AIM: To investigate clarithromycin resistance positions 2142, 2143 and 2144 of the 23 Sr RNA gene in Helicobacter pylori(H. pylori) by nested-allele specific primer-polymerase chain reaction(nested-ASP-PCR).METHODS: T...AIM: To investigate clarithromycin resistance positions 2142, 2143 and 2144 of the 23 Sr RNA gene in Helicobacter pylori(H. pylori) by nested-allele specific primer-polymerase chain reaction(nested-ASP-PCR).METHODS: The gastric tissue and saliva samples from 99 patients with positive results of the rapid urease test(RUT) were collected. The nested-ASP-PCR method was carried out with the external primers and inner allele-specific primers corresponding to the reference strain and clinical strains. Thirty gastric tissue and saliva samples were tested to determine the sensitivity of nested-ASP-PCR and ASP-PCR methods. Then, clarithromycin resistance was detected for 99 clinical samples by using different methods, including nestedASP-PCR, bacterial culture and disk diffusion. RESULTS: The nested-ASP-PCR method was successfully established to test the resistance mutation points 2142, 2143 and 2144 of the 23 SrR NA gene of H. pylori. Among 30 samples of gastric tissue and saliva, the H. pylori detection rate of nested-ASP-PCR was 90% and 83.33%, while the detection rate of ASP-PCR was just 63% and 56.67%. Especially in the saliva samples, nested-ASP-PCR showed much higher sensitivity in H. pylori detection and resistance mutation rates thanASP-PCR. In the 99 RUT-positive gastric tissue and saliva samples, the H. pylori-positive detection rate by nested-ASP-PCR was 87(87.88%) and 67(67.68%), in which there were 30 wild-type and 57 mutated strains in gastric tissue and 22 wild-type and 45 mutated strains in saliva. Genotype analysis showed that three-points mixed mutations were quite common, but different resistant strains were present in gastric mucosa and saliva. Compared to the high sensitivity shown by nested-ASP-PCR, the positive detection of bacterial culture with gastric tissue samples was 50 cases, in which only 26 drug-resistant strains were found through analyzing minimum inhibitory zone of clarithromycin. CONCLUSION: The nested-ASP-PCR assay showed higher detection sensitivity than ASP-PCR and drug sensitivity testing, which could be performed to evaluate clarithromycin resistance of H. pylori.展开更多
AIM: The interaction of mucosal addressin cell adhesion molecule 1 (MAdCAM-1) with integrin α4β7 mediates lymphocyte recruitment into mucosa-associated lymphoid tissue (MALT). Nodular gastritis is characterized by a...AIM: The interaction of mucosal addressin cell adhesion molecule 1 (MAdCAM-1) with integrin α4β7 mediates lymphocyte recruitment into mucosa-associated lymphoid tissue (MALT). Nodular gastritis is characterized by a unique military pattern on endoscopy representing increased numbers of lymphoid follicles with germinal center, strongly associated with H pylori infection. The purpose of this study was to address the implication of the MAdCAM-1/integrin β7 pathway in NG.METHODS: We studied 17 patients with NG and H pylori infection and 19 H pylori-positive and 14 H pylori-negative controls. A biopsy sample was taken from the antrum and snap-frozen for immunohistochemical analysis of MAdCAM1 and integrin β7. In simultaneous viewing of serial sections,the percentage of MAdCAM-1-positive to von Willebrand factor-positive vessels was calculated. We also performed immunostaining with anti-CD20, CD4, CD8 and CD68 antibodies to determine the lymphocyte subsets coexpressing integrin β7.RESULTS: Vascular endothelial MAdCAM-1 expression was more enhanced in gastric mucosa with than without H pylori infection. Of note, the percentages of MAdCAM-1-positive vessels were significantly higher in the lamina propria of NG patients than in H pylori-positive controls. Strong expression of MAdCAM-1 was identified adjacent to lymphoid follicles and dense lymphoid aggregates. Integrin β7-expressing mononuclear cells, mainly composed of CD20 and CD4 lymphocytes, were associated with vessels lined with MAdCAM-1-expressing endothelium.CONCLUSION: Our results suggest that the MAdCAM-1/integrin α4β7 homing system may participate in gastric inflammation in response to H pylori-infection and contributes to MALT formation, typically leading to the development of NG.展开更多
AIM: To estimate the frequency of microdeletions in the long arm of Y-chromosome of 20 infertile males from South India. METHODS: Polymerase chain reaction (PCR) amplification using Y-specific STS of azoospermia facto...AIM: To estimate the frequency of microdeletions in the long arm of Y-chromosome of 20 infertile males from South India. METHODS: Polymerase chain reaction (PCR) amplification using Y-specific STS of azoospermia factor (AZF) regions i.e., SY 84 for AZFa, SY 127 for AZFb and SY 254 for AZFc. RESULTS: Of the 20 infertile subjects 3 (15 %), one azoospermic and two oligozoospermic, showed microdeletions in the AZF region of Y-chromosome. CONCLUSION: The frequency of deletions involving AZF region of the Y-chromosome is 15 % in azoospermic and severely oligozoospermic infertile men. PCR amplification of AZF locus is useful for the diagnosis of microdeletions in the Y-chromosome.展开更多
