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1.
利用16S rDNA建立种特异性PCR快速检测鸭疫里默氏菌   总被引:13,自引:0,他引:13  
鸭疫里默氏菌感染是危害养鸭业的主要疾病,用表型指标鉴定鸭疫里默氏菌存在不足,因此有必要建立检测该菌的种特异性PCR法。利用已登录的鸭疫里默氏菌、大肠杆菌、沙门氏菌、多杀性巴氏杆菌的16S rDNA基因序列,设计了一对鸭疫里默氏菌16S rDNA基因的特异性引物190f和843r,分别以基因组DNA和菌落提取液为模板,从1~19型鸭疫里默氏菌参考菌株和代表亚型、变异株和可能新型的国内分离株共26株细菌中均扩增出大小为654bp的特异性片段,而扩增鸭大肠杆菌、鸭沙门氏菌和禽多杀性巴氏杆菌等感染鸭的常见细菌的结果均呈阴性。分别将鸭疫里默氏菌基因组DNA和菌落提取液进行10倍梯度稀释,基因组DNA的最小检出量为50pg,菌落最小检出量为15CFU/mL。结果说明,该PCR法具有较好的特异性和敏感性,可用于快速鉴定鸭疫里默氏菌。  相似文献   

2.
根据Gen Bank中鮰爱德华氏菌Edwardsiella ictaluri外膜微孔蛋白N(porin N)基因序列(Gen Bank No:NC_012779.2)设计了1对引物,预计目的片段大小为381 bp。通过对反应体系和条件的优化,并进行特异性试验、敏感性试验及人工感染组织样品检测,建立了一种快速检测鮰爱德华氏菌的PCR方法。结果表明,在所检测的鮰爱德华氏菌、迟缓爱德华氏菌、嗜水气单胞菌、温和气单胞菌、杀鲑气单胞菌、豚鼠气单胞菌、嗜麦芽寡养单胞菌、鲁氏耶尔森氏菌、海豚链球菌、不动杆菌、产气肠杆菌、大肠杆菌、拟态弧菌、荧光假单胞菌、弗氏柠檬酸杆菌15种细菌中仅鮰爱德华氏菌扩增出特异性条带;敏感性试验结果显示,该方法最小核酸检出量为9.35×10-3ng·μL-1;同时对人工感染的病料肝脏、细菌基因组DNA、细菌菌液及菌落进行扩增,结果显示4种材料均能检测出大小为381 bp的基因片段。本研究所建立的方法特异强、灵敏度高,适用于鮰爱德华氏菌感染病例的高效、快速检测。  相似文献   

3.
乳杆菌(Lactobacillus)是益生菌, 也是当前的研究热点之一。研究泡菜等样品中的乳杆菌需要快速的检出方法。根据已完成全基因组测序的14种乳杆菌的16S rDNA序列, 设计一对乳杆菌特异性引物。PCR检测结果表明该引物对乳杆菌和明串珠菌能扩增出800 bp的片段, 对表皮葡萄球菌、乳酸乳球菌和枯草芽胞杆菌却没有扩增条带, 具有一定的乳杆菌特异性。结合MRS乳杆菌半选择培养基和革兰氏染色, 运用菌落PCR技术, 可以快速高效地检出四川泡菜中的乳杆菌。再通过对PCR扩增片段测序, 可以将乳杆菌鉴定到种。从16份四川泡菜样品中检出了15株乳杆菌, 其中14株被鉴定为植物乳杆菌, 1株需进一步鉴定才能确定种。该方法可以检出乳杆菌新种。  相似文献   

4.
在杀鱼爱德华氏菌病原学研究中,在动物中制备杀鱼爱德华氏菌蛋白抗体耗时长,且获得的多克隆或多肽抗体在宿主细胞中特异性差,背景信号强。为解决这一问题,对在大肠杆菌(Escherichia coli)和沙门氏菌(Salmonella)中建立起来的λRed基因编辑方法进行调整和优化,建立了在杀鱼爱德华氏菌(Edwardsiella piscicida)基因组基因上添加HA标签序列的方法,为使用标签抗体研究杀鱼爱德华氏菌基因功能提供便利。λRed重组系统利用同源线性DNA片段与基因组DNA进行重组。即以质粒pSU315为模板,在引物上引入目的基因的特异性序列,扩增FRT序列和抗生素抗性基因;以获得的PCR产物转化携带pKD46的杀鱼爱德华氏菌,在pKD46表达的λ噬菌体的3个重组蛋白(Exo、Beta和Gam)作用下, PCR产物与杀鱼爱德华氏菌基因组发生同源重组,获得引入了抗生素抗性的靶基因缺失或靶基因携带标签序列的菌株;接着利用pKD46的温敏型特性,消除引入的pKD46;最后向杀鱼爱德华氏菌引入文章构建的表达Flp重组酶的质粒pKD46-flp,在FLP作用下,两个FRT位点之间发生重组,...  相似文献   

