CN103898006A - Goat source moraxella osloensis and specific segment for molecular identification of goat source moraxella osloensis as well as application of specific segment - Google Patents
Goat source moraxella osloensis and specific segment for molecular identification of goat source moraxella osloensis as well as application of specific segment Download PDFInfo
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- CN103898006A CN103898006A CN201410041790.8A CN201410041790A CN103898006A CN 103898006 A CN103898006 A CN 103898006A CN 201410041790 A CN201410041790 A CN 201410041790A CN 103898006 A CN103898006 A CN 103898006A
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Abstract
The invention belongs to the field of microbiology and in particular relates to goat source moraxella osloensis, a specific segment for molecular identification of the goat source moraxella osloensis, and a preparation method and application of the specific segment. The goat source moraxella osloensis has a biological collection number CCTCC (China Center for Type Culture Collection) M2014036 and is obtained by separating a festered part of the lung of a goat with pneumonia. A foundation is laid for further study on the physiological and biochemical characteristics of the goat source moraxella osloensis and the harm on animal bodies caused by the goat source moraxella osloensis. The preparation method of the specific segment for DNA (deoxyribonucleic acid) molecular identification of the goat source moraxella osloensis is simple, and the specific segment is obtained by amplifying a universal bacterial primer, can be used for quickly and accurately identifying goat source moraxella osloensis strains, and is good in specificity. The invention also relates to a kit for detecting the goat source moraxella osloensis, and the kit can be used for quickly screening the goat source moraxella osloensis.
Description
Technical field
The invention belongs to microbiology field, be specifically related to the specific fragment of goat source Moraxella osloensis and Molecular Identification thereof, also relate to preparation method and the application of described specific fragment.
Background technology
Moraxella osloensis (Moraxella osloensis) is that Henriksen found in Olso in 1967, this bacterium is the normal microflora on humans and animals mucous membrane, mainly in the time of Abwehrkraft des Koepers and immunizing power reduction, cause the infection of lung and urinary system aspect, may cause septicemia and other infection simultaneously.The detailed description of Moraxella osloensis is referring to website:
http://www.escience.gov.cn/MetaDataSiteMap/Crawler?resourceId=nongyewei_1544C0001000000596。
At present, research to Moraxella osloensis has made some progress, Zhao's californium is separated to a strain Moraxella osloensis from female's blood samples of patients, and study its resistance to penicillin, ceftazime, ceftriaxone, Ofloxacine USP 23, Ciprofloxacin, amikacin, Sulfamethoxazole Compound etc. [referring to document: isolate 1 strain Moraxella osloensis from blood, Chinese Journal of Nosocomiology, 2004,14(10): 1198].Lin Yanqing etc. detect Moraxella osloensis from nephrohydrosis, and it has been carried out to colony characteristics qualification [referring to document: detect Moraxella osloensis 1 example from nephrohydrosis, Chinese Journal of Nosocomiology, 2004,1(14): 14].At present, about Moraxella osloensis, the physiological function harm of goat be have not been reported.
Summary of the invention
In view of this, one of object of the present invention is to provide a kind of goat source Moraxella osloensis, for further studying from now on goat source Moraxella osloensis physio-biochemical characteristics and the harm of animal body being laid a good foundation; Specific fragment providing the Moraxella osloensis DNA molecular qualification of goat source and preparation method thereof is provided two of object of the present invention, this specific fragment can rapid detection, qualification goat source Moraxella osloensis, and described preparation method can specific amplification obtain the specific fragment of goat source Moraxella osloensis DNA molecular qualification; Three of object of the present invention is to provide the application of the specific fragment of goat source Moraxella osloensis DNA molecular qualification, utilizes this specific fragment can Rapid identification goat source Moraxella osloensis; Four of object of the present invention is to provide the test kit that detects goat source Moraxella osloensis, and this test kit can rapid screening goat source Moraxella osloensis.
For achieving the above object, technical scheme of the present invention is:
Goat source Moraxella osloensis, its biological deposit number is CCTCC M2014036.
