AIM: To design and establish a method of multiplex PCR normalization for simultaneously detecting HBV and HCV.METHODS: Two pairs of primers with a 20 bp joint sequence were used to amplify the target genes of HBV and ...AIM: To design and establish a method of multiplex PCR normalization for simultaneously detecting HBV and HCV.METHODS: Two pairs of primers with a 20 bp joint sequence were used to amplify the target genes of HBV and HCV by two rounds of amplification. After the two rounds of amplification all the products had the joint sequence. Then the joint sequence was used as primers to finish the last amplification. Finally multiplex PCR was normalized to a single PCR system to eliminate multiplex factor interference. Four kinds of nucleic acid extraction methods were compared and screened. A multiplex PCR normalization method was established and optimized by orthogonal design of 6 key factors. Then twenty serum samples were detected to evaluate the validity and authenticity of this method.RESULTS: The sensitivity, specificity, diagnostic index and efficiency were 83.3%, 70%, 153.3% and 72.2%,respectively for both HBsAg and anti-HCV positive patients,and were 78.6%, 80%, 258.6% and 79.2%, respectively for HBsAg positive patients, and were 75%, 90%, 165% and 83.3%, respectively for anti-HCV positive patients.CONCLUSION: The multiplex PCR normalization method shows a broad prospect in simultaneous amplification of multiple genes of different sources. It is practical, correct and authentic, and can be used to prevent and control HBV and HCV.展开更多
Objective To explore the optimal primer ratio and concentration of asymmetric polymerase chain reaction (A-PCR) in producing hepatitis B virus (HBV) single-stranded DNA (ssDNA) for pyrosequencing. Methods A-PCR was ca...Objective To explore the optimal primer ratio and concentration of asymmetric polymerase chain reaction (A-PCR) in producing hepatitis B virus (HBV) single-stranded DNA (ssDNA) for pyrosequencing. Methods A-PCR was carried out to generate HBV ssDNA with forward to reverse primers of different ratios (50∶1, 100∶1) and concentrations (13.0 pmol/25μL and 0.14 pmol/25μL, 19.5 pmol/25μL and 0.21 pmol/25μL), and the product yield and quality were compared respectively. Results The forward to reverse primer ratio of 50∶1 provided better yield and concentration of 19.5 pmol/25μL and 0.21 pmol//25μL generated a clearer band. Conclusion A simple and feasible method to produce HBV ssDNA for pyrosequencing in batch is established.展开更多
AIM:To evaluate the covalently closed circle DNA (cccDNA) level of hepatitis B virus (HBV) in patients' liver and sera. METHODS:HBV DNA was isolated from patients' liver biopsies and sera.A sensitive real-time...AIM:To evaluate the covalently closed circle DNA (cccDNA) level of hepatitis B virus (HBV) in patients' liver and sera. METHODS:HBV DNA was isolated from patients' liver biopsies and sera.A sensitive real-time PCR method,which is capable of differentiation of HBV viral genomic DNA and cccDNA,was used to quantify the total HBV cccDNA.The total HBV viral DNA was quantitated by real-time PCR using a HBV diagnostic kit (PG Biotech,LTD,Shenzhen,China) described previously. RESULTS:For the first time,we measured the level of HBV DNA and cccDNA isolated from ten HBV patients' liver biopsies and sera.In the liver biopsies,cccDNA was detected from all the biopsy samples.The copy number of cccDNA ranged from from 0.03 to 173.1 per cell,the copy number of total HBV DNA ranged from 0.08 to 3 717 per cell.The ratio of total HBV DNA to cccDNA ranged from 1 to 3 406.In the sera, cccDNA was only detected from six samples whereas HBV viral DNA was detected from all ten samples.The ratio of cccDNA to total HBV DNA ranged from 0 to 1.77%.To further investigate the reason why cccDNA could only be detected in some patients' sera,we performed longitudinal studies.The cccDNA was detected from the patients' sera with HBV reactivation but not from the patients' sera without HBV reactivation.The level of cccDNA in the sera was correlated with ALT and viral load in the HBV reactivation patients. CONCLUSION:HBV cccDNA is actively transcribed and replicated in some patients' hepatoo/tes,which is reflected by a high ratio of HBV total DNA vs cccDNA.Detection of cccDNA in the liver biopsy will provide an end-point for the anti-HBV therapy.The occurrence of cccDNA in the sera is an early signal of liver damage,which may be another important clinical parameter.展开更多
