer 1 is 10uM TAATTTAGAAGCTTTAAATGAGCGC, and primer 2 is 40uM
CATATCTAGGTCAATTAAGGCTTTG。
It is preferred that, 1.5uM KCl, 2.0uM MgCl are contained in the buffer solution2, 3uM glycerine.
(3) beneficial effect
The invention provides a kind of method of mycoplasma in quick detection milk frozen cattle semens, by using PCR method, base is set up
In the rapid technology of milk Mycoplasma bovis detection of nucleic acids, result can be obtained in 3 hours, greatly improve detection efficiency, for branch
Further the time has been won in diffusion for pathogen infection disease treatment and prevention mycoplasma.
Brief description of the drawings
Fig. 1 is that milk cow semen DNA extracts result, and Fig. 2 is milk cow detection of mycoplasma result.
Embodiment
To make the purpose, technical scheme and advantage of the embodiment of the present invention clearer, below in conjunction with the embodiment of the present invention,
Technical scheme in the embodiment of the present invention is clearly and completely described, it is clear that described embodiment is the present invention one
Divide embodiment, rather than whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art are not making
The every other embodiment obtained under the premise of creative work, belongs to the scope of protection of the invention.
Embodiment：
A kind of method of mycoplasma in quick detection milk frozen cattle semens, the solution that this method is used is as follows：
DNA extracts lysate：57.6g guanidine hydrochlorides, 50mL water-saturated phenols are taken, distilled water is added and is configured to 100mL solution.
PCR mixed liquors：2.5uM dNTP, 160uL primer 1,40uL primer 2s, 40uL Taq archaeal dna polymerases, 40uL is taken to delay
Fliud flushing, adds 640uL distilled water and is configured to 0.92mL mixed liquors.Wherein, primer 1 is 10uM
TAATTTAGAAGCTTTAAATGAGCGC, primer 2 is 40uM CATATCTAGGTCAATTAAGGCTTTG, is contained in buffer solution
1.5uM KCl、2.0uM MgCl2, 3uM glycerine.
Detecting step is as follows：
(1) milk frozen cattle semens are thawed 3min at normal temperatures, takes 50uL to be placed in 1.5mL centrifuge tubes after defrosting, in 12000r/
5min is centrifuged under min rotating speeds；(2) supernatant after centrifugation is abandoned, adds 0.5mL DNA and extract lysate, stand 5min；
(3) 100uL chloroforms are continuously added after standing, is well mixed, is centrifuged 3min under 12000r/min rotating speeds, supernatant is moved on into silica gel
In post, 1min is centrifuged under 12000r/min rotating speeds；(4) after centrifuging, 50uL distilled water eluted dnas are added；(5) take after 2uL elutions
DNA be added in 18uL PCR mixed liquors, enter performing PCR reaction；(6) agarose gel electrophoresis detection method, electrophoresis are selected
20min, observes result.
Observe result：As shown in Fig. 2 there is the generation of mycoplasma product in explanation milk frozen cattle semens.
To sum up, the embodiment of the present invention has the advantages that：
, can be 3 by using PCR method the invention provides a kind of method of mycoplasma in quick detection milk frozen cattle semens
Result is obtained in hour, detection efficiency is greatly improved, is conducive to treating mycoplasma infection diseases in time.
It should be noted that herein, such as first and second or the like relational terms are used merely to a reality
Body or operation make a distinction with another entity or operation, and not necessarily require or imply these entities or deposited between operating
In any this actual relation or order.Moreover, term " comprising ", "comprising" or its any other variant are intended to
Nonexcludability is included, so that process, method, article or equipment including a series of key elements not only will including those
Element, but also other key elements including being not expressly set out, or also include being this process, method, article or equipment
Intrinsic key element.In the absence of more restrictions, the key element limited by sentence "including a ...", it is not excluded that
Also there is other identical element in process, method, article or equipment including the key element.
The above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；Although with reference to the foregoing embodiments
The present invention is described in detail, it will be understood by those within the art that：It still can be to foregoing each implementation
Technical scheme described in example is modified, or carries out equivalent substitution to which part technical characteristic；And these modification or
Replace, the essence of appropriate technical solution is departed from the spirit and scope of various embodiments of the present invention technical scheme.
Claims (5)
1. a kind of method of mycoplasma in quick detection milk frozen cattle semens, it is characterised in that step is as follows：(1) milk frozen cattle semens are existed
Thawed 2-3min under normal temperature, takes 50uL to be placed in 1.5mL centrifuge tubes after defrosting, and 5min is centrifuged under 12000r/min rotating speeds；(2)
Supernatant after centrifugation is abandoned, 0.5mL DNA is added and extracts lysate, stand 5min；(3) 100uL is continuously added after standing
Chloroform, is well mixed, centrifuges 3min under 12000r/min rotating speeds, supernatant is moved on in silicagel column, under 12000r/min rotating speeds
Centrifuge 1min；(4) after centrifuging, 50uL distilled water eluted dnas are added；(5) take the DNA after 2uL elutions to be added to 18uL PCR to mix
Close in liquid, enter performing PCR reaction；(6) agarose gel electrophoresis detection method is selected, electrophoresis 20min observes result.