Biological preservation information explanation:
Be deposited in Chinese Typical Representative culture collection center on January 22nd, 2014, address is loujia hill belongs Wuhan University Chinese Typical Representative culture collection center in the school, deposit number is CCTCC M2014036, and Classification And Nomenclature is Morexella osloensis FLZ-0127 (Moraxella osloensis FLZ-0127).
Biological deposit number is the goat source Moraxella osloensis of CCTCC M2014036, is to separate and obtain from goat pneumonia case lungs suppuration position.
Further, described goat source Moraxella osloensis, described separation is to adopt aseptic streak inoculation to blood nutrient agar flat board at the lungs position of suppurating, 37 DEG C of constant temperature culture 24-48h separate and obtain pathogenic bacteria.
The specific fragment of goat source Moraxella osloensis DNA molecular qualification, sequence is as shown in SEQ ID NO:1.
The preparation method of the specific fragment of goat source Moraxella osloensis DNA molecular qualification, comprises the step of carrying out as follows:
(1) get the goat source Moraxella osloensis described in claim 2, extract genomic dna, prepare template DNA;
(2) get prepared template DNA in step (1), adopt the increase 16S rDNA of described goat source Moraxella osloensis of bacterium universal primer, obtain the specific fragment of goat source Moraxella osloensis DNA molecular qualification; Described universal primer is: upstream primer F27, and sequence is as shown in SEQ ID NO:2; Downstream primer R1492, sequence is as shown in SEQ ID NO:3.
Further, described preparation method, in described step (2), adopt bacterium universal primer to increase, PCR reaction system cumulative volume is 50 μ L, contains 2 × TaqPCR Master Mix25 μ L, the each 1 μ L of upstream and downstream primer, template DNA 2 μ L, surplus is water, described upstream and downstream primer concentration is 50 μ mol/L.
Further, described preparation method, described amplification for to increase in PCR instrument, and reaction conditions is 94 DEG C of denaturation 4min, 94 DEG C of sex change 45s, 55 DEG C of annealing 45s, 72 DEG C are extended 45s, 30 circulations, 72 DEG C are extended 10min, finish after reaction, and the PCR product of gained is in 4 DEG C of preservations; Adopt l0g/L agarose gel electrophoresis to detect described PCR product, PCR product sequence is as shown in SEQ ID NO:1.
The specific fragment of described goat source Moraxella osloensis DNA molecular qualification or the complementary sequence fragment of specific fragment be in the application detecting in the Moraxella osloensis of goat source, as specific molecular probe in detecting goat source Moraxella osloensis.
Detect the test kit of goat source Moraxella osloensis, comprise extracting genome DNA reagent and PCR reaction reagent, described extracting genome DNA reagent is to extract goat source Moraxella osloensis genomic dna, and described PCR reaction reagent comprises 2 × TaqPCR Master Mix, sequence upstream primer F27 and the downstream primer R1492 of sequence as shown in SEQ ID NO:3 as shown in SEQ ID NO:2; Described extracting genome DNA reagent is the genomic dna that extracts sample, described PCR reaction reagent is that the sample gene group DNA of gained is increased, obtaining amplified production is object fragment, and described PCR reaction reagent comprises 2 × TaqPCR Master Mix, sequence upstream primer F27 and the downstream primer R1492 of sequence as shown in SEQ ID NO:3 as shown in SEQ ID NO:2.
Detect the using method of the test kit of goat source Moraxella osloensis: get pathogenic bacteria sample to be checked, extract the genomic dna of pathogenic bacteria with extracting genome DNA reagent in test kit, prepare template DNA, then get template DNA, upstream primer F27, downstream primer R1492,2 × TaqPCR Master Mix and set up PCR reaction system and carry out pcr amplification, obtain amplified production; The amplified production of gained checks order, and sequence and the specific fragment of goat source Moraxella osloensis DNA molecular qualification or the complementary sequence fragment of specific fragment are compared, and then identify described pathogenic bacteria.