Objective:To establish a polymerase chain reaction(PCR) technique based on cytochrome b {cytb) gene of mitochondria DNA(mtDNA) for blood meal identification.Methods:The PCR technique was established based on published...Objective:To establish a polymerase chain reaction(PCR) technique based on cytochrome b {cytb) gene of mitochondria DNA(mtDNA) for blood meal identification.Methods:The PCR technique was established based on published information and validated using blood sample of laboratory animals of which their whole gene sequences are available in CenBank.PCR was next performed to compile gene sequences of different species of wild rodents.The primers used were complementary to the conserved region of the cytb gene of vertebrate's mtDNA.A total of 100 blood samples,both from laboratory animals and wild rodents were collected und analyzed.The obtained unknown sequences were compared with those in the GenBank database using BLAST program to identify the vertebrate animal species.Results:Gene sequences of 11 species of wild animals caught in 9 localities of Peninsular Malaysia were compiled using the established PCR. The animals involved were Rattus(rattus) tanezumi,Rattus tiomanicus,Leopoldamys sabanus, Tupaia glis,Tupaia minor,Niviventor cremoriventor,Rhinosciurus laticaudatus,Calloseiurus caniseps,Sundamys muelleri,Rattus rajah,and Maxomys whitelwadi.The BLAST results confirmed the host with exact or nearly exact matches(>89%identity).Ten new gene sequences have been deposited in CenBank database since September 2010.Conclusions:This study indicates that the PCR direct sequencing system using universal primer sets for vertebrate cytb gene is a promising technique for blood meal identification.展开更多
In order to investigate the association of genotypes of HLA-DRB1 and HLA-DQB1 alleles with the genetic susceptibility of chronic urticaria (CU), genotypes of HLA-DRB1 and HLA-DQB1 genes were detected by polymerase cha...In order to investigate the association of genotypes of HLA-DRB1 and HLA-DQB1 alleles with the genetic susceptibility of chronic urticaria (CU), genotypes of HLA-DRB1 and HLA-DQB1 genes were detected by polymerase chain reactions with sequence-specific primers (PCR-SSP) in 42 patients with CU (19 men and 23 women, mean age 30.67±12.45 y old as well as 193 racially matched healthy persons in ethnic Han from Hubei provinece. Gene frequencies of HLA-DRB1*12, *0901 (RR=3.11, χ2=7.579, P=0.006; RR=2.47, χ2=5.684, P=0.017) were significantly increased in CU patients as compared with that in healthy people. Gene frequencies of HLA-DQB1*05 (RR=0.26, χ2=6.683, P=0.01) were significantly decreased in CU patients. It was suggested that CU was found strongly associated with HLA-DRB1*12, *0901 and HLA-DQB1*05, the former might be the genetic markers for susceptibility to CU, but the latter might play a resistive role.展开更多
AIM: To determine whether one genotype (A or non-A genotypes of HBV) predominated over the other during the course of HBV infection.METHODS: Four baboons were inoculated with HBV. DNA was extracted from serum obtained...AIM: To determine whether one genotype (A or non-A genotypes of HBV) predominated over the other during the course of HBV infection.METHODS: Four baboons were inoculated with HBV. DNA was extracted from serum obtained at monthly intervals postinoculation for 52 weeks and HBV DNA was amplified using primers specific for the core region containing an insert characteristic of genotype A (nt 2 354-2 359, numbering from the EcoRI site). The amplicons were cloned into PCRScriptTM and a minimum of 15 clones per time point were sequenced in both directions.RESULTS: Both genotypes persisted for the entire followup period of 52 weeks. Genotype non-A predominated in two baboons and genotype A in one baboon. Neither genotype predominated in the fourth baboon, as shown at a 5 % level of testing.CONCLUSION: No conclusions concerning the dominance of either genotype or the natural progression or replication rates of HBV could be drawn because the pattern of the genotypes found may have been caused by sampling fluctuations at the time of DNA extraction and cloning as a result of the very low viral loads in the baboon sera.展开更多
