目的探索SYBR GreenI联合TaqMan荧光定量PCR检测HBV-DNA的意义。方法选择浓度为108.48、105.70和103.70copies/ml的3种HBV-DNA阳性血清和<1×103.0copies/ml的阴性血清各1份,在TaqMan-PCR混合反应体系中加入SYBR Green I组成双荧...目的探索SYBR GreenI联合TaqMan荧光定量PCR检测HBV-DNA的意义。方法选择浓度为108.48、105.70和103.70copies/ml的3种HBV-DNA阳性血清和<1×103.0copies/ml的阴性血清各1份,在TaqMan-PCR混合反应体系中加入SYBR Green I组成双荧光PCR(TaqMan+SYBR Green I组),同时进行TaqMan和SYBR Green I的单荧光PCR(分别为TaqMan组和SYBR Green I组),设置同一PCR和熔解曲线的循环参数,检测HBV-DNA含量及其Tm,每种方法一次检测每份血清5次。结果TaqMan+SYBR Green I组检测的HBV-DNA阳性血清均为阳性,其平均含量为108.55±、105.79±、103.81±,与TaqMan组的108.49±、105.69±、103.72±copies/ml0.320.290.300.310.300.25对应浓度值取10对数比较,无统计学意义(t=0.31、0.54和0.27,P>0.05);与SYBR Green I组的108.41±、0.35105.21±和103.26±copies/ml(不含未检出的两次血清)比较,除高浓度外,中低浓度有统计学意义(t=2.90和0.340.262.62,P<0.05)。TaqMan+SYBR Green I组和SYBR Green I组阳性血清均出现明显熔解曲线,熔解温度(Tm)分别为71.8℃、72℃和79.8℃,阴性血清未出现扩增曲线和Tm值。结论SYBR Green I联合TaqMan-PCR检测HBV-DNA时,具有能维持TaqMan-PCR的高灵敏度、特异性更强,并能同时检测HBV-DNATm的特点,为HBV的DNA多态性分析,尤其是在HBV基因分型方面提供了新的检测思路。展开更多
目的探讨SYBR Green I实时荧光定量多重PCR结合融解曲线分析方法快速检测常见超广谱β-内酰胺酶(ESBLs)耐药基因的可行性。方法选取经测序验证携带有tem、shv、ctx-m-1组、ctx-m-9组耐药基因的大肠埃希菌和肺炎克雷伯菌作为阳性菌株,建...目的探讨SYBR Green I实时荧光定量多重PCR结合融解曲线分析方法快速检测常见超广谱β-内酰胺酶(ESBLs)耐药基因的可行性。方法选取经测序验证携带有tem、shv、ctx-m-1组、ctx-m-9组耐药基因的大肠埃希菌和肺炎克雷伯菌作为阳性菌株,建立细菌耐药基因SYBR Green I实时荧光定量多重PCR检测体系,这四种耐药基因的扩增产物片段长度和融解温度(Tm值)都不同,可通过结合融解曲线分析加以区别。用此体系检测110株大肠埃希菌和94株肺炎克雷伯菌,根据特异性Tm值的有无和差别判断是否携带有耐药基因及携带耐药基因型别,结果与耐药表型、琼脂糖电泳、测序结果对照。结果经SYBR Green I实时荧光定量多重PCR结合融解曲线分析,分别有47株(42.7%)大肠埃希菌和34株(36.2%)肺炎克雷伯菌携带有这四种常见的ESBLs耐药基因,与表型确证实验和电泳结果比较达到100%的一致性;根据Tm值的不同,对耐药基因进行鉴别,携带单个耐药基因菌株的检测与电泳结果和测序结果比较达到100%的一致性;对携带多个耐药基因菌株的检测不能达到较好的分辨性。结论SYBR Green I实时荧光定量多重PCR结合融解曲线分析方法可以快速筛查常见超广谱β-内酰胺酶(ESBLs)耐药基因,对携带单个耐药基因的细菌的基因型能进行准确鉴定。这对指导临床医生合理使用抗生素进行临床治疗和对耐药细菌的流行病学监测具有重要价值。展开更多
BACKGROUND: TMS1/ASC is a bipartite protein comprising two protein-protein interactive domains: pyrin (PYD) and caspase recruitment domain (CARD). Proteins containing these domains play pivotal roles in regulating apo...BACKGROUND: TMS1/ASC is a bipartite protein comprising two protein-protein interactive domains: pyrin (PYD) and caspase recruitment domain (CARD). Proteins containing these domains play pivotal roles in regulating apoptosis and immune response pathways. The absence of TMS1/ ASC expression in some tumors is because methylation of the TMS1/ASC gene contributes to carcinogenesis and cancer development. We studied the methylation status of the TMS1/ASC gene and its clinical significance in cholangiocarcinoma. METHODS: Target DNA was modified by sodium bisulfite, coverting all unmethylated, but not methylated, cytosines to uracil, and subsequently by a nested amplification with primers specific for methylated versus unmethylated DNA. The PCR product was detected by gel electrophoresis and combined with the clinical records of patients. RESULTS: Aberrant methylation of the TMS1/ASC gene was detected in specimens of colorectal cancer tissues from 13 (36.1%) of 36 patients, and specimens of adjacent normal tissues from 3 patients (8.3%). No statistical differences were seen in the extent of differentiation and invasion, lymph node metastasis, and pathologic type between the methylated and unmethylated tissues (P】0.05). CONCLUSIONS: The frequency of TMS1/ASC gene methylation in cholangiocarcinoma is high, but it is not related to pathologic changes. The TMS1/ASC gene is probably suppressed by methylation, and is resistant to apoptosis and immunological surveillance. The gene epigenetically affected in methylated tissues could be associated with carcinogenesis of cholangiocarcinoma.展开更多