Beneficial effect of the present invention:
Goat of the present invention source Moraxella osloensis, for further studying goat source Moraxella osloensis physio-biochemical characteristics and the harm of animal body being laid a good foundation; The specific fragment of goat of the present invention source Moraxella osloensis DNA molecular qualification, its preparation method is simple, and described specific fragment can be identified goat source Moraxella osloensis bacterial strain fast and accurately, and specificity is good; The test kit of detection of the present invention goat source Moraxella osloensis, this test kit can rapid screening goat source Moraxella osloensis.
Brief description of the drawings
Fig. 1 bacterial isolate bacterium bacterium colony picture (10 times of object lens).
Fig. 2 gramstaining result.
Fig. 3 PCR product agarose gel electrophoresis result.
The phylogenetic tree shape figure (Moraxella oslooensis (#) be bacterial isolate bacterium) of Fig. 4 based on 16S rDNA sequence construct.
Fig. 5 PCR product sequencing result.
Embodiment
Illustrated embodiment is in order better content of the present invention to be described, but is not that content of the present invention only limits to illustrated embodiment.So those of ordinary skill in the art carry out nonessential improvement and adjustment according to foregoing invention content to embodiment, still belong to protection scope of the present invention.In order to make the object, technical solutions and advantages of the present invention clearer, below in conjunction with drawings and Examples, the invention will be further described.
The experimental technique of unreceipted actual conditions in preferred embodiment, conventionally according to normal condition, for example molecular cloning experiment guide (third edition, J. the work such as Pehanorm Brooker, Huang Peitang etc. translate, Science Press, 2002) described in condition, or the condition of advising according to manufacturer is carried out.The test materials that the present invention is used, if no special instructions, is commercially available purchase product.
In addition, in embodiment, the sequential write of related nucleotide sequence is defaulted as from 5 ' end to 3 ' end.
Screening and the qualification of embodiment 1 goat source Moraxella osloensis
1. reagent
Nutrient agar, purchased from Hangzhou microorganism reagent company limited;
Blood nutrient agar, is prepared by Chongqing Academy of Animal Sciences's veterinary institute.Method is as follows: sterilizing adds the triangular flask of granulated glass sphere, through jugular vein aseptic collection goat blood, and at the uniform velocity defiber sheep blood is made in concussion, is cooled to the ordinary nutrient agar substratum of 45-50 DEG C after adding sterilizing in 6% ratio, after shaking up gently, be sub-packed in while hot and in plate, make flat board;
The extraction of genomic dna adopts DNA extraction agent box, purchased from TIANGEN Biotech (Beijing) Co., Ltd.;
PCR reaction reagent 2xTaq PCR Master Mix is purchased from TIANGEN Biotech (Beijing) Co., Ltd.;
Sepharose reclaims test kit purchased from Dongsheng bio tech ltd, Guangzhou;
The pcr amplification primer of 16S rDNA is universal primer, and expection amplified fragments size is 1.5kb left and right, and by Dalian, precious biotechnology company limited is synthetic.
Primer sequence is: F27:5'-AGAGTTTGATCCTGG-CTCAG-3';
R1492：5'-ACGGCTACCTTGTTACGACTT-3'。
Electrophoresis standard substance DNA Marker, after electrophoresis, its stripe size is followed successively by 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp.
2. screening
Goat is the pneumonia case of Chongqing City's sheep field censorship, censorship pneumonia case goat is dissected in laboratory, aseptic line is carried out in lungs suppuration position to be inoculated into respectively on plain agar flat board and blood nutrient agar flat board, in 37 DEG C of constant temperature culture, in this process, will examine the growing state of bacterium, phenomenon shows, the bacterial strain being separated to is grown very slow on plain agar flat board, after 96h, still do not observe bacterium colony, illustrate that this bacterium is higher to nutritional needs; On blood nutrient agar, grow after 48h, can be observed diameter is the viscosity bacterium colony of 2.0～2.5mm, as shown in Figure 1.The pathogenic bacteria that separation is obtained carries out purifying cultivation at LB substratum, the purifying pathogenic bacteria bacterial strain obtaining is carried out to gramstaining, microscopic examination result as shown in Figure 2, the bacterial strain that purifying is obtained carries out finding that this bacterium is Gram-negative bacteria after gramstaining, be shaft-like, short and wide, closely oval, grow up to or be short chain shape.
