Effect of transcranial direct current stimulation combined with contralateral control functional electrical stimulation on upper limb function of stroke patients / 中国康复理论与实践

ObjectiveTo investigate the effect of anodal transcranial direct current stimulation (atDCS) combined with contralaterally controlled functional electrical stimulation (CCFES) on upper limb motor function of stroke patients. MethodsFrom January to December, 2022, 60 stroke patients from Zhejiang Provincial People's Hospital were randomly divided into atDCS group (n = 20), CCFES group (n = 20) and combined group (n = 20). All the groups accepted routine rehabilitation, while atDCS group accepted atDCS on the primary motor (M1) area of the damaged hemisphere, CCFES group accepted CCFES on the triceps brachii and extensors carpi muscles, and the combined group accepted atDCS on the M1 area of damaged hemisphere and CCFES on triceps brachii and extensors carpi muscles, for six weeks. They were assessed with Fugl-Meyer Assessment-Upper Extremities (FMA-UE), Wolf Motor Function Test (WMFT), and the electromyography root mean square (RMS) ratio of bilateral triceps brachii muscles and extensor carpi muscles, before and after treatment. ResultsThe FMA-UE score, WMFT score, and the RMS ratio of the triceps brachii muscles and extensor carpi muscles improved in all the groups after treatment (|t| > 5.007, P < 0.001), and improved the most in the combined group (F > 14.492, P < 0.001). ConclusionatDCS combined with CCFES can effectively improve upper limb motor function of stroke patients.

Construction of a medical service experience system based on staff role play / 中华医院管理杂志

Objective:To build a medical service experience system based on role play of hospital staff, and explore its contribution to upgrading medical service, elevating medical service quality, and improving patients′ satisfaction.Methods:An indicator system for medical service experience was developed via literature review. 20 newly recruited hospital employees in 2019 were sampled randomly to form a service experience team, who were arranged to experience the full medical service process of outpatients and inpatients based on role play. The questionnaire for service quality based on patient perception and expectation was used to survey the expectation and perception values of those role players on their medical service experiences, and summarize the defects found on such six dimensions as hospital reliability, responsiveness, tangibility, assurance, empathy and cost-effectiveness. After due corrections of these defects, further experiences and patient satisfaction survey were made to assess the effects of such actions taken. Paired t test was used respectively in statistical analysis of service experience value and actual perception value, as well as the perception values before and after the actions taken, and χ2 test was used for a statistical analysis of patient satisfaction. Results:Role players experienced respectively at the emergency, outpatient and inpatient departments of the hospital for a week in August 2019. Statistics of their experiences indicated≥0.50 points difference between the mean expectation and perception values on doctor-patient communication, patient help responsiveness, outpatient process, examination report delivery duration, service attitude, patient trust on medical workers, medical environment and patient privacy protection.Following a 3-month reform, role players found improvements in such aspects as hospital reliability, responsiveness, tangibility, assurance, empathy and cost-effectiveness; patient satisfaction improved sizably in December 2019 over January of the same year( P<0.05). Conclusions:The construction and application of the medical service experience system based on role play of hospital staff prove highly useful in upgrading medical service actions, improving medical service quality, and improving patient satisfaction.

Clinical emergence features and implications of hepatitis B virus rtA181T mutation / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To determine the mutational profile and clinical implications of the viral reverse-transcriptase (rt)A 181T mutation in hepatitis B virus (HBV) through population-based analysis of clinical samples.</p><p><b>METHODS</b>Serum samples from 3, 013 patients who visited The 302 Hospital (Beijing, China) were investigated.HBV DNA was extracted and HBV mutations and genotypes were determined by direct sequencing.Recombinant plasmids harboring the rtA181T/sW172* mutant or wild type sequence were constructed and transfected into the HepG2 cell line. The levels of HBsAg in culture supernatants were compared and statistically analyzed.</p><p><b>RESULTS</b>The incidence of rtA181T across the study population was 4.1% (165/3, 013), and most of the rtAl 81T-positive patients had received adefovir and/or lamivudine.Forty percent (66/165) of the rtA 181T cases were single mutants and treatment responsive, 46.1% (76/165) included the adefovir-resistant mutation rtA 181 V/N236T, 12.1% (20/165) included the lamivudine-resistant mutation rtM204V/rtM2041, and 1.8% (3/165) included multidrug-resistant mutations.Interestingly, 73.9% (122/165) of the rtA181T-positive samples were detected with co-existing wild-type nucleotides at the site. The rates of HBV/C to HBV/B were 92.1% to 7.9% in the rtA181T-positive patients, but 82.1% to 17.9% in the rtA181T-negative paticnts (P less than 0.01).Almost all (98.2%; 129/165) of the rtA181T led to sW172*, while only 1.8% of the rtA181T (3/165) led to sW172L or sW172S.HBsAg secretion in vitro was reduced from the rtA181T/ sW172* strain, but there was no significant difference observed in the average serum HBsAg and HBV DNA levels of patients who carried or did not carry the mutant.</p><p><b>CONCLUSION</b>The HBV rtA181T mutation is closely associated with adefovir and lamivudine exposure.rtA181T may led to sW172*, culminating in suppression of HBsAg secretion.However, co-existence of the mutant with wild-type sequences was common among our patient population, suggesting that the mutation had little impact on serum HBsAg and HBV DNA levels across the clinical study population.</p>