文摘目的探索SYBR GreenI联合TaqMan荧光定量PCR检测HBV-DNA的意义。方法选择浓度为108.48、105.70和103.70copies/ml的3种HBV-DNA阳性血清和<1×103.0copies/ml的阴性血清各1份,在TaqMan-PCR混合反应体系中加入SYBR Green I组成双荧光PCR(TaqMan+SYBR Green I组),同时进行TaqMan和SYBR Green I的单荧光PCR(分别为TaqMan组和SYBR Green I组),设置同一PCR和熔解曲线的循环参数,检测HBV-DNA含量及其Tm,每种方法一次检测每份血清5次。结果TaqMan+SYBR Green I组检测的HBV-DNA阳性血清均为阳性,其平均含量为108.55±、105.79±、103.81±,与TaqMan组的108.49±、105.69±、103.72±copies/ml0.320.290.300.310.300.25对应浓度值取10对数比较,无统计学意义(t=0.31、0.54和0.27,P>0.05);与SYBR Green I组的108.41±、0.35105.21±和103.26±copies/ml(不含未检出的两次血清)比较,除高浓度外,中低浓度有统计学意义(t=2.90和0.340.262.62,P<0.05)。TaqMan+SYBR Green I组和SYBR Green I组阳性血清均出现明显熔解曲线,熔解温度(Tm)分别为71.8℃、72℃和79.8℃,阴性血清未出现扩增曲线和Tm值。结论SYBR Green I联合TaqMan-PCR检测HBV-DNA时,具有能维持TaqMan-PCR的高灵敏度、特异性更强,并能同时检测HBV-DNATm的特点,为HBV的DNA多态性分析,尤其是在HBV基因分型方面提供了新的检测思路。
文摘目的探讨SYBR Green I实时荧光定量多重PCR结合融解曲线分析方法快速检测常见超广谱β-内酰胺酶(ESBLs)耐药基因的可行性。方法选取经测序验证携带有tem、shv、ctx-m-1组、ctx-m-9组耐药基因的大肠埃希菌和肺炎克雷伯菌作为阳性菌株,建立细菌耐药基因SYBR Green I实时荧光定量多重PCR检测体系,这四种耐药基因的扩增产物片段长度和融解温度(Tm值)都不同,可通过结合融解曲线分析加以区别。用此体系检测110株大肠埃希菌和94株肺炎克雷伯菌,根据特异性Tm值的有无和差别判断是否携带有耐药基因及携带耐药基因型别,结果与耐药表型、琼脂糖电泳、测序结果对照。结果经SYBR Green I实时荧光定量多重PCR结合融解曲线分析,分别有47株(42.7%)大肠埃希菌和34株(36.2%)肺炎克雷伯菌携带有这四种常见的ESBLs耐药基因,与表型确证实验和电泳结果比较达到100%的一致性;根据Tm值的不同,对耐药基因进行鉴别,携带单个耐药基因菌株的检测与电泳结果和测序结果比较达到100%的一致性;对携带多个耐药基因菌株的检测不能达到较好的分辨性。结论SYBR Green I实时荧光定量多重PCR结合融解曲线分析方法可以快速筛查常见超广谱β-内酰胺酶(ESBLs)耐药基因,对携带单个耐药基因的细菌的基因型能进行准确鉴定。这对指导临床医生合理使用抗生素进行临床治疗和对耐药细菌的流行病学监测具有重要价值。
文摘BACKGROUND: TMS1/ASC is a bipartite protein comprising two protein-protein interactive domains: pyrin (PYD) and caspase recruitment domain (CARD). Proteins containing these domains play pivotal roles in regulating apoptosis and immune response pathways. The absence of TMS1/ ASC expression in some tumors is because methylation of the TMS1/ASC gene contributes to carcinogenesis and cancer development. We studied the methylation status of the TMS1/ASC gene and its clinical significance in cholangiocarcinoma. METHODS: Target DNA was modified by sodium bisulfite, coverting all unmethylated, but not methylated, cytosines to uracil, and subsequently by a nested amplification with primers specific for methylated versus unmethylated DNA. The PCR product was detected by gel electrophoresis and combined with the clinical records of patients. RESULTS: Aberrant methylation of the TMS1/ASC gene was detected in specimens of colorectal cancer tissues from 13 (36.1%) of 36 patients, and specimens of adjacent normal tissues from 3 patients (8.3%). No statistical differences were seen in the extent of differentiation and invasion, lymph node metastasis, and pathologic type between the methylated and unmethylated tissues (P】0.05). CONCLUSIONS: The frequency of TMS1/ASC gene methylation in cholangiocarcinoma is high, but it is not related to pathologic changes. The TMS1/ASC gene is probably suppressed by methylation, and is resistant to apoptosis and immunological surveillance. The gene epigenetically affected in methylated tissues could be associated with carcinogenesis of cholangiocarcinoma.