3. Molecular Identification
The bacterium liquid of getting pure culture in 1.5mL step 2 is centrifugal, extracts genome by DNA extraction agent box specification sheets.What pcr amplification was selected is bacterium universal primer, in PCR reaction system is: 2 × TaqPCR Master Mix25 μ L, the each 1 μ L(50 μ mol/L of upstream and downstream primer), template DNA 2 μ L, add ddH
2o to 50 μ L increases in PCR instrument.Reaction conditions is: 94 DEG C of denaturation 4min, and 94 DEG C of sex change 45s, 55 DEG C of annealing 45s, 72 DEG C are extended 45s, 30 circulations, 72 DEG C are extended 10min, finish the rear 4 DEG C of preservations of reaction.PCR product adopts l0g/L agarose gel electrophoresis to detect, and electrophoresis result as shown in Figure 3.
Associated nucleic acid order known in the pathogenic bacteria bacterial strain 16S rDNA sequence of separation and purification and GenBank is analysed and compared, and utilize biosoftware MEGA5 constructing system evo-devo tree.
The 16SrDNA sequence of this bacterium is carried out to sequence alignment at GenBank, find that institute's calling sequence and Moraxella osloensis (Moraxella osloensis logs in and is numbered JN084136) homology are up to 99.9%.The representative strain 16S rDNA of 4 kinds of Pseudomonas such as institute's calling sequence and the higher eisseria (Neisseria) of its dependency, Moraxella (Moraxella), Lampropedia (Lampropedia) and paracoccus (Paracoccus) is carried out to sequence alignment analysis, each representative strain relevant information is as table 1, and utilize biosoftware MEGA5 constructing system to grow evolutionary tree, phylogenetic evolution result is as shown in Figure 4.
The representative strain of table 1 phylogenetic tree and 16rDNA sequence information table
Can find out from table 1 and Fig. 4, the comparison of the homology based on 16S rDNA sequence shows, the pathogenic bacteria being separated to and the homology of each representative strain are all higher, wherein with bacterial strain JN084136 homology up to 99.9%; Grow evolutionary tree by constructing system, can find that the representative strain of testing isolated strains and Moraxella gathers for cluster, Neisseria and Lampropedia are positioned at another branch, the bacterial strain nearest with experiment isolated strains evolutionary distance is Moraxella osloensis (JN084136), can determine that thus this isolate is Moraxella osloensis.
The purifying of 4.PCR product and order-checking
Utilize universal primer this pathogenic bacteria to be carried out to the amplification of 16SrDNA, PCR product adopts l0g/L agarose gel electrophoresis to detect, consistent with expected results; The PCR product conforming to expection object stripe size, after glue reclaims test kit purifying, is sent to Dalian precious biotechnology company limited and carries out DNA sequencing, obtain the sequencing result of amplified production as shown in Figure 5.
The success of Moraxella osloensis bacterial strain is separated into further to be studied from now on its physio-biochemical characteristics and the harm of animal body is laid a good foundation.
The preparation of the specific fragment of embodiment 2 goat source Moraxella osloensis DNA molecular qualifications
The specific fragment of goat of the present invention source Moraxella osloensis DNA molecular qualification, can be used as specific probe rapid detection goat source Moraxella osloensis, for foundation and this sick study on prevention of the PCR fast diagnosis method of Moraxella osloensis pneumonia provide the foundation from now on.
Finally explanation is, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although the present invention is had been described in detail with reference to preferred embodiment, those of ordinary skill in the art is to be understood that, can modify or be equal to replacement technical scheme of the present invention, and not departing from aim and the scope of technical solution of the present invention, it all should be encompassed in the middle of claim scope of the present invention.