5.
珙桐内生细菌的分离鉴定及系统发育分析   总被引:2,自引:0,他引:2       下载免费PDF全文
用微生物学传统分离培养方法从珙桐茎、叶及叶柄中分离到内生细菌56株,选取17株内生菌,提取其基因组DNA,并以此为模板,PCR扩增其16S rDNA,扩增出约1 500 bp大小的DNA条带,对15株内生菌16S rDNA测序,用BLAST软件对测序结果进行相似性比对,发现11株为Bacillus属,相似性为95%-98%;2株为Lysinibacillus属,相似性为97%和99%;1株为Bordetella属,相似性为95%;1株为非培养细菌的同源菌,相似性96%。芽孢杆菌为珙桐内生细菌优势菌属。通过构建系统发育树发现这15株内生菌明显聚为2大支。  相似文献   

6.
PCR在鸭瘟临床诊断和免疫及致病机理研究中的初步应用   总被引:13,自引:0,他引:13  
根据GenBank文献,应用Oligo 6.0分析软件合成了用于扩增鸭瘟病毒(duck plague virus,DPV)EcoR Ⅰ 765bp片段的1对引物,上游引物(P1)位于EcoR Ⅰ片段的246~266nt,下游引物(P2)位于EcoR Ⅰ片段的727~744nt,以DPV CHa株DNA为模板,筛选PCR最佳反应条件,建立了检测DPV的PCR方法。应用该方法对强毒株DPV鸭胚培养物和弱毒株的鸡胚培养物进行扩增,均可获得498bp的DNA片段。而对正常鸭(鸡)胚和鸡马立克氏病毒、鸡传染性喉气管炎病毒、鸭病毒性肝炎病毒、多杀性巴氏杆菌、大肠杆菌、鸭副伤寒沙门氏菌和鸭疫里默氏杆菌培养物进行检测,结果均呈阳性。扩增产物测序结果与文献报道一致,证明了PCR方法的特异性。对DPV CHa株鸡胚毒提取物DNA进行检测,其最低检出量为10fg。用病毒分离、Dot—ELISA和PCR三种方法分别检测1990~2002年期间送检的临床样品,对所获得的结果进行χ^2分析,证明PCR检出率明显高于前2种方法。CHa株免疫雏鸭后对血液、心、肝、脾、肺、肾、十二指肠、直肠、法氏囊、胸腺、胰腺、延脑、大脑、小脑、舌头、肌肉、骨髓、食道共18种组织和粪便进行PCR检测,结果表明:①皮下接种4h后,心、肝、脾、肾、法氏囊、胸腺、胰腺、延脑、大脑和小脑为阳性,8h后18种组织和粪便均为阳性;②口服接种4h后舌头和食道为阳性,8h后,心、肝、脾、肾、胸腺、胰腺、延脑、大脑、小脑、舌头、食道和血液均为阳性;③滴鼻接种4h后无阳性组织,8h后检测心、肝、脾、肾、胸腺、延脑、大脑、小脑、舌头、食道和血液,均为阳性;④三种途径免疫的鸭,于12h至第21天均检测出DPV DNA。DPV强毒SC1株人工感染成年鸭2h后,即能从脑、肝、脾、法氏囊和胸腺中检出DPV DNA。12h后和死亡鸭的心、肝、脾、肺、肾、十二指肠、直肠、法氏囊、胸腺、胰腺、脑、胸肌、食道、腺胃、血液、舌头、皮肤、骨髓等组织器官和口腔分泌物及粪便中,均检测到DPV的DNA。该研究为阐明鸭瘟病毒在体内分布提供了重要的数据。  相似文献   