A novel HBV integration site involved in hepatocarcinogenesis was investigated. The HBV DNA integration sites were detected by Alu-PCR in hepatocellular carcinoma tissues, matched surrounding liver tissues in 30 patie...A novel HBV integration site involved in hepatocarcinogenesis was investigated. The HBV DNA integration sites were detected by Alu-PCR in hepatocellular carcinoma tissues, matched surrounding liver tissues in 30 patients with hepatitis B-related hepatocellular carcinoma (HCC) and 3 cases of normal liver tissues. The integration sites and flanking sequences in human genome were sequenced and blasted, and the expression of integrated HBV genes was determined by reverse transcriptase-polymerase chain reaction (RT-PCR). The influence of the up-regulated expression of integrated genes on hepatocarcinogenesis was analyzed. Nineteen integration sites of HBV DNA into HCC tissues were obtained by RT-PCR and sequencing. These genes encoding proteins were: LOC51030, LOC157777, minichromosome maintenance complex component 3 associated protein (MCM3AP), MCTP1, SH3 and multiple ankyrin repeat domains 2 isoform 2, CCDC40, similar to HCG2033532, mitochondrial ribosomal S5 pseudogene 4. One of them was integrated into the intron of MCM3AP. RT-PCR demonstrated that the expression levels of MCM3AP mRNA in HCC tissues, matched surrounding liver tissues and normal liver tissues were in a descendent order. The ratio of MCM3AP mRNA to the GAPDH mRNA in these three tissues was 1.07375, 0.21573, 0.06747 respectively, with the difference being statistically significant among them (P<0.05). Meanwhile, the expression levels of MCM3AP mRNA from HCC tissues in which HBV DNA integrated into MCM3AP were still significantly higher than those without HBV DNA integrated into MCM3AP. It was concluded that the HBV DNA integration sites into human genome were random, and MCM3AP was a new site. The up-regulated MCM3AP mRNA may affect flanking sequences which promote the hepatocarcinogenesis.展开更多
Objective: To amplify HBV-RNase H gene fragment and express RNase H for further use in the studiesof HBV associated liver diseases. Methods: The encoding gene of HBV-RNase H was separately amplified for thefirst half ...Objective: To amplify HBV-RNase H gene fragment and express RNase H for further use in the studiesof HBV associated liver diseases. Methods: The encoding gene of HBV-RNase H was separately amplified for thefirst half and second half (H1 and H2)by PCR from full length HBV gene and cloned into pT7Blue-T vector.Clones were first screened by digestion with Xbal and HindⅢ enzyme for the correct size, and analyzed further byDNA sequencing. The RNase H1 and H2 fragments isolated from XbaⅠ and HindⅢ digestion products of pT7 BlueRNaseH plasmid were ligated to the GSTag expressing vectors separately, and expressed in E. coli BL21. The expressed proteins were checked by PAGE gel and Western blot. Results: Both H1 and H2 nucleotide seqences wereconsisted with the known genes and the proteins, with correct size, were further confirmed by western blot to bethe GST and RNaseHl or H2 fusion proteins. Conclusion: The successful cloning and expression of HBV-RNase Hwill contribute to further research and application in HBV associated diseases.展开更多
In the present study 100 cardiac patients were randomly selected from the cardiology ward, Allied Hospital Faisalabad, Pakistan. All the selected cases were analysed for different parameters like Hepatitis B surface A...In the present study 100 cardiac patients were randomly selected from the cardiology ward, Allied Hospital Faisalabad, Pakistan. All the selected cases were analysed for different parameters like Hepatitis B surface Antigen (HbsAg), Bilirubin, Alkaline phosphatase, serum glutamic pyruvic transaminase, and serum glutamic-oxaloacetic transaminase. Out of total 16% patients were lying in the age of 21-30 year, 25% in the age of 31-40 year, 35% in the age of 41-50 year, 19% in the age of 51-60 years and 5% patients in the age of 61-70 years. No subject was found positive in age group 21-30 years patients. 35% patients have higher value of SGPT while, other 26% were with higher value of SGOT. Rest of the 32 and 24% have higher ALP and Bilirubin levels, respectively. Assay profile revealed that ALP level was increased with increasing age, body mass index, Creactive protein, diabetes, smoking, sex, serum uric acid, lead, cadmium, hypercholesterolemia, lesion of liver and cardiovascular disease. The serum of the eight HbsAg positive cases were tested for the presence of HBV through PCR and no sample was found positive. At the end of the study, PCR amplified samples were run on 1.5% agarose gel to confirm the case.展开更多
