AIM: To develop a Fok-I nested polymerase chain reaction(PCR)-restriction fragment length polymorphism analysis(PRA) method for the detection of hepatitis B virus X region(HBx) V5 M mutation.METHODS: Nested PCR was ap...AIM: To develop a Fok-I nested polymerase chain reaction(PCR)-restriction fragment length polymorphism analysis(PRA) method for the detection of hepatitis B virus X region(HBx) V5 M mutation.METHODS: Nested PCR was applied into DNAs from 198 chronic patients at 2 different stages [121 patients with hepatocellular carcinoma(HCC) and 77 carrier patients]. To identify V5 M mutants, digestion of nested PCR amplicons by the restriction enzyme Fok-I(GGA TGN9↓) was done. For size comparison, the enzymetreated products were analyzed by electrophoresis on 2.5% agarose gels, stained with ethidium bromide, and visualized on a UV transilluminator.RESULTS: The assay enabled the identification of 69 patients(sensitivity of 34.8%; 46 HCC patients and 23 carrier patients). Our data also showed that V5 M prevalence in HCC patients was significantly higher than in carrier patients(47.8%, 22/46 patients vs 0%, 0/23 patients, P < 0.001), suggesting that HBx Ag V5 M mutation may play a pivotal role in HCC generation in chronic patients with genotype C infections.CONCLUSION: The Fok-I nested PRA developed in this study is a reliable and cost-effective method to detect HBx Ag V5 M mutation in chronic patients with genotype C2 infection.展开更多
Objective:To investigate the polyadenylation of mRNA in E. Coli. Methods: The mRNA of E. Coli was enriched from the total RNA with oligo(dT)-cellulose, prior to reverse transcription using oligo(dT)18as the primer. Do...Objective:To investigate the polyadenylation of mRNA in E. Coli. Methods: The mRNA of E. Coli was enriched from the total RNA with oligo(dT)-cellulose, prior to reverse transcription using oligo(dT)18as the primer. Double-stranded cDNA was subsequently synthesized, which was subjected to digestion with Sau3A I to produce multiple gene fragments for ligation with the adapters. PCR was carried out in 10 groups according to 10 different pairs of the selective primers, and the PCR products were then cloned into T-vectors. Results: More than 100 gene fragments had been cloned, 30 of which were sequenced. Conclusion:Polyadenylation of E. Coli mRNA may not be a biochemical curiosity but a general attribute of bacterial mRNA.展开更多
Objective: To investigate the association between the diversity of urease gene and urease activity of clinical isolates of Helicobacter pylori (H. pylori). Methods: Polymerase chain reaction-restriction fragment lengt...Objective: To investigate the association between the diversity of urease gene and urease activity of clinical isolates of Helicobacter pylori (H. pylori). Methods: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of urease gene and rapid urease activity test were used to study the urease activity of different clinical isolates of H. pylori. Results: H. pylori clinical isolates were divided into 4 types according to their PCR-RFLP results of urease gene and urease activity. Type Ⅰ , possessing strong urease activity (0. 11) and presented 1 fragment of 1. 7 kb by PCR-RFLP, had close relations with gastric ulcer; type Ⅱ , with the weakest urease activity (0. 07) and 2 fragments (1. 3 and 0. 4 kb respectively) , was associated with duodenal bulb ulcer; type Ⅲ , with the strongest urease activity (0. 12) and 2 fragments (0. 4 and 0. 17 kb) with or without 1 fragment (0. 23 or 0. 37 kb) , was responsible for gastritis; type Ⅳ , with weak urease activity (0. 09) and 2展开更多
Objective To understand the deletion in the survival motor neuron gene (SMN) of childhood onset spinal muscular atrophy (SMA) in Chinese, and the value of diagnosis of SMA using polymerase chain reaction restric...Objective To understand the deletion in the survival motor neuron gene (SMN) of childhood onset spinal muscular atrophy (SMA) in Chinese, and the value of diagnosis of SMA using polymerase chain reaction restriction fragment length polymorphism (PCR RFLP)method. Methods\ Deletions of SMN gene of exon 7 and 8 in 10 cases of presumed SMA, and 20 normal controls from 6 families and 30 unrelated controls were performed by PCR RFLP analysis. Results\ Deletions of SMN gene detected in 9 of 10 (90%) cases of presumed SMA . No deletions of SMN in the telomere were found in the other members of families and controls.Conclusion\ PCR RFLP is a sensitive, specific and simple method in diagnosis of SMA.\;展开更多