7.
【目的】建立添加有扩增内标(IAC,Internal amplification control)的沙门氏菌EvaGreen荧光定量PCR检测体系,提高PCR检测可靠性。【方法】通过比较已有沙门氏菌属细菌的基因组序列,筛选沙门氏菌属特异检测靶点,设计特异引物;再用复合引物法构建扩增内标,优化参数,建立沙门氏菌内标PCR检测体系,利用特异性和灵敏度实验评价体系的检测性能。【结果】筛选得到的新特异靶点基因编码III型分泌系统蛋白(ssaQ)。针对该基因设计特异引物(SsaQ6),建立了添加有扩增内标的常规PCR和EvaGreen荧光定量PCR检测体系;二者对151株沙门氏菌和34株非沙门氏菌的检测符合率均达100%,对基因组DNA的检测下限达14.9拷贝/PCR和2.76拷贝/PCR;人工污染牛奶样品(初始染菌量:4-6 cfu/10 mL),増菌10 h和8 h后分别可检出沙门氏菌。【结论】本研究发掘的新靶点基因ssaQ特异性强,基于这一新靶点建立的添加有扩增内标的EvaGreen荧光定量PCR比常规内标PCR的检测限更低,重复性更好,快速方便,在12 h内即可得出检测结果,并且定量准确,有利于推进沙门氏菌PCR检测方法的标准化应用。  相似文献   

8.
从纳豆中分离纯化得到1株β-葡萄糖苷酶高产菌株,命名为N1菌,利用16S rDNA分析对该菌作了系统进化鉴定.首先提取N1菌基因组DNA,根据不同种属细菌的16S rDNA序列两端的保守性设计通用引物,对N1菌16S rDNA片断进行PCR扩增,对扩增得到的目标片段测序,利用BLAST对测序结果进行比对,构建系统进化树进行分析,初步确定该菌属于枯草芽孢杆菌.研究表明以16S rDNA分析对该菌鉴定完全可行.  相似文献   

9.
PCR在鸭瘟病诊断和免疫及致病机理研究中的初步应用   总被引:13,自引:2,他引:11  
根据GenBank文献,应用Oligo 6.0分析软件合成了用于扩增鸭瘟病毒(duck plague virus,DPV)EcoRⅠ765bp片段的1对引物,上游引物(P1)位于EcoR Ⅰ片段的246~266nt,下游引物(P2)位于EcoRⅠ片段的727~744nt,以DPV CHa株DNA为模板,筛选PCR最佳反应条件,建立了检测DPV的PCR方法.应用该方法对强毒株DPV鸭胚培养物和弱毒株的鸡胚培养物进行扩增,均可获得498bp的DNA片段.而对正常鸭(鸡)胚和鸡马立克氏病毒、鸡传染性喉气管炎病毒、鸭病毒性肝炎病毒、多杀性巴氏杆菌、大肠杆菌、鸭副伤寒沙门氏菌和鸭疫里默氏杆菌培养物进行检测,结果均呈阳性.扩增产物测序结果与文献报道一致,证明了PCR方法的特异性.对DPV CHa株鸡胚毒提取物DNA进行检测,其最低检出量为10fg.用病毒分离、Dot-ELISA和PCR三种方法分别检测1990~2002年期间送检的临床样品,对所获得的结果进行χ2分析,证明PCR检出率明显高于前2种方法.CHa株免疫雏鸭后对血液、心、肝、脾、肺、肾、十二指肠、直肠、法氏囊、胸腺、胰腺、延脑、大脑、小脑、舌头、肌肉、骨髓、食道共18种组织和粪便进行PCR检测,结果表明:①皮下接种4h后,心、肝、脾、肾、法氏囊、胸腺、胰腺、延脑、大脑和小脑为阳性,8h后18种组织和粪便均为阳性;②口服接种4h后舌头和食道为阳性,8h后,心、肝、脾、肾、胸腺、胰腺、延脑、大脑、小脑、舌头、食道和血液均为阳性;③滴鼻接种4h后无阳性组织,8h后检测心、肝、脾、肾、胸腺、延脑、大脑、小脑、舌头、食道和血液,均为阳性;④三种途径免疫的鸭,于12h至第21天均检测出DPV DNA.DPV强毒SC1株人工感染成年鸭2h后,即能从脑、肝、脾、法氏囊和胸腺中检出DPV DNA.12h后和死亡鸭的心、肝、脾、肺、肾、十二指肠、直肠、法氏囊、胸腺、胰腺、脑、胸肌、食道、腺胃、血液、舌头、皮肤、骨髓等组织器官和口腔分泌物及粪便中,均检测到DPV的DNA.该研究为阐明鸭瘟病毒在体内分布提供了重要的数据.  相似文献   