2. the method for mycoplasma in quick detection milk frozen cattle semens as claimed in claim 1, it is characterised in that step (2) is described
DNA extract lysate compound method be：57.6g guanidine hydrochlorides, 50mL water-saturated phenols are taken, distilled water is added and is configured to 100mL's
Solution.
3. the method for mycoplasma in quick detection milk frozen cattle semens as claimed in claim 1, it is characterised in that step (5) is described
PCR mixed liquors are formulated as：2.5uM dNTP, 160uL primer 1,40uL primer 2s, 40uL Taq archaeal dna polymerases, 40uL is taken to delay
Fliud flushing, adds 640uL distilled water and is configured to 0.92mL mixed liquors.
4. the method for mycoplasma in quick detection milk frozen cattle semens as claimed in claim 3, it is characterised in that the primer 1 is
10uM TAATTTAGAAGCTTTAAATGAGCGC, primer 2 is 40uM CATATCTAGGTCAATTAAGGCTTTG.
5. the method for mycoplasma in quick detection milk frozen cattle semens as claimed in claim 3, it is characterised in that in the buffer solution
Contain 1.5uM KCl, 2.0uM MgCl2, 3uM glycerine.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710191035.1A CN107012214A (en) | 2017-03-28 | 2017-03-28 | A kind of method of mycoplasma in quick detection milk frozen cattle semens |
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| CN201710191035.1A CN107012214A (en) | 2017-03-28 | 2017-03-28 | A kind of method of mycoplasma in quick detection milk frozen cattle semens |
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| CN107012214A true CN107012214A (en) | 2017-08-04 |
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| CN201710191035.1A Pending CN107012214A (en) | 2017-03-28 | 2017-03-28 | A kind of method of mycoplasma in quick detection milk frozen cattle semens |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0436200A (en) * | 1990-05-31 | 1992-02-06 | Iatron Lab Inc | Method for detecting mycobacterium tuberculosis |
| WO2000070086A1 (en) * | 1999-05-14 | 2000-11-23 | Enterprise Ireland (Trading As Bioresearch Ireland) | Nucleic acid probe-based diagnostic assays for prokaryotic and eukaryotic organisms |
| CN101736085A (en) * | 2009-11-18 | 2010-06-16 | 华中农业大学 | Loop-mediated isothermal amplification detection method of mycoplasma bovis |
-
2017
- 2017-03-28 CN CN201710191035.1A patent/CN107012214A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0436200A (en) * | 1990-05-31 | 1992-02-06 | Iatron Lab Inc | Method for detecting mycobacterium tuberculosis |
| WO2000070086A1 (en) * | 1999-05-14 | 2000-11-23 | Enterprise Ireland (Trading As Bioresearch Ireland) | Nucleic acid probe-based diagnostic assays for prokaryotic and eukaryotic organisms |
| CN101736085A (en) * | 2009-11-18 | 2010-06-16 | 华中农业大学 | Loop-mediated isothermal amplification detection method of mycoplasma bovis |
Non-Patent Citations (8)
| Title |
|---|
| S.SUBRAMANIAM 等: "Species identification of Mycoplasma bovis and Mycoplasma agalactiae based on the uvrC genes by PCR", 《MOLECULAR AND CELLULAR PROBES》 * |
| YUMIKO HIGA 等: "An improved loop-mediated isothermal amplification assay for the detection of Mycoplasma bovis", 《THE JOURNAL OF VETERINARY MEDICAL SCIENCE》 * |
| ZHIDI BAI 等: "Development of a loop-mediated isothermal amplification assay for sensitive and rapid detection of Mycoplasma bovis", 《AFRICAN JOURNAL OF BIOTECHNOLOGY》 * |
| 尹伯元: "《标记免疫学》", 30 April 1998, 原子能出版社 * |
| 张申 等: "《分子生物学检验 新版》", 31 January 2017, 华中科技大学电子音像出版社 * |
| 彭清洁 等: "牛支原体引起奶牛乳房炎的诊断", 《中国奶牛》 * |
| 杜军 等: "《现代药学生物技术综合实验教程》", 31 December 2014, 中山大学出版社 * |
| 高金波: "《全国普通高等医学院校药学类专业十三五规划教材 分析化学》", 31 January 2016, 中国医药科技出版社 * |
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Application publication date: 20170804 |
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