Claims (9)
1. goat source Moraxella osloensis, is characterized in that, biological deposit number is CCTCC M2014036.
2. biological deposit number is the goat source Moraxella osloensis of CCTCC M2014036, it is characterized in that, is to separate and obtain from goat pneumonia case lungs suppuration position.
3. goat according to claim 2 source Moraxella osloensis, is characterized in that, described separation is to adopt aseptic streak inoculation to the clear agar plate of blood at the lungs position of suppurating, and 37 DEG C of constant temperature culture 24-48h separate and obtain pathogenic bacteria.
4. the specific fragment of goat source Moraxella osloensis DNA molecular qualification, sequence is as shown in SEQ ID NO:1.
5. the preparation method of the specific fragment of goat source Moraxella osloensis DNA molecular qualification, is characterized in that, comprises the step of carrying out as follows:
(1) get the goat source Moraxella osloensis described in claim 2, extract genomic dna, prepare template DNA;
(2) get prepared template DNA in step (1), adopt the increase 16S rDNA of described goat source Moraxella osloensis of bacterium universal primer, obtain the specific fragment of goat source Moraxella osloensis DNA molecular qualification; Described universal primer is: upstream primer F27, and sequence is as shown in SEQ ID NO:2; Downstream primer R1492, sequence is as shown in SEQ ID NO:3.
6. preparation method according to claim 5, it is characterized in that, in described step (2), adopt bacterium universal primer to increase, PCR reaction system cumulative volume is 50 μ L, contains 2 × TaqPCR Master Mix25 μ L, the each 1 μ L of upstream and downstream primer, template DNA 2 μ L, surplus is water, described upstream and downstream primer concentration is 50 μ mol/L.
7. preparation method according to claim 6, it is characterized in that, described amplification for to increase in PCR instrument, and reaction conditions is 94 DEG C of denaturation 4min, 94 DEG C of sex change 45s, 55 DEG C of annealing 45s, 72 DEG C are extended 45s, 30 circulations, and 72 DEG C are extended 10min, finish after reaction, the PCR product of gained is in 4 DEG C of preservations; Adopt l0g/L agarose gel electrophoresis to detect described PCR product, PCR product sequence is as shown in SEQ ID NO:1.
8. the specific fragment of goat claimed in claim 4 source Moraxella osloensis DNA molecular qualification or the complementary sequence fragment of specific fragment are in the application detecting in the Moraxella osloensis of goat source.
9. detect the test kit of goat source Moraxella osloensis, it is characterized in that, comprise extracting genome DNA reagent and PCR reaction reagent, described extracting genome DNA reagent is to extract goat source Moraxella osloensis genomic dna, and described PCR reaction reagent comprises 2 × TaqPCR Master Mix, sequence upstream primer F27 and the downstream primer R1492 of sequence as shown in SEQ ID NO:3 as shown in SEQ ID NO:2.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022121864A1 (en) * | 2020-12-08 | 2022-06-16 | 复旦大学 | Marker combination for skin typing and use thereof |
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| WO2013180302A1 (en) * | 2012-05-29 | 2013-12-05 | Kao Corporation | Method of evaluating inhibitory effect on damp-dry malodor |
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| US5262186A (en) * | 1991-06-07 | 1993-11-16 | Rhone Poulenc Specialty Chemicals Co. | Process for treating fish and shellfish to control bacterial contamination and/or growth |
| WO2013180302A1 (en) * | 2012-05-29 | 2013-12-05 | Kao Corporation | Method of evaluating inhibitory effect on damp-dry malodor |
Non-Patent Citations (2)
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| 张京文等: "从脓液、血培养中分离奥斯陆莫拉菌", 《中华医史杂志》, no. 02, 28 April 1996 (1996-04-28), pages 121 * |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2022121864A1 (en) * | 2020-12-08 | 2022-06-16 | 复旦大学 | Marker combination for skin typing and use thereof |
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Application publication date: 20140702 |