10.
食品中沙门氏菌分子检测靶点的筛选与评价   总被引:3,自引:1,他引:2  
[目的]发掘新的沙门氏菌分子检测靶点,筛选检测性能优秀的引物.[方法]利用BLAST程序比较沙门氏菌属内基因组DNA序列的同源性以及沙门氏菌与非沙门氏菌基因组DNA序列之间的特异性,发掘出100多个检测沙门氏菌属的特异性片段,并从中随机挑选出15个片段作为候选靶点,一共设计了27对引物(FS1~FS27),对它们的特异性、灵敏度加以评价,从中筛选检测性能最好的引物.[结果]在27对引物中,检测性能最优的引物为FS23,采用该引物对供试菌株的相应检测靶点进行PCR扩增,44株沙门氏菌都能扩增到一条492 bp特异性片段,而22株非沙门氏菌则不能扩增出这一特异性片段.以FS23为引物建立PCR方法检测猪霍乱沙门氏菌基因组DNA的灵敏度为11.9 fg/μL,细菌纯培养物灵敏度为4.9×102cfu/mL;用猪霍乱沙门氏菌人工污染牛奶样品,如果接种起始菌量为100 cfu/25 mL时,只需要增菌5 h,采用上述方法即能检测出沙门氏菌.[结论]引物FS23对应的基因序列是一个性能优良的新分子检测靶点,具备很高的特异性和灵敏性,能够广泛应用于食品中沙门氏菌的快速检测.  相似文献   

11.
【目的】从疑似鸭疫里默氏杆菌病的病死雏鹅分离病原菌进行鉴定。【方法】根据细菌培养特性、生化特性、动物试验、血清型鉴定及分子生物学特性对分离菌株进行鉴定。【结果】分离菌株为革兰氏阴性菌,不发酵糖类和醇类,尿素酶试验和氧化酶还原试验为阳性,致病,不同分离株的16S rRNA基因经多重序列比对分析,结果显示鹅源分离株与鸭源鸭疫里默氏杆菌处于同一进化支上,与鸡源鸭疫里默氏杆菌进化关系稍远,血清型鉴定为1型。【结论】分离菌株为血清1型的鸭疫里默氏杆菌,对鸭和鹅均有高致病性,自家疫苗能够较好地保护雏鹅。  相似文献   

12.
Model samples of Campylobacter jejuni for polymerase chain reaction (PCR) were prepared by rapid and simple procedures consisting of centrifugation, proteinase K treatment, Chelex 100 treatment, and boiling lyses. A PCR based on specific amplification of the variable sequence of 16S rRNA gene was performed using Tth DNA polymerase and the PCR products were visualized by agarose gel electrophoresis. The assay allowed the detection of 10 CFU/mL C. jejuni in the physiological saline and 100 CFU/mL in the basic Park and Sanders broth.  相似文献   

13.
We established a Chelex 100-Microwave method for the purification of bacterial genomic DNA (gDNA) in less than 20 min with high yield and good quality, useful for multiple purposes. It combines Chelex 100, proteinase K, RNase A and heating in a microwave oven. The resulting gDNA was used directly to identify bacterial species of the Order Lactobacillales by means of PCR amplification of their 16S rDNA gene, isolated from sediments on the Yucatan Peninsula, Mexico. This method produced gDNA free of phenolic and protein residual contaminants from 100 of these isolated bacteria. 16S rDNA amplification and sequencing showed Pediococcus acidilactici to prevail in inland lagoons, and Pediococcus pentosaceus, Lactobacillus plantarum, Lactobacillus sp., and Lactobacillus fermentum to be most abundant in the soils of livestock farms. The combination of Chelex 100, enzymes and microwave heating used in the Chelex 100-Microwave method produced large amounts of highly pure gDNA from Gram-positive and Gram-negative bacteria, in less than 20 min.  相似文献   