Significance and progress in detection of HBV cccDNA in peripheral blood mononuclear cells / 临床肝胆病杂志

Hepatitis B virus (HBV)covalently closed circular DNA (cccDNA)is an intermediate replicative episome which is stable and not easy to eliminate.cccDNA serves as the template for HBV replication and plays an important role in persistence of HBV infection in hep-atocytes,and it is also a biomarker of HBV activity.Studies suggested that HBV has extrahepatic infectivity and cccDNA could be detected in peripheral blood mononuclear cells (PBMC).The predictive values of HBV cccDNA in PBMC for HBV recurrence after liver transplanta-tion and antiviral therapy and mother-to -child transmission of HBV in patients with hepatitis B,as well as the possible mechanism of cccDNA involved in extrahepatic HBV infection,are summarized.Therefore,the detection of HBV cccDNA is of great significance for the study and treatment of HBV.

Establishment of a PCR-product direct sequencing for the detection of HBV YMDD mutation / 中华检验医学杂志

Objective To develop an assay of PCR-produet direct sequencing to detect hepatitis B virus (HBV) YMDD mutation, and compare the results gained by the sequencing and traditional real-time fluorescent PCR assays. Methods Serum samples were collected from 103 patients with chronic hepatitis B. HBV DNA were extracted from sers. YMDD mutation was detected by a commercial real-time PCR assay. Meanwhile, HBV reverse transcriptase-encoding gene was amplified by a nested PCR assay. The PCR products were directly subjected to sequencing at two directions, and the sequencing results were analyzed by NTI program. Using Kappa test, comparison was made between the results of rtM204-site mutations obtained by the direct sequencing and YMDD mutations by the real-time fluorescent PCR. Results The direct sequencing assay proved to be highly effective with bread range of detection in viral load from 500 to 1010copies/ml. And it may simultaneously avoid inhibitory effect caused by high viral load. The coincidence rates between two assays were 100% for YIDD, 97. 1% for YVDD, 76. 2% for YIDD/YVDD coexistence (Kappa = 0. 853, P < 0. 01). Conclusions The direct sequencing assay for HBV drug-resistant mutation detection is highly sensitive with broad dynamic range. It has high coincidence rate with real-time fluorescent PCR assay with advantage of detecting YMDD, YIDD and YVDD mutations simultaneously.

Effects of Divalent Mg2+ and Monovalent Na+ on Cleavage Reactions by Multiribozyme System in vitro / 中国生物化学与分子生物学报

To better understand the cleavage efficiency of muhiribozyme system on its RNA substrate in the presence and absence of divalent magnesium and monovalent sodium ions.we constructed pGEM-Coat'A,pGEM-Coat'A196Rz plasmids and pGEM-MDRl target plasmid.They were applied to transcribe RNAs with SP6/T7 transcription kit.Cleavage reactions were carried out in cell-free system and reaction products were analyzed by electrophoresis on 6% denaturing polyacrylamide gels in TBS buffer.The gels were dried and exposed to X-ray films for autoradiography.The Image J software was employed to analyze the dried gels.The results indicated that the cleavage efficiency of the muhiribozyme was dependent on the concentration of divalent Mg2+.The cleavage products increased with the concentrations of divalent Mg2+ and were Mg2+ concentration and time dependent.No cleavage product was obtained in the presence of lower than 200 mmol/L Na+ alone.On the contrary,monovalent Na+ inhibited the Mg2+ -induced cleavage reaction in Na+ and Mg2+ coexistance.The cleavage rate was significantly lower than that observed with divalent Mg2+ alone.These results suggested that divalent Mg2+ was required for muhiribozyme on substrate cleavage reaction in the physical condition,whereas monovalent Na+ was not.