AIM: Little has been known about the pathogenesis of nonerosive reflux disease (NERD). Recent studies have implicated interleukin 8 (IL-8) in the development and progression of gastroesophgeal reflux disease (GERD). T...AIM: Little has been known about the pathogenesis of nonerosive reflux disease (NERD). Recent studies have implicated interleukin 8 (IL-8) in the development and progression of gastroesophgeal reflux disease (GERD). The purpose of this study was to determine IL-8 RNA expression levels in NERD patients with or without subtle mucosal changes. METHODS: We studied 26 patients with NERD and 13 asymptomatic controls. Biopsy sample was taken from the esophagus 3 cm above the gast^oesophageal junction and snap frozen for measurement of IL-8 mRNA levels by real-time quantitative polymerase chain reaction (PCR). We also examined mRNA expression of IL-8 receptors, CXCR-1 and -2 by reverse transcriptase PCR. The patients were endoscopically classified into grade M (mucosal color changes without visible mucosal break) and N (neither minimal involvement nor mucosal break) of the modified Los Angeles classification. RESULTS: The relative IL-8 mRNA expression levels were significantly higher in esophageal mucosa of NERD patients than those in esophageal mucosa of the controls. There was a significant difference in IL-8 mRNA levels between grades M and N. The CXCR-1 and -2 mRNAs were consUtutdvely expressed in esophageal mucosa. CONCLUSION: Our results suggest that high IL-8 levels in esophageal mucosa may be involved in the pathogenesis of NERD through interaction with its receptors. NERD seems to be composed of a heterogeneous population in terms of not only endoscopically minimal involvement but also immune and inflammatory processes.展开更多
Contagious bovine pleuropneumonia is a disease caused by Mycoplasma mycoides subspecie mycoides, a transboundary animal disease causing serious devastation to cattle producers in Africa. The study was designed to iden...Contagious bovine pleuropneumonia is a disease caused by Mycoplasma mycoides subspecie mycoides, a transboundary animal disease causing serious devastation to cattle producers in Africa. The study was designed to identify and characterize the pathogenic member of mycoplasma cluster the Mycoplasma mycoides subspecie mycoides (Mmm) isolated from cattle infected with the disease. Three hundred (300) samples of nasal swabs and pleural fluid from cattle showing signs of CBPP were analyzed using culture and biochemical identification techniques and polymerase chain reaction (PCR) using specific primers to determine the prevalence of contagious bovine pleuropneumonia in Nasarawa State, Nigeria. Isolation recorded a prevalence of 4% and PCR recorded a prevalence of 67.7%. Isolates subjected to PCR analysis produced an amplicon size of 548 bp and 1.1 k bp respectively for the Mycoplasma mycoides cluster and Mycoplasma mycoides subspecie mycoides. Sequencing of the 16 S rRNA gene blast search revealed 96% to 99% sequence homology of Mycoplasma mycoides subspecie mycoides compared with 14 available sequences in the gen bank at NCBI. Based on this investigation mass vaccination of cattle is recommended, isolation and PCR techniques could be used as diagnostic tools for CBPP disease in three agro ecological zones of Nasarawa state, Nigeria.展开更多
基金the Hospital of Qixia in Nanjing for providing sufficient financial support for this study
文摘AIM: To investigate clarithromycin resistance positions 2142, 2143 and 2144 of the 23 Sr RNA gene in Helicobacter pylori(H. pylori) by nested-allele specific primer-polymerase chain reaction(nested-ASP-PCR).METHODS: The gastric tissue and saliva samples from 99 patients with positive results of the rapid urease test(RUT) were collected. The nested-ASP-PCR method was carried out with the external primers and inner allele-specific primers corresponding to the reference strain and clinical strains. Thirty gastric tissue and saliva samples were tested to determine the sensitivity of nested-ASP-PCR and ASP-PCR methods. Then, clarithromycin resistance was detected for 99 clinical samples by using different methods, including nestedASP-PCR, bacterial culture and disk diffusion. RESULTS: The nested-ASP-PCR method was successfully established to test the resistance mutation points 2142, 2143 and 2144 of the 23 SrR NA gene of H. pylori. Among 30 samples of gastric tissue and saliva, the H. pylori detection rate of nested-ASP-PCR was 90% and 83.33%, while the detection rate of ASP-PCR was just 63% and 56.67%. Especially in the saliva samples, nested-ASP-PCR showed much higher sensitivity in H. pylori detection and resistance mutation rates thanASP-PCR. In the 99 RUT-positive gastric tissue and saliva samples, the H. pylori-positive detection rate by nested-ASP-PCR was 87(87.88%) and 67(67.68%), in which there were 30 wild-type and 57 mutated strains in gastric tissue and 22 wild-type and 45 mutated strains in saliva. Genotype analysis showed that three-points mixed mutations were quite common, but different resistant strains were present in gastric mucosa and saliva. Compared to the high sensitivity shown by nested-ASP-PCR, the positive detection of bacterial culture with gastric tissue samples was 50 cases, in which only 26 drug-resistant strains were found through analyzing minimum inhibitory zone of clarithromycin. CONCLUSION: The nested-ASP-PCR assay showed higher detection sensitivity than ASP-PCR and drug sensitivity testing, which could be performed to evaluate clarithromycin resistance of H. pylori.
