应用RAPD对申克孢子丝菌线粒体类别进行4型内及种群分析

发布时间：2018-05-01 18:43

本文选题：申克孢子丝菌 + 随机扩增DNA多态性　；参考：《中国医科大学》2007年硕士论文

【摘要】： 目的应用RAPD方法对36株mtDNA-4型申克孢子丝菌的基因组DNA进行多态性分析，比较此型内部菌株间的差异；通过对24株mtDNA1～24型申克孢子丝菌代表菌株的基因组DNA多态性分析，初步探讨mtDNA分型和RAPD分型之间的关系。材料和方法一、实验材料实验菌株：36株来自中国北方地区的申克孢子丝菌临床分离菌株，为mtDNA-4型。24株来自不同国家、地区申克孢子丝菌代表菌株，为mtDNA1～24型。二、实验试剂 1、基因组DNA提取主要试剂 (1)基因组DNA提取液 2％(W／V)CTAB(hexadecyltrimethylammonium bromide十六烷基三甲基溴化铵) (2) Tris饱和酚 (3)氯仿 (4)异戊醇 (5) 0.5mol／L乙酸铵 (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主要试剂 (1)真菌随机引物 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、主要实验仪器 (1)蒸汽高压消毒箱(美国Tuttnauer 2540MK型) (2) PH4000AB型电热恒温培养箱(天津市泰斯特仪器有限公司) (3)紫外凝胶成像系统(英国Gel worker eD，UVP GDS 8000) (4)全能型高性能台式冷冻离心机(德国Heaeus，Biofuge Stratos) (5) 05RP-22离心机(Hitachi) (6) HZO-X100震荡培养箱(哈尔滨市东联电子开发有限公司) (7)电泳仪(北京六一仪器厂，DYY-Ⅲ2型) (8)电泳槽(北京六一仪器厂，DYY-Ⅲ2型) (9) PCR扩增仪(德国Biometra，T-Gradient) 四、实验方法 1、菌种基因组DNA的提取采用CTAB法提取36株来自我国北方地区的临床分离菌株(mtDNA-4型)及24株来自不同国家地区的保存菌株(mtDNA1～24型)的基因组DNA，调整浓度至100ng／ul以备PCR扩增。 2、PCR扩增 (1)利用8个随机引物分别进行扩增，筛选出对所有的菌株具有良好扩增片段的引物OPB07、OPBG14、OP02进行正式实验，其中应用OP02对mtDNA1～24型菌株进行扩增。 (2) PCR反应体系：总体积为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反应条件： 94℃5min预变性； 45个循环：94℃1min变性、36℃1min复性、72℃1min延伸；后延伸72℃7min。 (4)结果检测PCR产物在质量分数为1.5％的琼脂糖凝胶上80伏特电压下电泳并在紫外透射仪下观察并用凝胶一次成像仪照相。 3、数据处理 (1)数据转换首先用PCR Marker作为分子量标记，确定各反应条带在凝胶成像上的相对位置。在同一分子量水平上，有反应条带记作“1”，无反应条带记作“0”。将所有菌株的反应条带都转换为“1”，“0”组成的数据组。 (2)单匹配系数SM的计算公式为SM=2xnxy／(nx+ny)×100％，nxy：两菌株共有的DNA条带数，nx：x菌株的DNA条带数，ny：y菌株的DNA条带数。 (3)聚类分析统计学处理根据PCR产物带型，用NTSYS-PC2.02版软件包中的UPGMA聚类分析法进行单匹配系数SM的计算和树状图自动生成。结果 1、mtDNA-4型 (1)引物OPB07对36株mtDNA-4型的申克孢子丝菌临床分离菌株PCR扩增后，可以将其分成2个电泳带型。 (2)引物OPBG14可以将36株mtDNA-4型的申克孢子丝菌临床分离菌株分成4个电泳带型。 (3)引物OP02可以将将36株mtDNA-4型的申克孢子丝菌临床分离菌株分为3个电泳带型。 2、mtDNA 1～24型(引物OP02) 引物OP02对来自不同国家地区的mtDNA 1～24型的代表菌株进行PCR扩增，B群菌株(mtDNA4～10、12、13、20、21和24型)株间差异较小，其中4型与6型，7型与20型，8、9、10、13与21型带型完全相同，5型，12型及24型带型与其它皆不相同。A群菌株(mtDNA 1～3、11、14～19、22和23型)株间差异较大，，无带型完全相同菌株。 3、聚类分析结果 36株菌的相似系数在0.87～1.0之间，表明mtDNA-4型菌株之间存在较小的遗传差异，在SM=0.92相似水平上36株mtDNA-4型菌可分为4个亚型。在树状图SM=0.57相似水平上mtDNA 1～24型也可分成两群，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，与mtDNA分型的A、B群仅有型22的差异。结论应用RAPD方法可对mtDNA-4型的申克孢子丝菌进一步分为4个亚型，为孢子丝菌病的分子流行病学研究提供依据：初步提示RAPD分型与mtDNA分型之间具有一定的一致性。
[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.

【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R379

【参考文献】