Adenovirus-mediated transfer of anti-MDR1 ribozyme in the treatment of multidrug-resistant human lymphoma in SCID mice / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To evaluate the effect of adenovirus-mediated transfer of anti-MDR1 ribozyme on restoring drug sensitivity of multidrug-resistant human lymphoma both in vitro and in SCID mice.</p><p><b>METHODS</b>A recombinant adenovirus expressing ribozyme against codon 196 of MDR1 mRNA (Ad-196MDR1-Rz) was developed through cotransfection of shuttle vector pCA14 containing 196MDR1-Rz and rescue vector pJM17 into human embryonic kidney cell line 293. In vitro Daudi/MDR20, a MDR1-mediated drug-resistant human lymphoma cell line, was transduced by Ad-196MDR1-Rz at MOI of 400 pfu/cell. RT-PCR and FACS analyses were used to evaluate the MDR1 expression in both transcriptional and translational levels. MTT assay was used for analysis of drug resistance. In vivo, SCID mice were inoculated subcutaneously by 5 x 10(6) Daudi/MDR20 or parental Daudi/wt cells. Adenovirus was injected locally. Vincristine (VCR) was given intraperitoneally.</p><p><b>RESULTS</b>In vitro transduction of Ad-196MDR1-Rz to Daudi/MDR20 cells was able to interrupt MDR1 transcription, inhibit P-gp expression and restore drug sensitivity to VCR. Of SCID mice bearing Daudi/MDR20 cells, tumor free rate and long term survival were 66.7% (6/9) and > 120 days in the therapeutic group of Ad-196MDR1 + VCR vs 12.5% (7/8) and none survived > 120 days in the control groups of Ad-Mock + VCR or VCR alone. The difference was very statistically significant.</p><p><b>CONCLUSION</b>Ad-mediated transfer of 196MDR1-Rz can revert drug resistance of MDR tumor cells both in vitro and in vivo. Ad-196MDR1-Rz may be helpful as an adjuvant in the chemotherapy of P-gp mediated MDR human tumor.</p>

DYNAMIC CHANGES IN PERIPHERAL BLOOD LYMPHOCYTE AND PLASMA CYTOKINE LEVELS IN PATIENTS WITH SEVERE ACUTE RESPIRATORY SYNDROME (SARS) / 解放军医学杂志

Objective To investigate the immunological status of patients with SARS by monitoring the dynamic changes in peripheral blood lymphocyte count and plasma levels of cytokines. Methods Levels of IFN ?,TNF ?, IFN ?, IL 12 and IL 10 in plasma of 21 SARS patients were sequentially assayed with enzyme linked immunosorbent assay (ELISA) Lymphocyte count of peripheral blood was also determined. Results Peripheral blood lymphocyte counts in 66 7% of patients, plasma IFN ? level in 38 1% of patients, and plasma TNF ? level in 61 9% of patients showed inverted V shaped profiles, with peaks in the middle of SARS course. Production of Th2 cytokine IL 10 was not influenced in the present study in all patients, while high levels of Th1 cytokines IL 12 and IFN ? were observed during the observation period in some patients. The first detected values of IFN ?. In 85 7% of patients, the levels of IFN ? at the onset of symptoms were much lower than those in normal controls (867 18?306 50pg/ml; P

Immunohistochemistry study on the over expression of ERBB3 in gastric cancer / 解放军医学杂志

Objective To investigate the expression of ERBB3 protein,which encoded by ERBB3 gene,in gastric cancer(intestinal type and diffuse type)as well as the normal tissue,and to elucidate the possible role of ERBB3 gene in the progress of gastric cancer.Methods 65 samples of gastric cancer tissue(of which 43 samples were intestinal type and 22 were diffuse type,classified according to the Lauren patho-classification)and 65 samples of normal tissue(control group)were investigated immunohistochemically.The expression of ERBB3 in both control group and gastric cancer group was determined.Comparisons were made according to different pathological types and TNM clinical stages within and between groups.Results 7 of 65 samples(10.8%)in gastric cancer group were found to over-express the ERBB3 protein,while only 1 of 65 samples(1.6%)in control group was found to over-express the ERBB3 protein.Statistical differences(P

