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
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 establish a multiple detection method based on comparative polymerase chain reaction (cPCR) and ligase detection reaction (LDR)/ligase chain reaction (LCR) to quantify the intestinal bacterial components. METH...AIM: To establish a multiple detection method based on comparative polymerase chain reaction (cPCR) and ligase detection reaction (LDR)/ligase chain reaction (LCR) to quantify the intestinal bacterial components. METHODS: Comparative quantification of 16S rDNAs from different intestinal bacterial components was used to quantify multiple intestinal bacteria. The 16S rDNAs of different bacteria were amplified simultaneously by cPCR. The LDR/LCR was examined to actualize the genotyping and quantification. Two beneficial (Bifidobacterium , Lactobacillus ) and three conditionally pathogenic bacteria (Enterococcus , Enterobacterium and Eubacterium ) were used in this detection. With cloned standard bacterial 16S rDNAs, standard curves were prepared to validate the quantitative relations between the ratio of original concentrations of two templates and the ratio ofthe fluorescence signals of their final ligation products. The internal controls were added to monitor the whole detection flow. The quantity ratio between two bacteria was tested. RESULTS: cPCR and LDR revealed obvious linear correlations with standard DNAs, but cPCR and LCR did not. In the sample test, the distributions of the quantity ratio between each two bacterial species were obtained. There were significant differences among these distributions in the total samples. But these distributions of quantity ratio of each two bacteria remained stable among groups divided by age or sex. CONCLUSION: The detection method in this study can be used to conduct multiple intestinal bacteria genotyping and quantification, and to monitor the human intestinal health status as well.展开更多
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.
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 establish a multiple detection method based on comparative polymerase chain reaction (cPCR) and ligase detection reaction (LDR)/ligase chain reaction (LCR) to quantify the intestinal bacterial components. METHODS: Comparative quantification of 16S rDNAs from different intestinal bacterial components was used to quantify multiple intestinal bacteria. The 16S rDNAs of different bacteria were amplified simultaneously by cPCR. The LDR/LCR was examined to actualize the genotyping and quantification. Two beneficial (Bifidobacterium , Lactobacillus ) and three conditionally pathogenic bacteria (Enterococcus , Enterobacterium and Eubacterium ) were used in this detection. With cloned standard bacterial 16S rDNAs, standard curves were prepared to validate the quantitative relations between the ratio of original concentrations of two templates and the ratio ofthe fluorescence signals of their final ligation products. The internal controls were added to monitor the whole detection flow. The quantity ratio between two bacteria was tested. RESULTS: cPCR and LDR revealed obvious linear correlations with standard DNAs, but cPCR and LCR did not. In the sample test, the distributions of the quantity ratio between each two bacterial species were obtained. There were significant differences among these distributions in the total samples. But these distributions of quantity ratio of each two bacteria remained stable among groups divided by age or sex. CONCLUSION: The detection method in this study can be used to conduct multiple intestinal bacteria genotyping and quantification, and to monitor the human intestinal health status as well.