14.
Complete 23S and almost complete 16S rRNA gene sequences were determined for the type strains of the validly described Enterococcus species, Melissococcus pluton and Tetragenococcus halophilus. A comprehensive set of rRNA targeted specific oligonucleotide hybridization probes was designed according to the multiple probe concept. In silico probe design and evaluation was performed using the respective tools of the ARB program package in combination with the ARB databases comprising the currently available 16S as well as 23S rRNA primary structures. The probes were optimized with respect to their application for reverse hybridization in microplate format. The target comprising 16S and 23S rDNA was amplified and labeled by PCR (polymerase chain reaction) using general primers targeting a wide spectrum of bacteria. Alternatively, amplification of two adjacent rDNA fragments of enterococci was performed by using specific primers. In vitro evaluation of the probe set was done including all Enterococcus type strains, and a selection of other representatives of the gram-positive bacteria with a low genomic DNA G+C content. The optimized probe set was used to analyze enriched drinking water samples as well as original samples from waste water treatment plants.  相似文献   

15.
The flora on the surface of smear-ripened cheeses is composed of numerous species of bacteria and yeasts that contribute to the production of the desired organoleptic properties. Due to the absence of selective media, it is very difficult to quantify cheese surface bacteria, and, consequently, the ecology of the cheese surface microflora has not been extensively investigated. We developed a SYBR green I real-time PCR method to quantify Corynebacterium casei, a major species of smear-ripened cheeses, using primers designed to target the 16S rRNA gene. It was possible to recover C. casei genomic DNA from the cheese matrix with nearly the same yield that C. casei genomic DNA is recovered from cells recovered by centrifugation from liquid cultures. Quantification was linear over a range from 10(5) to 10(10) CFU per g of cheese. The specificity of the assay was demonstrated with DNA from species related to C. casei and from other bacteria and yeasts belonging to the cheese flora. Nine commercial cheeses were analyzed by real-time PCR, and six of them were found to contain more than 10(5) CFU equivalents of C. casei per g. In two of them, the proportion of C. casei in the total bacterial flora was nearly 40%. The presence of C. casei in these samples was further confirmed by single-strand conformation polymorphism analysis and by a combined approach consisting of plate counting and 16S rRNA gene sequencing. We concluded that SYBR green I real-time PCR may be used as a reliable species-specific method for quantification of bacteria from the surface of cheeses.  相似文献   

16.
Yuan J  Liu W  Sun M  Song S  Cai J  Hu S 《Journal of bacteriology》2011,193(11):2896-2897
Riemerella anatipestifer is a well-described pathogen of waterfowl and other avian species which can cause a great loss to the poultry industry. Here we obtained the complete genome sequence of R. anatipestifer strain RA-GD, which was isolated from an infected duck in Guangzhou, China, and was cultivated in our laboratory.  相似文献   

17.
To explore the bacterial microbiota in Chilean oyster (Tiostrea chilensis), a molecular approach that permits detection of different bacteria, independently of their capacity to grow in culture media, was used. Bacterial diversity was assessed by analysis of both the 16S rDNA and the 16S-23S intergenic region, obtained by PCR amplifications of DNA extracted from depurated oysters. RFLP of the PCR amplified 16S rDNA showed a prevailing pattern in most of the individuals analyzed, indicating that a few bacterial species were relatively abundant and common in oysters. Cloning and sequencing of the 16S rDNA with the prevailing RFLP pattern indicated that this rRNA was most closely related to Arcobacter spp. However, analysis by the size of the amplified 16S-23S rRNA intergenic regions revealed not Arcobacter spp. but Staphylococcus spp. related bacteria as a major and common component in oyster. These different results may be caused by the absence of target for one of the primers employed for amplification of the intergenic region. Neither of the two bacteria species found in large abundance was recovered after culturing under aerobic, anaerobic, or microaerophilic conditions. This result, however, is expected because the number of bacteria recovered after cultivation was less than 0.01% of the total. All together, these observations suggest that Arcobacter-related strains are probably abundant and common in the Chilean oyster bacterial microbiota.  相似文献   