Analysis of variation in CTL epitope of HBV popular in China / 解放军医学杂志

Objective To analyze the variation in CTL epitope of hepatitis B virus (HBV) popular in China. Methods Amino acid sequences of 13 cytotoxic T lymphocyte (CTL) epitopes previously reported abroad were taken as reference sequences, and comparison was made draw between these reference sequences and both the 45 CTL-epitopic sequences obtained from the Genbank and also 5 CTL-epitopic sequences from native patients with chronic hepatitis B, using Vector NTI software. Results Sequence variations were observed frequently within several CTL eptitopes of HBV genotype B and C compared with the reference sequences. In the 13 analyzed CTL epitopes, 4 showed variation accounting over 70% in genotype B, and 2 have variance over 70% in occurrence in genotype C. Among these, C18-27 (FLPSDFFPSV), HBcAg-derived CTL epitope which was the most frequently studied previously, is showed in variation more frequently in HBV genotype B and C popular in China, reaching 71% and 97%, respectively. Conlusion Some CTL epitopic sequences of HBV genotype B and C popular in China have their own charactiristic difference from previously reported ones, which should be considered in the study of CTL response of Chinese subjects.

Transcriptional activation function of a new protein-XTP11 in yeast cells / 解放军医学杂志

Objective To construct the yeast expression vector of a new gene transactivated by hepatitis B virus X protein (XTP11), and to explore the feasibility of cloning the hepatocyte proteins interacting with XTP11 protein by yeast two hybrid system. Methods The XTP11 gene was amplified by polymerase chain reaction (PCR) with specific primers, and the amplified fragment was subcloned into the Nco I/BamH I sites (5′ ends) of yeast expression vector pGBKT7,which was named as pGBKT7(-)-XTP11 encoding fusion proteins of full length of XTP11 and DNA binding domain of yeast protein GAL4. The pGBKT7 (-)-XTP11 plasmid was transformed in the yeast cells AH109 and the expression of XTP11 protein in yeast cells was detected by Western blotting assay. Then the yeast cells were plated on synthetic dropout nutrient medium (SD/-Trp and SD/-Trp-His-Ade) containing X-?-gal, and their autonomous activation was tested. Results The yeast expression vector of XTP11 gene was constructed and the fusion protein in yeast cells was examined. The yeast cells transformed with pGBKT7-XTP11 plasmid could grew well on both of the media, and turned blue on medium containing X-?-gal for the production X-?-galactosidase. This phenomenon suggested that XTP11 protein acted to substitute the functional yeast protein GAL4 activation domain (AD) to activate transcription of reporter genes (ADE2, HIS3, MEL1 and LacZ). Conclusion The XTP11 protein fused to the GAL4 DNA-binding domain functioned as a transcriptional activation domain in yeast, and the transcriptional activation function of XTP11 protein in yeast might restrain researchers to use yeast two hybrid system to clone hepatocyte proteins interacting with XTP11 protein.

Investigation of FoxP3 expression in peripheral blood and liver tissue of patients with hepatitis B / 解放军医学杂志

Objective To investigate forkhead/winged helix transcription factor (FoxP3) expression in peripheral blood and liver-infiltrating lymphocytes (LIL) of patients with hepatitis B. Methods Flow cytometry, immunohistochemistry and real-time RT-PCR were employed for Fox P3 analysis of blood or live tissue obtained from patients with acute hepatitis B (AHB), chronic hepatitis B (CHB) and chronic severe hepatitis B (CSHB), as well as health controls. Results Fox P3 mRNA and protein expressions were specifically identified in CD4+CD25+ regulatory T cells. The level of Fox P3 mRNA in CD4+ T cells was identical with that in CD4+CD25+ regulatory T cell in peripheral blood. In CSHB patients a significant increase of Fox P3 mRNA expression was found in peripheral blood. The mean relative FoxP3 mRNA levels of CD4+ T cells in CSHB patients, AHB patients, CHB patients, and healthy controls were 0.199?0.174, 0.053?0.017, 0.059?0.053, and 0.056?0.021, respectively (CSHB group vs. other groups, P all

Dynamic analysis of anti-Cov antibody and SARS coronavirus in the plasma of 25 SARS patients / 解放军医学杂志

Objective To observe dynamic responsive regularity of specific IgM and IgG antibodies in SARS patients. Methods 145 specimens of plasma from 25 cases of clinically diagnosed SARS patients were examined in the study. ELISA was employed to detect IgM and IgG antibodies against SARS coronaviral antigens. Nested RT-PCR was used to qualitatively determine SARS coronavirus (SARS-CoV). Results 49.0% (71/145) and 54.5% (79/145) of the samples were positive for IgM and IgG antibodies, respectively. Both antibodies were found to be detected in 84.0% of the patients. The antibodies were found to be detectable from the 2nd to 4th week after the onset of disease in most patients, and there was a fendeney of sising in positive rate until the 5th week after the onset. Thereafter, the detectable rate of IgM antibody began to decline, while that of IgG antibody remained to rise. Positive rate for serum SARS-CoV was 15.8% (18/114) for all samples or 40.0% (10/25) of patients. Most virus-positive samples were those which were collected within 4 weeks after disease onset. Conclusions Anti-SARS-CoV IgM/IgG antibody and detection of virus in plasma could serve as practical diagnostic indicators for SARS. In most cases, when the serum was pasitive for antibody the serum virus positive rate would soon declined. However, concomitant existence of antibody and viral sequence in plasma was observed in a few patients.

