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
It is difficult to make a precise diagnosis of intestinal tuberculosis and to differentiate it from Crohn's disease. For evaluating Polymerase Chain Reaction (PCR) assay in these two aspects, 36 specimens of intes...It is difficult to make a precise diagnosis of intestinal tuberculosis and to differentiate it from Crohn's disease. For evaluating Polymerase Chain Reaction (PCR) assay in these two aspects, 36 specimens of intestinal tuberculosis from surgical resections and endoscopic biopsies and 26 Crohn's disease samples were subjected to PCR assay. 21 specimens of normal colon tissue surrounding cancer were used as the control. Oligonucleotides derived from the IS 6110 sequence, which is repeated in M. tuberculosis chromosome and highly specific for the M. tuberculosis complex, were used as a primer. The amplified PCR products were detected by examination of ethidium-bromide-stained polyacrylamide gels. The specificity of PCR products was confirmed by digestion with Sal 1 restrictive endonuclease and southern blot hybridization using digoxigenin-labeled probe. The results showed that the M. tuberculosis DNA was identified in 27 / 36 intestinal tuberculosis, but none of 26 Crohn's disease. Acid fast bacilli were only found in 16 / 36 intestinal tuberculosis. In conclusion, as a rapid, sensitive, and specific pathogenic method in diagnosis of intestinal tuberculosis, PCR assay has been developed in this study, and is considered valuable in the differentiation between intestinal tuberculosis and Crohn's disease.展开更多
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.
文摘It is difficult to make a precise diagnosis of intestinal tuberculosis and to differentiate it from Crohn's disease. For evaluating Polymerase Chain Reaction (PCR) assay in these two aspects, 36 specimens of intestinal tuberculosis from surgical resections and endoscopic biopsies and 26 Crohn's disease samples were subjected to PCR assay. 21 specimens of normal colon tissue surrounding cancer were used as the control. Oligonucleotides derived from the IS 6110 sequence, which is repeated in M. tuberculosis chromosome and highly specific for the M. tuberculosis complex, were used as a primer. The amplified PCR products were detected by examination of ethidium-bromide-stained polyacrylamide gels. The specificity of PCR products was confirmed by digestion with Sal 1 restrictive endonuclease and southern blot hybridization using digoxigenin-labeled probe. The results showed that the M. tuberculosis DNA was identified in 27 / 36 intestinal tuberculosis, but none of 26 Crohn's disease. Acid fast bacilli were only found in 16 / 36 intestinal tuberculosis. In conclusion, as a rapid, sensitive, and specific pathogenic method in diagnosis of intestinal tuberculosis, PCR assay has been developed in this study, and is considered valuable in the differentiation between intestinal tuberculosis and Crohn's disease.