Background Nontuberculous mycobacterium （NTM） had been reported to cause cutaneous infections which are difficult to interpret due to the variability of the clinical manifestations. Among NTM infections, Mycobacteri...Background Nontuberculous mycobacterium （NTM） had been reported to cause cutaneous infections which are difficult to interpret due to the variability of the clinical manifestations. Among NTM infections, Mycobacterium marinum （M. marinum） are mostly seen to cause skin infection. It is therefore important to establish a rapid approach for detection and identification of M. marinum from lesions of patients with suspected M. marinum infections. Methods Specimens were obtained from 5 patients with swimming pool granuloma. DNA was extracted and polymerase chain reaction （PCR） was performed. PCR products were digested with Hae III and BstE II, then analysed by pattern restriction analysis to detect heat shock protein （hsp） 65 kD gene. Results The 65 kD hsp gene was found in all specimens from patients with swimming pool granuloma. PCR restriction analysis （PRA） identified all 5 samples to be M. marinum infections, and the result was consistent with that of routine bacteriological identification. The lesions subsided or markedly improved upon treatment. Conclusions PRA is a sensitive, specific and rapid method in identification of mycobacteria. Application of this method will be helpful for early diagnosis of mycobacterial skin infections.展开更多
INTRODUCTIONLeprosy caused by Mvcobacterium leprae （M. leprae）, is a chronic granulomatous disease affecting the skin and peripheral nervous system, which is transmitted through direct contact with nontreated or ina...INTRODUCTIONLeprosy caused by Mvcobacterium leprae （M. leprae）, is a chronic granulomatous disease affecting the skin and peripheral nervous system, which is transmitted through direct contact with nontreated or inadequate treatment patients. Diagnosis of leprosy depends on the clinical signs and symptoms and slit skin smear positivity. However, it＇s sometimes similar with other granulomatous disease caused by mycobacterial infection. Early stage leprosy is difficult to diagnose by clinical criterion alone because the sensitivity of acid-fast bacilli staining is quite low. The polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP） shows the great advantage in rapid identification and diagnosis for early cases and has a differentiation between leprosy and nonleprosy cases.展开更多
AIM: To evaluate tumor necrosis factor-α converting enzyme(TACE) methylation status in patients with chronic hepatitis B(CHB).METHODS: Eighty patients with hepatitis B e antigen(HBe Ag)-positive CHB, 80 with HBe Ag-n...AIM: To evaluate tumor necrosis factor-α converting enzyme(TACE) methylation status in patients with chronic hepatitis B(CHB).METHODS: Eighty patients with hepatitis B e antigen(HBe Ag)-positive CHB, 80 with HBe Ag-negative CHB, and 40 healthy controls(HCs) were randomly enrolled in this study. Genomic DNA was extracted from peripheral blood mononuclear cells and methylation status of TACE promoter was determined by methylation-specific polymerase chain reaction. The clinical and laboratory parameters were collected.RESULTS: One hundred and thirty of 160 patients with CHB(81.25%) and 38 of 40 HCs(95%) displayed TACE promoter methylation. The difference was significant(χ2 = 4.501, P < 0.05). TACE promoter methylation frequency in HBe Ag-positive CHB(58/80, 72.5%) was significantly lower than that in HBe Ag-negative CHB(72/80, 90%; χ2 = 8.041, P < 0.01) and HCs(χ2 = 8.438, P < 0.01). However, no significant difference was observed in the methylation frequency between HBe Agnegative CHB and HCs(χ2 = 0.873, P > 0.05). In the HBe Ag-positive group, TACE methylation frequency was significantly negatively correlated with HBe Ag(r =-0.602, P < 0.01), alanine aminotransferase(r =-0.461, P < 0.01) and aspartate aminotransferase(r =-0.329, P < 0.01). CONCLUSION: Patients with HBe Ag-positive CHB have aberrant demethylation of the TACE promoter, which may potentially serve as a biomarker for HBe Ag seroconversion.展开更多
We report here a simple and efficient method for site-directed mutagenesis using polymer-ase chain reaction (PCR). In constructing a new expression plasmid for the EcoRI restrictiongene, we made two point mutations. W...We report here a simple and efficient method for site-directed mutagenesis using polymer-ase chain reaction (PCR). In constructing a new expression plasmid for the EcoRI restrictiongene, we made two point mutations. While one created a new Sall site prior to the SDsequence, the other replaced Glu144 with Lys. A 1.5 kb Sall-PstI fragment isolated frompER101 was used as the template. Two 25 mer oligonucleotide primers containing the de-sired mutations were synthesized and used to direct PCR amplification with Taq DNA poly-merase. About 0.5μg of the 0.49 kb fragment was obtained from 0.05 μ of the 1.5 kb frag-ment by carrying out polymerase chain reaction for 30 cycles. As calculated theoretically,99% of the product contained the desired mutations. The product was cloned into pUC19using Sall and PstI, two of the transformed colonies were randomly chosen for sequence anal-ysis, and both of them were shown to contain the desired mutations. Finally, the amplifiedfragment was cloned into pER304 to place展开更多
基金Supported by a National Research Foundation(NRF)of Korea grant funded by the Korean government(Ministry of EducationScience+2 种基金and TechnologyMEST)Grant No.2013-005810