文摘AIM: The interaction of mucosal addressin cell adhesion molecule 1 (MAdCAM-1) with integrin α4β7 mediates lymphocyte recruitment into mucosa-associated lymphoid tissue (MALT). Nodular gastritis is characterized by a unique military pattern on endoscopy representing increased numbers of lymphoid follicles with germinal center, strongly associated with H pylori infection. The purpose of this study was to address the implication of the MAdCAM-1/integrin β7 pathway in NG.METHODS: We studied 17 patients with NG and H pylori infection and 19 H pylori-positive and 14 H pylori-negative controls. A biopsy sample was taken from the antrum and snap-frozen for immunohistochemical analysis of MAdCAM1 and integrin β7. In simultaneous viewing of serial sections,the percentage of MAdCAM-1-positive to von Willebrand factor-positive vessels was calculated. We also performed immunostaining with anti-CD20, CD4, CD8 and CD68 antibodies to determine the lymphocyte subsets coexpressing integrin β7.RESULTS: Vascular endothelial MAdCAM-1 expression was more enhanced in gastric mucosa with than without H pylori infection. Of note, the percentages of MAdCAM-1-positive vessels were significantly higher in the lamina propria of NG patients than in H pylori-positive controls. Strong expression of MAdCAM-1 was identified adjacent to lymphoid follicles and dense lymphoid aggregates. Integrin β7-expressing mononuclear cells, mainly composed of CD20 and CD4 lymphocytes, were associated with vessels lined with MAdCAM-1-expressing endothelium.CONCLUSION: Our results suggest that the MAdCAM-1/integrin α4β7 homing system may participate in gastric inflammation in response to H pylori-infection and contributes to MALT formation, typically leading to the development of NG.
文摘AIM: To estimate the frequency of microdeletions in the long arm of Y-chromosome of 20 infertile males from South India. METHODS: Polymerase chain reaction (PCR) amplification using Y-specific STS of azoospermia factor (AZF) regions i.e., SY 84 for AZFa, SY 127 for AZFb and SY 254 for AZFc. RESULTS: Of the 20 infertile subjects 3 (15 %), one azoospermic and two oligozoospermic, showed microdeletions in the AZF region of Y-chromosome. CONCLUSION: The frequency of deletions involving AZF region of the Y-chromosome is 15 % in azoospermic and severely oligozoospermic infertile men. PCR amplification of AZF locus is useful for the diagnosis of microdeletions in the Y-chromosome.
基金financially supported by a grant(JPP-IMR Code:09-030) from the Ministry of Health,Malaysia
文摘Objective:To establish a polymerase chain reaction(PCR) technique based on cytochrome b {cytb) gene of mitochondria DNA(mtDNA) for blood meal identification.Methods:The PCR technique was established based on published information and validated using blood sample of laboratory animals of which their whole gene sequences are available in CenBank.PCR was next performed to compile gene sequences of different species of wild rodents.The primers used were complementary to the conserved region of the cytb gene of vertebrate's mtDNA.A total of 100 blood samples,both from laboratory animals and wild rodents were collected und analyzed.The obtained unknown sequences were compared with those in the GenBank database using BLAST program to identify the vertebrate animal species.Results:Gene sequences of 11 species of wild animals caught in 9 localities of Peninsular Malaysia were compiled using the established PCR. The animals involved were Rattus(rattus) tanezumi,Rattus tiomanicus,Leopoldamys sabanus, Tupaia glis,Tupaia minor,Niviventor cremoriventor,Rhinosciurus laticaudatus,Calloseiurus caniseps,Sundamys muelleri,Rattus rajah,and Maxomys whitelwadi.The BLAST results confirmed the host with exact or nearly exact matches(>89%identity).Ten new gene sequences have been deposited in CenBank database since September 2010.Conclusions:This study indicates that the PCR direct sequencing system using universal primer sets for vertebrate cytb gene is a promising technique for blood meal identification.