基金Supported by the National Key Technology Research and Development Program of China during the 9th Five-Year Plan Period, No.96C020117
文摘AIM: To design and establish a method of multiplex PCR normalization for simultaneously detecting HBV and HCV.METHODS: Two pairs of primers with a 20 bp joint sequence were used to amplify the target genes of HBV and HCV by two rounds of amplification. After the two rounds of amplification all the products had the joint sequence. Then the joint sequence was used as primers to finish the last amplification. Finally multiplex PCR was normalized to a single PCR system to eliminate multiplex factor interference. Four kinds of nucleic acid extraction methods were compared and screened. A multiplex PCR normalization method was established and optimized by orthogonal design of 6 key factors. Then twenty serum samples were detected to evaluate the validity and authenticity of this method.RESULTS: The sensitivity, specificity, diagnostic index and efficiency were 83.3%, 70%, 153.3% and 72.2%,respectively for both HBsAg and anti-HCV positive patients,and were 78.6%, 80%, 258.6% and 79.2%, respectively for HBsAg positive patients, and were 75%, 90%, 165% and 83.3%, respectively for anti-HCV positive patients.CONCLUSION: The multiplex PCR normalization method shows a broad prospect in simultaneous amplification of multiple genes of different sources. It is practical, correct and authentic, and can be used to prevent and control HBV and HCV.
基金supported by the National Natural Science Foundation of China (No.60878056)the Doctoral Foundation of Xi’an Jiaotong University (DFXJTU2004-12)
文摘Objective To explore the optimal primer ratio and concentration of asymmetric polymerase chain reaction (A-PCR) in producing hepatitis B virus (HBV) single-stranded DNA (ssDNA) for pyrosequencing. Methods A-PCR was carried out to generate HBV ssDNA with forward to reverse primers of different ratios (50∶1, 100∶1) and concentrations (13.0 pmol/25μL and 0.14 pmol/25μL, 19.5 pmol/25μL and 0.21 pmol/25μL), and the product yield and quality were compared respectively. Results The forward to reverse primer ratio of 50∶1 provided better yield and concentration of 19.5 pmol/25μL and 0.21 pmol//25μL generated a clearer band. Conclusion A simple and feasible method to produce HBV ssDNA for pyrosequencing in batch is established.
基金SuppoSed by CRCG grant from the University of Hong KongCERG grant from University Grant Council of Hong Kong Research Fund from Science and Technology Commission of Shanghai,China
文摘AIM:To evaluate the covalently closed circle DNA (cccDNA) level of hepatitis B virus (HBV) in patients' liver and sera. METHODS:HBV DNA was isolated from patients' liver biopsies and sera.A sensitive real-time PCR method,which is capable of differentiation of HBV viral genomic DNA and cccDNA,was used to quantify the total HBV cccDNA.The total HBV viral DNA was quantitated by real-time PCR using a HBV diagnostic kit (PG Biotech,LTD,Shenzhen,China) described previously. RESULTS:For the first time,we measured the level of HBV DNA and cccDNA isolated from ten HBV patients' liver biopsies and sera.In the liver biopsies,cccDNA was detected from all the biopsy samples.The copy number of cccDNA ranged from from 0.03 to 173.1 per cell,the copy number of total HBV DNA ranged from 0.08 to 3 717 per cell.The ratio of total HBV DNA to cccDNA ranged from 1 to 3 406.In the sera, cccDNA was only detected from six samples whereas HBV viral DNA was detected from all ten samples.The ratio of cccDNA to total HBV DNA ranged from 0 to 1.77%.To further investigate the reason why cccDNA could only be detected in some patients' sera,we performed longitudinal studies.The cccDNA was detected from the patients' sera with HBV reactivation but not from the patients' sera without HBV reactivation.The level of cccDNA in the sera was correlated with ALT and viral load in the HBV reactivation patients. CONCLUSION:HBV cccDNA is actively transcribed and replicated in some patients' hepatoo/tes,which is reflected by a high ratio of HBV total DNA vs cccDNA.Detection of cccDNA in the liver biopsy will provide an end-point for the anti-HBV therapy.The occurrence of cccDNA in the sera is an early signal of liver damage,which may be another important clinical parameter.
