發(fā)布時(shí)間：2018-05-01 18:43

  本文選題：申克孢子絲菌 + 隨機(jī)擴(kuò)增DNA多態(tài)性��； 參考：《中國(guó)醫(yī)科大學(xué)》2007年碩士論文

【摘要】： 目的 應(yīng)用RAPD方法對(duì)36株mtDNA-4型申克孢子絲菌的基因組DNA進(jìn)行多態(tài)性分析，比較此型內(nèi)部菌株間的差異；通過(guò)對(duì)24株mtDNA1～24型申克孢子絲菌代表菌株的基因組DNA多態(tài)性分析，初步探討mtDNA分型和RAPD分型之間的關(guān)系。 材料和方法 一、實(shí)驗(yàn)材料 實(shí)驗(yàn)菌株：36株來(lái)自中國(guó)北方地區(qū)的申克孢子絲菌臨床分離菌株，為mtDNA-4型。24株來(lái)自不同國(guó)家、地區(qū)申克孢子絲菌代表菌株，為mtDNA1～24型。 二、實(shí)驗(yàn)試劑 1、基因組DNA提取主要試劑 (1)基因組DNA提取液 2％(W／V)CTAB(hexadecyltrimethylammonium bromide十六烷基三甲基溴化銨) (2) Tris飽和酚 (3)氯仿 (4)異戊醇 (5) 0.5mol／L乙酸銨 (6) 99％乙醇 (7) 75％乙醇 (8) TE(trisaminomethane-ethylenediaminetetraacetic acid) 10 mmol／L Tris.Cl(ph8.0) 1mmol／L EDTA(ph8.0) 2、PCR主要試劑 (1)真菌隨機(jī)引物 OPBG01 5′-GTGGCTCTCC-3′ OPBG14 5′-GACCAGCCCA-3′ OPBG19 5′-GGTCTCGCTC-3′ OPB07 5′-GGTGACGCAG-3′ OPAA11 5′-ACCCGACCTG-3′ OPD18 5′-GAGAGCCAAC-3′ OP02 5′-(GACA)_4-3′ OP08 5′-TGCCGAGCTG-3′ (2) 5U／ul TaKaRa Taq(DNA聚合酶) 三、主要實(shí)驗(yàn)儀器 (1)蒸汽高壓消毒箱(美國(guó)Tuttnauer 2540MK型) (2) PH4000AB型電熱恒溫培養(yǎng)箱(天津市泰斯特儀器有限公司) (3)紫外凝膠成像系統(tǒng)(英國(guó)Gel worker eD，UVP GDS 8000) (4)全能型高性能臺(tái)式冷凍離心機(jī)(德國(guó)Heaeus，Biofuge Stratos) (5) 05RP-22離心機(jī)(Hitachi) (6) HZO-X100震蕩培養(yǎng)箱(哈爾濱市東聯(lián)電子開發(fā)有限公司) (7)電泳儀(北京六一儀器廠，DYY-Ⅲ2型) (8)電泳槽(北京六一儀器廠，DYY-Ⅲ2型) (9) PCR擴(kuò)增儀(德國(guó)Biometra，T-Gradient) 四、實(shí)驗(yàn)方法 1、菌種基因組DNA的提取 采用CTAB法提取36株來(lái)自我國(guó)北方地區(qū)的臨床分離菌株(mtDNA-4型)及24株來(lái)自不同國(guó)家地區(qū)的保存菌株(mtDNA1～24型)的基因組DNA，調(diào)整濃度至100ng／ul以備PCR擴(kuò)增。 2、PCR擴(kuò)增 (1)利用8個(gè)隨機(jī)引物分別進(jìn)行擴(kuò)增，篩選出對(duì)所有的菌株具有良好擴(kuò)增片段的引物OPB07、OPBG14、OP02進(jìn)行正式實(shí)驗(yàn)，其中應(yīng)用OP02對(duì)mtDNA1～24型菌株進(jìn)行擴(kuò)增。 (2) PCR反應(yīng)體系：總體積為25ul 引物(100pmol／ul) 1ul TaKaRaTaq(5U／ul) 0.25ul 10×buffer(含MgCl_2) 4ul dNTP(dATP、dCTP、dGTP和dTTP各2.5mmol／L) 4ul template DNA(100ng／ul) 1ul (3) PCR反應(yīng)條件： 94℃5min預(yù)變性； 45個(gè)循環(huán)：94℃1min變性、36℃1min復(fù)性、72℃1min延伸； 后延伸72℃7min。 (4)結(jié)果檢測(cè)PCR產(chǎn)物在質(zhì)量分?jǐn)?shù)為1.5％的瓊脂糖凝膠上80伏特電壓下電泳并在紫外透射儀下觀察并用凝膠一次成像儀照相。 3、數(shù)據(jù)處理 (1)數(shù)據(jù)轉(zhuǎn)換首先用PCR Marker作為分子量標(biāo)記，確定各反應(yīng)條帶在凝膠成像上的相對(duì)位置。在同一分子量水平上，有反應(yīng)條帶記作“1”，無(wú)反應(yīng)條帶記作“0”。將所有菌株的反應(yīng)條帶都轉(zhuǎn)換為“1”，“0”組成的數(shù)據(jù)組。 (2)單匹配系數(shù)SM的計(jì)算公式為SM=2xnxy／(nx+ny)×100％，nxy：兩菌株共有的DNA條帶數(shù)，nx：x菌株的DNA條帶數(shù)，ny：y菌株的DNA條帶數(shù)。 (3)聚類分析統(tǒng)計(jì)學(xué)處理根據(jù)PCR產(chǎn)物帶型，用NTSYS-PC2.02版軟件包中的UPGMA聚類分析法進(jìn)行單匹配系數(shù)SM的計(jì)算和樹狀圖自動(dòng)生成。 結(jié)果 1、mtDNA-4型 (1)引物OPB07對(duì)36株mtDNA-4型的申克孢子絲菌臨床分離菌株P(guān)CR擴(kuò)增后，可以將其分成2個(gè)電泳帶型。 (2)引物OPBG14可以將36株mtDNA-4型的申克孢子絲菌臨床分離菌株分成4個(gè)電泳帶型。 (3)引物OP02可以將將36株mtDNA-4型的申克孢子絲菌臨床分離菌株分為3個(gè)電泳帶型。 2、mtDNA 1～24型(引物OP02) 引物OP02對(duì)來(lái)自不同國(guó)家地區(qū)的mtDNA 1～24型的代表菌株進(jìn)行PCR擴(kuò)增，B群菌株(mtDNA4～10、12、13、20、21和24型)株間差異較小，其中4型與6型，7型與20型，8、9、10、13與21型帶型完全相同，5型，12型及24型帶型與其它皆不相同。A群菌株(mtDNA 1～3、11、14～19、22和23型)株間差異較大，，無(wú)帶型完全相同菌株。 3、聚類分析結(jié)果 36株菌的相似系數(shù)在0.87～1.0之間，表明mtDNA-4型菌株之間存在較小的遺傳差異，在SM=0.92相似水平上36株mtDNA-4型菌可分為4個(gè)亞型。在樹狀圖SM=0.57相似水平上mtDNA 1～24型也可分成兩群，Ⅰ群包括型1～3、11、14～19和23，Ⅱ群包括型4～10、12、13、20～22和24，與mtDNA分型的A、B群僅有型22的差異。 結(jié)論 應(yīng)用RAPD方法可對(duì)mtDNA-4型的申克孢子絲菌進(jìn)一步分為4個(gè)亞型，為孢子絲菌病的分子流行病學(xué)研究提供依據(jù)：初步提示RAPD分型與mtDNA分型之間具有一定的一致性。
[Abstract]:objective
The genomic DNA of 36 strains of mtDNA-4 type sporospore spsporum was analyzed by RAPD method, and the differences among the strains were compared. The relationship between the mtDNA genotyping and the RAPD typing was preliminarily discussed by analyzing the genomic DNA polymorphism of 24 strains of mtDNA1 ~ 24.
Materials and methods
First, experimental materials
Experimental strains: 36 strains of spores shinshinus from northern China, mtDNA-4.24 strains from different countries, the representative strains of spidium spore from different countries, mtDNA1 to 24.
Two, experimental reagents
1, genomic DNA extracts the main reagents
(1) genomic DNA extract
2% (W / V) CTAB (hexadecyltrimethylammonium bromide sixteen alkyl three methyl ammonium bromide)
(2) Tris saturated phenol
(3) chloroform
(4) isoamyl alcohol
(5) 0.5mol / L ammonium acetate
(6) 99% ethanol
(7) 75% ethanol
(8) TE (trisaminomethane-ethylenediaminetetraacetic acid)
10 mmol / L Tris.Cl (ph8.0)
1mmol / L EDTA (ph8.0)
2, PCR major reagents
(1) random primers of fungi
OPBG01 5 '-GTGGCTCTCC-3'
OPBG14 5 '-GACCAGCCCA-3'
OPBG19 5 '-GGTCTCGCTC-3'
OPB07 5 '-GGTGACGCAG-3'
OPAA11 5 '-ACCCGACCTG-3'
OPD18 5 '-GAGAGCCAAC-3'
OP02 5 '- (GACA) _4-3'
OP08 5 '-TGCCGAGCTG-3'
(2) 5U / UL TaKaRa Taq (DNA polymerase)
Three, the main experimental instruments
(1) steam high pressure sterilizer (American Tuttnauer 2540MK)
(2) PH4000AB type electrothermal incubator (Tianjin TST Instrument Co., Ltd.)