文摘In order to investigate the association of genotypes of HLA-DRB1 and HLA-DQB1 alleles with the genetic susceptibility of chronic urticaria (CU), genotypes of HLA-DRB1 and HLA-DQB1 genes were detected by polymerase chain reactions with sequence-specific primers (PCR-SSP) in 42 patients with CU (19 men and 23 women, mean age 30.67±12.45 y old as well as 193 racially matched healthy persons in ethnic Han from Hubei provinece. Gene frequencies of HLA-DRB1*12, *0901 (RR=3.11, χ2=7.579, P=0.006; RR=2.47, χ2=5.684, P=0.017) were significantly increased in CU patients as compared with that in healthy people. Gene frequencies of HLA-DQB1*05 (RR=0.26, χ2=6.683, P=0.01) were significantly decreased in CU patients. It was suggested that CU was found strongly associated with HLA-DRB1*12, *0901 and HLA-DQB1*05, the former might be the genetic markers for susceptibility to CU, but the latter might play a resistive role.
文摘AIM: To determine whether one genotype (A or non-A genotypes of HBV) predominated over the other during the course of HBV infection.METHODS: Four baboons were inoculated with HBV. DNA was extracted from serum obtained at monthly intervals postinoculation for 52 weeks and HBV DNA was amplified using primers specific for the core region containing an insert characteristic of genotype A (nt 2 354-2 359, numbering from the EcoRI site). The amplicons were cloned into PCRScriptTM and a minimum of 15 clones per time point were sequenced in both directions.RESULTS: Both genotypes persisted for the entire followup period of 52 weeks. Genotype non-A predominated in two baboons and genotype A in one baboon. Neither genotype predominated in the fourth baboon, as shown at a 5 % level of testing.CONCLUSION: No conclusions concerning the dominance of either genotype or the natural progression or replication rates of HBV could be drawn because the pattern of the genotypes found may have been caused by sampling fluctuations at the time of DNA extraction and cloning as a result of the very low viral loads in the baboon sera.
文摘AIM: Little has been known about the pathogenesis of nonerosive reflux disease (NERD). Recent studies have implicated interleukin 8 (IL-8) in the development and progression of gastroesophgeal reflux disease (GERD). The purpose of this study was to determine IL-8 RNA expression levels in NERD patients with or without subtle mucosal changes. METHODS: We studied 26 patients with NERD and 13 asymptomatic controls. Biopsy sample was taken from the esophagus 3 cm above the gast^oesophageal junction and snap frozen for measurement of IL-8 mRNA levels by real-time quantitative polymerase chain reaction (PCR). We also examined mRNA expression of IL-8 receptors, CXCR-1 and -2 by reverse transcriptase PCR. The patients were endoscopically classified into grade M (mucosal color changes without visible mucosal break) and N (neither minimal involvement nor mucosal break) of the modified Los Angeles classification. RESULTS: The relative IL-8 mRNA expression levels were significantly higher in esophageal mucosa of NERD patients than those in esophageal mucosa of the controls. There was a significant difference in IL-8 mRNA levels between grades M and N. The CXCR-1 and -2 mRNAs were consUtutdvely expressed in esophageal mucosa. CONCLUSION: Our results suggest that high IL-8 levels in esophageal mucosa may be involved in the pathogenesis of NERD through interaction with its receptors. NERD seems to be composed of a heterogeneous population in terms of not only endoscopically minimal involvement but also immune and inflammatory processes.
文摘Contagious bovine pleuropneumonia is a disease caused by Mycoplasma mycoides subspecie mycoides, a transboundary animal disease causing serious devastation to cattle producers in Africa. The study was designed to identify and characterize the pathogenic member of mycoplasma cluster the Mycoplasma mycoides subspecie mycoides (Mmm) isolated from cattle infected with the disease. Three hundred (300) samples of nasal swabs and pleural fluid from cattle showing signs of CBPP were analyzed using culture and biochemical identification techniques and polymerase chain reaction (PCR) using specific primers to determine the prevalence of contagious bovine pleuropneumonia in Nasarawa State, Nigeria. Isolation recorded a prevalence of 4% and PCR recorded a prevalence of 67.7%. Isolates subjected to PCR analysis produced an amplicon size of 548 bp and 1.1 k bp respectively for the Mycoplasma mycoides cluster and Mycoplasma mycoides subspecie mycoides. Sequencing of the 16 S rRNA gene blast search revealed 96% to 99% sequence homology of Mycoplasma mycoides subspecie mycoides compared with 14 available sequences in the gen bank at NCBI. Based on this investigation mass vaccination of cattle is recommended, isolation and PCR techniques could be used as diagnostic tools for CBPP disease in three agro ecological zones of Nasarawa state, Nigeria.