18.
目的:在生物浸出中,微生物群落结构分析有着重要意义,而群落分析的基础是提取纯度高、损失少的基因组DNA。为了解决这一问题,本实验通过比较两种较常用的DNA提取方法,煮沸裂解法和试剂盒法,寻找一种灵敏、快速、经济实用的制备浸矿细菌基因组DNA的方法。方法:分别用煮沸裂解法和试剂盒法提取6种浸矿菌的基因组DNA,从所提取的基因组DNA浓度、纯度、回收率和对PCR扩增反应的影响方面比较了两种方法的提取效果;用两种方法来处理不同浓度梯度的一种菌,通过实时定量PCR来比较两种方法的灵敏性。结果:相同处理量(108个)的革兰氏阳性菌(1株)、革兰氏阴性菌(4株)、古菌(1株)经两种方法提取的基因组DNA差异较大,煮沸裂解法所得的6组基因组DNA更纯,其OD260/OD280的值更接近1.8-2.0(纯DNA的OD260/OD280在1.8-2.0之间),前者所提DNA回收率最大可达后者的16.7倍;煮沸裂解法只需较少菌(102个)便能让实时定量PCR检测到所提DNA模板浓度,比试剂盒法灵敏。结论:两种方法提取的基因组DNA均可用于后续的PCR扩增,此外,前者提取的DNA浓度随细菌浓度增加而呈线性增大,而后者随菌浓度增大,所提DNA量增加有限,因此,在生物浸出中微生物基因组DNA的提取可直接采用简单快速的煮沸提取法,为实验节约成本和时间。  相似文献   

19.
鸭疫里默氏杆菌外膜蛋白生物学特性研究   总被引:1,自引:0,他引:1  
血清2型鸭疫里默氏杆菌强毒菌株体外传200代获得了无毒力无免疫原性菌株,采用超声波裂解和超速离心法提取二株菌的外膜蛋白, 以比较分析鸭疫里默氏杆菌外膜蛋白的生物学特性。电镜观察细菌超微结构显示传代菌株外膜膜密度降低, 外膜泡的数量明显减少, 细胞质不均匀、内有空泡产生;免疫印迹结果表明二株菌的外膜蛋白免疫原性多肽存在明显区别;原代菌株的外膜蛋白仅与2型RA抗体出现特异性凝集, 而传代菌株的外膜蛋白与 1、2、10与11型RA抗体均出现凝集;二株菌的外膜蛋白均可诱导雏鸭产生抗体, 但原代菌株外膜蛋白诱导雏鸭产生抗体滴度显著高于200代次菌株;原代菌株外膜蛋白免疫鸭对同源RA菌株的攻击可产生100%的免疫保护, 而传代菌株外膜蛋白免疫鸭对同源RA菌株的攻击不产生免疫保护。序列分析显示两者的外膜蛋白A同源性达到99.9%。结果表明强毒菌株的外膜蛋白为良好的亚单位疫苗候选, 体外连续传代对RA外膜蛋白生物学特性影响显著。  相似文献   

20.
鸭疫里默氏杆菌外膜蛋白生物学特性研究   总被引:1,自引:0,他引:1  
血清2型鸭疫里默氏杆菌强毒菌株体外传200代获得了无毒力无免疫原性菌株,采用超声波裂解和超速离心法提取二株菌的外膜蛋白, 以比较分析鸭疫里默氏杆菌外膜蛋白的生物学特性。电镜观察细菌超微结构显示传代菌株外膜膜密度降低, 外膜泡的数量明显减少, 细胞质不均匀、内有空泡产生;免疫印迹结果表明二株菌的外膜蛋白免疫原性多肽存在明显区别;原代菌株的外膜蛋白仅与2型RA抗体出现特异性凝集, 而传代菌株的外膜蛋白与 1、2、10与11型RA抗体均出现凝集;二株菌的外膜蛋白均可诱导雏鸭产生抗体, 但原代菌株外膜蛋白诱导雏鸭产生抗体滴度显著高于200代次菌株;原代菌株外膜蛋白免疫鸭对同源RA菌株的攻击可产生100%的免疫保护, 而传代菌株外膜蛋白免疫鸭对同源RA菌株的攻击不产生免疫保护。序列分析显示两者的外膜蛋白A同源性达到99.9%。结果表明强毒菌株的外膜蛋白为良好的亚单位疫苗候选, 体外连续传代对RA外膜蛋白生物学特性影响显著。  相似文献   

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