The BRCA1 （Breast Cancer Anti-estrogen resistance-I）, early-onset gene is expressed in cells of breast and other tissue and helps to repair damaged DNA or destroy cells in cases DNA cannot be repaired. When the BRCA...The BRCA1 （Breast Cancer Anti-estrogen resistance-I）, early-onset gene is expressed in cells of breast and other tissue and helps to repair damaged DNA or destroy cells in cases DNA cannot be repaired. When the BRCA1 gene is damaged, then the DNA is not repaired appropriately and this enhances the risk for cancer. Fluorescence and UV-visible thermal studies were performed for WT （wild type） and MT （mutant type targets） full systems. The target DNAs used were in the form of short oligonucleotides, genomic DNA. The probe system was used for detection of WT and SNP alleles of human BRCAI [（170-190, G---~T） and （290-310, G---~T）]. The Cy5 dye attached to a probe oligonucleotide （10-mer） undergoes a fluorescence intensity change on hybridisation of the probe to the WT compared to MT targets. Our results indicate that the system consisting of the target sequence and the one probe oligonucleotides bearing the Cy5 dye assemble correctly at the specified target. Once the full system （probe and target） is arranged under suitable conditions, a red-shift emission and change in fluorescence intensity are seen at a suitable wavelength. Thermal studies also showed significant differences in T,, between WT and MT. The results suggest that the differences in the fluorescence intensity at 665 nm and the spectrophotometric T,,,cs） for the WT and MT can be attributed to the type of binding of the probe to the target. The systems were sensitive to single nucleotide polymorphisms and this may help in high throughput applications in genetic testing and molecular diagnostics.展开更多
目的建立PCR反向线点杂交技术(PCR-RLB)快速检测和鉴定常见念珠菌的方法。方法以念珠菌属间隔序列Ⅱ(ITS2)为靶基因设计通用引物,用生物素标记反义引物,PCR扩增白念珠菌、热带念珠菌、克柔念珠菌、光滑念球菌、近平滑念珠菌、都柏林念珠...目的建立PCR反向线点杂交技术(PCR-RLB)快速检测和鉴定常见念珠菌的方法。方法以念珠菌属间隔序列Ⅱ(ITS2)为靶基因设计通用引物,用生物素标记反义引物,PCR扩增白念珠菌、热带念珠菌、克柔念珠菌、光滑念球菌、近平滑念珠菌、都柏林念珠菌DNA,然后与通过氨基标记固定在尼龙膜上的各特异性寡核苷酸探针杂交,并进行临床标本和分离株的检测。结果念珠菌标准菌株可扩增出302～441 bp DNA片段,6种特异性寡核苷酸探针可分别与相应念珠菌PCR产物杂交,其敏感性为10 cfu/mL。通过对100例分离株的检测,然后与培养法鉴定(Merieux Vitek)的结果比较,有97株与培养结果一致,另外3株通过DNA测序结果证实与PCR-RLB测定结果一致。通过对200例女性阴道拭子进行检测,PCR-RLB阳性率为49%,而涂片法和培养法阳性率分别为27%和39%,明显高于涂片法和培养法(P<0.05)。结论该方法可快速、敏感、准确鉴定临床上常见的念珠菌。展开更多
文摘The BRCA1 （Breast Cancer Anti-estrogen resistance-I）, early-onset gene is expressed in cells of breast and other tissue and helps to repair damaged DNA or destroy cells in cases DNA cannot be repaired. When the BRCA1 gene is damaged, then the DNA is not repaired appropriately and this enhances the risk for cancer. Fluorescence and UV-visible thermal studies were performed for WT （wild type） and MT （mutant type targets） full systems. The target DNAs used were in the form of short oligonucleotides, genomic DNA. The probe system was used for detection of WT and SNP alleles of human BRCAI [（170-190, G---~T） and （290-310, G---~T）]. The Cy5 dye attached to a probe oligonucleotide （10-mer） undergoes a fluorescence intensity change on hybridisation of the probe to the WT compared to MT targets. Our results indicate that the system consisting of the target sequence and the one probe oligonucleotides bearing the Cy5 dye assemble correctly at the specified target. Once the full system （probe and target） is arranged under suitable conditions, a red-shift emission and change in fluorescence intensity are seen at a suitable wavelength. Thermal studies also showed significant differences in T,, between WT and MT. The results suggest that the differences in the fluorescence intensity at 665 nm and the spectrophotometric T,,,cs） for the WT and MT can be attributed to the type of binding of the probe to the target. The systems were sensitive to single nucleotide polymorphisms and this may help in high throughput applications in genetic testing and molecular diagnostics.
文摘目的建立PCR反向线点杂交技术(PCR-RLB)快速检测和鉴定常见念珠菌的方法。方法以念珠菌属间隔序列Ⅱ(ITS2)为靶基因设计通用引物,用生物素标记反义引物,PCR扩增白念珠菌、热带念珠菌、克柔念珠菌、光滑念球菌、近平滑念珠菌、都柏林念珠菌DNA,然后与通过氨基标记固定在尼龙膜上的各特异性寡核苷酸探针杂交,并进行临床标本和分离株的检测。结果念珠菌标准菌株可扩增出302～441 bp DNA片段,6种特异性寡核苷酸探针可分别与相应念珠菌PCR产物杂交,其敏感性为10 cfu/mL。通过对100例分离株的检测,然后与培养法鉴定(Merieux Vitek)的结果比较,有97株与培养结果一致,另外3株通过DNA测序结果证实与PCR-RLB测定结果一致。通过对200例女性阴道拭子进行检测,PCR-RLB阳性率为49%,而涂片法和培养法阳性率分别为27%和39%,明显高于涂片法和培养法(P<0.05)。结论该方法可快速、敏感、准确鉴定临床上常见的念珠菌。