文摘AIM: To develop a Fok-I nested polymerase chain reaction(PCR)-restriction fragment length polymorphism analysis(PRA) method for the detection of hepatitis B virus X region(HBx) V5 M mutation.METHODS: Nested PCR was applied into DNAs from 198 chronic patients at 2 different stages [121 patients with hepatocellular carcinoma(HCC) and 77 carrier patients]. To identify V5 M mutants, digestion of nested PCR amplicons by the restriction enzyme Fok-I(GGA TGN9↓) was done. For size comparison, the enzymetreated products were analyzed by electrophoresis on 2.5% agarose gels, stained with ethidium bromide, and visualized on a UV transilluminator.RESULTS: The assay enabled the identification of 69 patients(sensitivity of 34.8%; 46 HCC patients and 23 carrier patients). Our data also showed that V5 M prevalence in HCC patients was significantly higher than in carrier patients(47.8%, 22/46 patients vs 0%, 0/23 patients, P < 0.001), suggesting that HBx Ag V5 M mutation may play a pivotal role in HCC generation in chronic patients with genotype C infections.CONCLUSION: The Fok-I nested PRA developed in this study is a reliable and cost-effective method to detect HBx Ag V5 M mutation in chronic patients with genotype C2 infection.
文摘Objective:To investigate the polyadenylation of mRNA in E. Coli. Methods: The mRNA of E. Coli was enriched from the total RNA with oligo(dT)-cellulose, prior to reverse transcription using oligo(dT)18as the primer. Double-stranded cDNA was subsequently synthesized, which was subjected to digestion with Sau3A I to produce multiple gene fragments for ligation with the adapters. PCR was carried out in 10 groups according to 10 different pairs of the selective primers, and the PCR products were then cloned into T-vectors. Results: More than 100 gene fragments had been cloned, 30 of which were sequenced. Conclusion:Polyadenylation of E. Coli mRNA may not be a biochemical curiosity but a general attribute of bacterial mRNA.
基金Supported by National Natural Science Foundation of China (No. 39570334)
文摘Objective: To investigate the association between the diversity of urease gene and urease activity of clinical isolates of Helicobacter pylori (H. pylori). Methods: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of urease gene and rapid urease activity test were used to study the urease activity of different clinical isolates of H. pylori. Results: H. pylori clinical isolates were divided into 4 types according to their PCR-RFLP results of urease gene and urease activity. Type Ⅰ , possessing strong urease activity (0. 11) and presented 1 fragment of 1. 7 kb by PCR-RFLP, had close relations with gastric ulcer; type Ⅱ , with the weakest urease activity (0. 07) and 2 fragments (1. 3 and 0. 4 kb respectively) , was associated with duodenal bulb ulcer; type Ⅲ , with the strongest urease activity (0. 12) and 2 fragments (0. 4 and 0. 17 kb) with or without 1 fragment (0. 23 or 0. 37 kb) , was responsible for gastritis; type Ⅳ , with weak urease activity (0. 09) and 2
文摘Objective To understand the deletion in the survival motor neuron gene (SMN) of childhood onset spinal muscular atrophy (SMA) in Chinese, and the value of diagnosis of SMA using polymerase chain reaction restriction fragment length polymorphism (PCR RFLP)method. Methods\ Deletions of SMN gene of exon 7 and 8 in 10 cases of presumed SMA, and 20 normal controls from 6 families and 30 unrelated controls were performed by PCR RFLP analysis. Results\ Deletions of SMN gene detected in 9 of 10 (90%) cases of presumed SMA . No deletions of SMN in the telomere were found in the other members of families and controls.Conclusion\ PCR RFLP is a sensitive, specific and simple method in diagnosis of SMA.\;
文摘Background Nontuberculous mycobacterium （NTM） had been reported to cause cutaneous infections which are difficult to interpret due to the variability of the clinical manifestations. Among NTM infections, Mycobacterium marinum （M. marinum） are mostly seen to cause skin infection. It is therefore important to establish a rapid approach for detection and identification of M. marinum from lesions of patients with suspected M. marinum infections. Methods Specimens were obtained from 5 patients with swimming pool granuloma. DNA was extracted and polymerase chain reaction （PCR） was performed. PCR products were digested with Hae III and BstE II, then analysed by pattern restriction analysis to detect heat shock protein （hsp） 65 kD gene. Results The 65 kD hsp gene was found in all specimens from patients with swimming pool granuloma. PCR restriction analysis （PRA） identified all 5 samples to be M. marinum infections, and the result was consistent with that of routine bacteriological identification. The lesions subsided or markedly improved upon treatment. Conclusions PRA is a sensitive, specific and rapid method in identification of mycobacteria. Application of this method will be helpful for early diagnosis of mycobacterial skin infections.