(3) UV Gel imaging system (Gel worker eD, UVP GDS 8000)
(4) all-purpose high performance desktop refrigerated centrifuge (Heaeus, Biofuge Stratos, Germany)
(5) 05RP-22 centrifuge (Hitachi)
(6) HZO-X100 shock incubator (Harbin Dong Lian Electronic Development Co., Ltd.)
(7) electrophoretic apparatus (Beijing 61 instrument factory, DYY- III 2)
(8) electrophoretic trough (Beijing 61 instrument factory, DYY- III 2)
(9) PCR amplification apparatus (Biometra, T-Gradient, Germany)
Four, experimental method
1, extraction of bacterial genome DNA
36 strains of clinical isolates (type mtDNA-4) from northern China and 24 strains of genomic DNA from different countries (mtDNA1 ~ 24) were extracted by CTAB method, and the concentration of DNA was adjusted to 100ng / UL for PCR amplification.
2, PCR amplification
(1) amplified by 8 random primers, the primers OPB07, OPBG14, and OP02, which had good amplified fragments of all the strains, were screened for formal experiments, in which OP02 was used to amplify the strains of mtDNA1 to 24.
(2) the PCR reaction system: the total volume is 25ul
Primers (100pmol / UL) 1ul
TaKaRaTaq (5U / UL) 0.25ul
10 x buffer (including MgCl_2) 4ul
DNTP (dATP, dCTP, dGTP and dTTP each 2.5mmol / L) 4ul
Template DNA (100ng / UL) 1ul
(3) PCR reaction conditions:
5min predenaturation at 94 C;
45 cycles: 94 1min denaturation, 36 1min renaturation, 72 1min extension;
After extension 72 C 7min.
(4) the results showed that the PCR products were electrophoretic at 80 volt voltage on the agarose gel with a mass fraction of 1.5% and were observed under the ultraviolet transmittance and were photographed with the gel one time imager.
3, data processing
(1) the data conversion first uses PCR Marker as a molecular weight marker to determine the relative position of each reaction strip on the gel imaging. At the same molecular weight level, the reaction strip is recorded as "1" and the non reactive strip is recorded as "0". All the strain bands of all strains are converted to "1" and "0" data groups.
(2) the formula for the single matching coefficient SM is SM=2xnxy / (nx+ny) x 100%, nxy: the number of DNA bands shared by two strains, the number of DNA bands of the nx:x strain, and the DNA band number of ny:y strain.
(3) clustering analysis and statistical processing based on the PCR product band type, using the UPGMA clustering analysis method in the NTSYS-PC2.02 software package to calculate the single matching coefficient SM and automatically generate the tree pattern.
Result
1, mtDNA-4 type
(1) primer OPB07 can be divided into 2 electrophoretic bands by amplifying 36 strains of mtDNA-4 type of spores isolated from clinical isolates of SHK spora. PCR.
(2) primer OPBG14 can divide 36 isolates of mtDNA-4 type of spores of SHK spores into 4 electrophoretic bands.
(3) primer OP02 can divide 36 isolates of mtDNA-4 type of spores of SHK spores into 3 electrophoresis bands.
2, type mtDNA 1~24 (primer OP02)
Primer OP02 was used to amplify PCR from the representative strains of mtDNA 1~24 from different countries. The difference between the strains of B group (mtDNA4 to 10,12,13,20,21 and type 24) was small, including type 4 and 6, 7 and 20, 8,9,10,13 and type 21, and 5, 12 and 24 with other.A group strains (mtDNA 1 to 3,11,14 to 19,22) There was a great difference between the 23 strains and the same strains without bands.
3, the result of cluster analysis
The similarity coefficients of the 36 strains were between 0.87 and 1, indicating that there was a small genetic difference between mtDNA-4 strains, and 36 mtDNA-4 strains could be divided into 4 subtypes at the similar level of SM=0.92. The mtDNA 1~24 could be divided into two groups at the similar level of the tree figure SM=0.57, and the group I included the type 1 to 19 and 23, and the group II group included the 4 to 10,12,13,20. Between 22 and 24, there was only 22 difference between group A and group B of mtDNA.
conclusion
The RAPD method can be further divided into 4 subtypes of mtDNA-4 type spidium spore, which provides the basis for the molecular epidemiological study of sporofiliosis: it is suggested that there is a certain consistency between the RAPD typing and the mtDNA typing.

【學(xué)位授予單位】：中國(guó)醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R379

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