An efficient method was developed to detect point mutations using oligonucleotide microarrays and the ligase detection reaction (LDR). Allele-specific LDR primers were immobilized on polylysine-coated glass slides to ...An efficient method was developed to detect point mutations using oligonucleotide microarrays and the ligase detection reaction (LDR). Allele-specific LDR primers were immobilized on polylysine-coated glass slides to perform LDR on a chip. The spotting concentration and detection limit were analyzed using a synthesized oligonucleotide as a template. The optimal primer spotting concentration was 20 mmol/L and the lowest detectable template concentration was 0.33 nmol/L. The method was successfully employed to iden-tify malignant mutations of hypertrophic cardiomyopathy. Asymmetric polymerase chain reaction was em-ployed to prepare single stranded DNA as LDR templates from cloned plasmids. The discrimination ratios for AC, TC, GT, TT, GA, and AA mismatches are 32.82, 44.24, 17.75, 18.34, 11.66, and 8.91, respectively. This method may allow construction of multiple mutation detection systems.展开更多
AIM: To compare the ligase detection reaction (LDR) and real-time PCR for detection of low abundant YMDD mutants in patients with chronic hepatitis B infection.METHODS: Mixtures of plasmids and serum samples from 52 c...AIM: To compare the ligase detection reaction (LDR) and real-time PCR for detection of low abundant YMDD mutants in patients with chronic hepatitis B infection.METHODS: Mixtures of plasmids and serum samples from 52 chronic hepatitis B patients with low abundant lamivudine-resistant mutations were tested with LDR and real-time PCR. Time required and reagent cost for both assays were evaluated.RESULTS: Real-time PCR detected 100, 50, 10, 1 and 0.1% of YIDD plasmid, whereas LDR detected 100, 50, 10, 1, 0.1, and 0.01% of YIDD plasmid, in mixtures with YMDD plasmid of 106 copies/mL. Among the 52 clinical serum samples, completely concordant results were obtained for all samples by both assays, and 39 YIDD, 9 YVDD, and 4 YIDD/YVDD were detected. Cost and time required for LDR and real-time PCR are 60/80 CNY (8/10.7 US dollars) and 4.5/2.5 h, respectively.CONCLUSION: LDR and real-time PCR are both sensitive and inexpensive methods for monitoring low abundant YMDD mutants during lamivudine therapy in patients with chronic hepatitis B. LDR is more sensitive and less expensive, while real-time PCR is more rapid.展开更多
基金the National High-Tech Research and Devel-opment (863) Program of China (No. 2002AA2Z2011)
文摘An efficient method was developed to detect point mutations using oligonucleotide microarrays and the ligase detection reaction (LDR). Allele-specific LDR primers were immobilized on polylysine-coated glass slides to perform LDR on a chip. The spotting concentration and detection limit were analyzed using a synthesized oligonucleotide as a template. The optimal primer spotting concentration was 20 mmol/L and the lowest detectable template concentration was 0.33 nmol/L. The method was successfully employed to iden-tify malignant mutations of hypertrophic cardiomyopathy. Asymmetric polymerase chain reaction was em-ployed to prepare single stranded DNA as LDR templates from cloned plasmids. The discrimination ratios for AC, TC, GT, TT, GA, and AA mismatches are 32.82, 44.24, 17.75, 18.34, 11.66, and 8.91, respectively. This method may allow construction of multiple mutation detection systems.
文摘AIM: To compare the ligase detection reaction (LDR) and real-time PCR for detection of low abundant YMDD mutants in patients with chronic hepatitis B infection.METHODS: Mixtures of plasmids and serum samples from 52 chronic hepatitis B patients with low abundant lamivudine-resistant mutations were tested with LDR and real-time PCR. Time required and reagent cost for both assays were evaluated.RESULTS: Real-time PCR detected 100, 50, 10, 1 and 0.1% of YIDD plasmid, whereas LDR detected 100, 50, 10, 1, 0.1, and 0.01% of YIDD plasmid, in mixtures with YMDD plasmid of 106 copies/mL. Among the 52 clinical serum samples, completely concordant results were obtained for all samples by both assays, and 39 YIDD, 9 YVDD, and 4 YIDD/YVDD were detected. Cost and time required for LDR and real-time PCR are 60/80 CNY (8/10.7 US dollars) and 4.5/2.5 h, respectively.CONCLUSION: LDR and real-time PCR are both sensitive and inexpensive methods for monitoring low abundant YMDD mutants during lamivudine therapy in patients with chronic hepatitis B. LDR is more sensitive and less expensive, while real-time PCR is more rapid.
文摘目的研究安徽合肥地区原发性高血压(essential hypertension,EH)与血管紧张素转化酶(angiotensinconverting enzyme,ACE)基因多态性的关系,分析年龄、性别、吸烟、饮酒、饮食、家族史等因素对EH的影响。方法选择2022年3月至2022年5月安徽中医药大学第一附属医院心内科就诊的EH患者400例作为EH组,同期健康体检者100例作为对照组,采用多重高温连接酶检测反应(improved multiple ligase detection reaction,imLDR)技术检测ACE基因rs12709426多态性;利用问卷调查表采集患者一般情况及各项影响因素,采用全自动生化分析仪检测相关指标。结果ACE基因rs12709426只有纯合子AA基因型,无基因位点多态性;EH组基因型多态性频率与对照组比较,差异无统计学意义(χ^(2)=0,P>0.999)。两组人群饮酒、饮食、家族史、体质量指数(body mass index,BMI)、谷氨酸(glutamate,GLU)、低密度脂蛋白胆固醇(low density lipoprotein cholesterin,LDL⁃C)、高密度脂蛋白胆固醇(high density lipoprotein cholesterin,HDL⁃C)等因素比较,差异有统计学意义(P<0.05),其余差异均无统计学意义(P>0.05)。结论安徽合肥地区EH患者ACE基因rs12709426只有纯合子AA基因型,与EH发病无明显相关性;饮酒、饮食、家族史、BMI、GLU、LDL⁃C、HDL⁃C与EH发病具有明显相关性。