基金supported by grants from the National Natural Sciences Foundation of China (No. 30872237)the National Program on Key Basic Research Project (973 Program) (No. 2007CB512900)
文摘A novel HBV integration site involved in hepatocarcinogenesis was investigated. The HBV DNA integration sites were detected by Alu-PCR in hepatocellular carcinoma tissues, matched surrounding liver tissues in 30 patients with hepatitis B-related hepatocellular carcinoma (HCC) and 3 cases of normal liver tissues. The integration sites and flanking sequences in human genome were sequenced and blasted, and the expression of integrated HBV genes was determined by reverse transcriptase-polymerase chain reaction (RT-PCR). The influence of the up-regulated expression of integrated genes on hepatocarcinogenesis was analyzed. Nineteen integration sites of HBV DNA into HCC tissues were obtained by RT-PCR and sequencing. These genes encoding proteins were: LOC51030, LOC157777, minichromosome maintenance complex component 3 associated protein (MCM3AP), MCTP1, SH3 and multiple ankyrin repeat domains 2 isoform 2, CCDC40, similar to HCG2033532, mitochondrial ribosomal S5 pseudogene 4. One of them was integrated into the intron of MCM3AP. RT-PCR demonstrated that the expression levels of MCM3AP mRNA in HCC tissues, matched surrounding liver tissues and normal liver tissues were in a descendent order. The ratio of MCM3AP mRNA to the GAPDH mRNA in these three tissues was 1.07375, 0.21573, 0.06747 respectively, with the difference being statistically significant among them (P<0.05). Meanwhile, the expression levels of MCM3AP mRNA from HCC tissues in which HBV DNA integrated into MCM3AP were still significantly higher than those without HBV DNA integrated into MCM3AP. It was concluded that the HBV DNA integration sites into human genome were random, and MCM3AP was a new site. The up-regulated MCM3AP mRNA may affect flanking sequences which promote the hepatocarcinogenesis.
文摘Objective: To amplify HBV-RNase H gene fragment and express RNase H for further use in the studiesof HBV associated liver diseases. Methods: The encoding gene of HBV-RNase H was separately amplified for thefirst half and second half (H1 and H2)by PCR from full length HBV gene and cloned into pT7Blue-T vector.Clones were first screened by digestion with Xbal and HindⅢ enzyme for the correct size, and analyzed further byDNA sequencing. The RNase H1 and H2 fragments isolated from XbaⅠ and HindⅢ digestion products of pT7 BlueRNaseH plasmid were ligated to the GSTag expressing vectors separately, and expressed in E. coli BL21. The expressed proteins were checked by PAGE gel and Western blot. Results: Both H1 and H2 nucleotide seqences wereconsisted with the known genes and the proteins, with correct size, were further confirmed by western blot to bethe GST and RNaseHl or H2 fusion proteins. Conclusion: The successful cloning and expression of HBV-RNase Hwill contribute to further research and application in HBV associated diseases.
文摘In the present study 100 cardiac patients were randomly selected from the cardiology ward, Allied Hospital Faisalabad, Pakistan. All the selected cases were analysed for different parameters like Hepatitis B surface Antigen (HbsAg), Bilirubin, Alkaline phosphatase, serum glutamic pyruvic transaminase, and serum glutamic-oxaloacetic transaminase. Out of total 16% patients were lying in the age of 21-30 year, 25% in the age of 31-40 year, 35% in the age of 41-50 year, 19% in the age of 51-60 years and 5% patients in the age of 61-70 years. No subject was found positive in age group 21-30 years patients. 35% patients have higher value of SGPT while, other 26% were with higher value of SGOT. Rest of the 32 and 24% have higher ALP and Bilirubin levels, respectively. Assay profile revealed that ALP level was increased with increasing age, body mass index, Creactive protein, diabetes, smoking, sex, serum uric acid, lead, cadmium, hypercholesterolemia, lesion of liver and cardiovascular disease. The serum of the eight HbsAg positive cases were tested for the presence of HBV through PCR and no sample was found positive. At the end of the study, PCR amplified samples were run on 1.5% agarose gel to confirm the case.