基金The study was supported by a grant from the National Natural Science Foundation of China （No. 81271761）.
文摘INTRODUCTIONLeprosy caused by Mvcobacterium leprae （M. leprae）, is a chronic granulomatous disease affecting the skin and peripheral nervous system, which is transmitted through direct contact with nontreated or inadequate treatment patients. Diagnosis of leprosy depends on the clinical signs and symptoms and slit skin smear positivity. However, it＇s sometimes similar with other granulomatous disease caused by mycobacterial infection. Early stage leprosy is difficult to diagnose by clinical criterion alone because the sensitivity of acid-fast bacilli staining is quite low. The polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP） shows the great advantage in rapid identification and diagnosis for early cases and has a differentiation between leprosy and nonleprosy cases.
基金Supported by Key Project of Chinese Ministry of Science and Technology,No.2012ZX10002007 and No.2013ZX10002001National Natural Science Foundation of China,No.81171579,No.81201287 and No.81371832Science and Technology Development Plan of Shandong Province,China,No.2014GSF118068
文摘AIM: To evaluate tumor necrosis factor-α converting enzyme(TACE) methylation status in patients with chronic hepatitis B(CHB).METHODS: Eighty patients with hepatitis B e antigen(HBe Ag)-positive CHB, 80 with HBe Ag-negative CHB, and 40 healthy controls(HCs) were randomly enrolled in this study. Genomic DNA was extracted from peripheral blood mononuclear cells and methylation status of TACE promoter was determined by methylation-specific polymerase chain reaction. The clinical and laboratory parameters were collected.RESULTS: One hundred and thirty of 160 patients with CHB(81.25%) and 38 of 40 HCs(95%) displayed TACE promoter methylation. The difference was significant(χ2 = 4.501, P < 0.05). TACE promoter methylation frequency in HBe Ag-positive CHB(58/80, 72.5%) was significantly lower than that in HBe Ag-negative CHB(72/80, 90%; χ2 = 8.041, P < 0.01) and HCs(χ2 = 8.438, P < 0.01). However, no significant difference was observed in the methylation frequency between HBe Agnegative CHB and HCs(χ2 = 0.873, P > 0.05). In the HBe Ag-positive group, TACE methylation frequency was significantly negatively correlated with HBe Ag(r =-0.602, P < 0.01), alanine aminotransferase(r =-0.461, P < 0.01) and aspartate aminotransferase(r =-0.329, P < 0.01). CONCLUSION: Patients with HBe Ag-positive CHB have aberrant demethylation of the TACE promoter, which may potentially serve as a biomarker for HBe Ag seroconversion.
文摘We report here a simple and efficient method for site-directed mutagenesis using polymer-ase chain reaction (PCR). In constructing a new expression plasmid for the EcoRI restrictiongene, we made two point mutations. While one created a new Sall site prior to the SDsequence, the other replaced Glu144 with Lys. A 1.5 kb Sall-PstI fragment isolated frompER101 was used as the template. Two 25 mer oligonucleotide primers containing the de-sired mutations were synthesized and used to direct PCR amplification with Taq DNA poly-merase. About 0.5μg of the 0.49 kb fragment was obtained from 0.05 μ of the 1.5 kb frag-ment by carrying out polymerase chain reaction for 30 cycles. As calculated theoretically,99% of the product contained the desired mutations. The product was cloned into pUC19using Sall and PstI, two of the transformed colonies were randomly chosen for sequence anal-ysis, and both of them were shown to contain the desired mutations. Finally, the amplifiedfragment was cloned into pER304 to place