Dynamic detection of RNA of SARS-associated coronavirus in blood and excreta of SARS patients / 解放军医学杂志

Objective To serially examine virus carrying condition of clinical specimens collected from SARS patients, in order to evaluate the dynamic changes of virus in the bady and its clinical significance. Methods A total of 494 samples, including plasma, urine, feces, sputum and throat swab, obtained from 84 cases of clinically diagnesed SARS patients at different time-points were examined for the study. Nested RT-PCR was employed for the detection of SARS-CoV using 2 pairs of primers targeting P and N region of the viral genome. The amplified viral genomic fragments were confirmed by DNA sequence analysis. Results Specific bands for SARS-CoV were amplified from various specimens of some SARS patients. Highest positive rate was found in sputum compared with the others. However, simultaneous examination of more than two specimens increased detectable rate of the virus. Moreover, dynamic examination revealed that the highest positive rate occurred in the 2nd week after the onset of the disease and next to the highest during the 3rd to 4th week. However, dynamic detectable rates for different specimens were not identical. Conclusion Nested RT-PCR is a practicable method for the detection of SARS-CoV and helpful for the early diagnosis or confirmation of the disease. Dynamic examinations for SARS-CoV by combined employment of the N and P primers and by involving multiple specimens may significantly increase the positive detectable rate, therefore would be helpful in diagnosis and eratuation of therapeutic effect of SARS.

Screening the down-regulated genes in HepG2 cells transfected by IFN-? / 解放军医学杂志

Objective To screen the down-regulated genes in HepG2 cells transfected by pcDNA3.1(-)-IFN-? to further investigate the biological mechanism of IFN-?. Methods pcDNA3.1(-)-IFN-? was transfected into HepG2 cells as treatment group and the pcDNA3.1(-) transfected cells as the control to perform suppression subtractive hybridization (SSH). The subtractive library for down-regulated genes was obtained when pcDNA3.1(-) transfected HepG2 as tester and pcDNA3.1(-)-IFN-? transfected cells as driver. A housekeeping gene, G3PDH, was used to estimate the subtractive efficiency. Genes with lower expressions in IFN-? transfected HepG2 cells were obtained from the library after being randomly sequenced and analyzed. Among the obtained genes, regulation of IFN-? on heat shock 90kD (Hsp90) mRNA was further investigated by RT-PCR. Results G3PDH was subtracted efficiently indicating that the subtractive library was constructed successfully. 50 positive clones were randomly isolated for PCR identification. Results showed that most of the plasmids in the clones contained 200-1 000bp inserts. The down-regulated genes obtained were as follows: eukaryotic translation elongation factor 1 beta, ferritin, RAD23 homolog B, signal sequence receptor 2 (SSR2), tissue factor pathway inhibitor, heat shock 90kD protein 1, and adenosylmethionine decarboxylase 1. RT-PCR showed that hsp90 mRNA had a reduced expression in pcDNA3.1 (-)-IFN-? transfected HepG2 cells. Conclusion The down-regulated cDNA subtractive library in HepG2 cells after pcDNA3.1 (-)-IFN-? transfection was constructed successfully. IFN-? could down-regulate the hsp90 mRNA expression.

Screening of mimotopes of swine influenza virus A (H1N1) by phage display technology / 解放军医学杂志

Objective To screen the specific antigen mimotopes of influenza A(H1N1)virus by phage display technology,in order to pave a way to develop novel influenza vaccine.Methods Using human convalescent serum of pandemic influenza A(H1N1)in 2009 as solid-phase selective molecule,an artificial synthesized phage randomly displaying cyclic 7-mer peptide was screened with three rounds of "absorption-elution-amplification" selection.At the end of the third round selection,32 clones were randomly chosen from the top-agar phage plaques and placed onto E.coli ER2378 in logarithmic growth phase.After culturing for 5 hours,the positive clones were identified by enzyme linked immunosorbent assay(ELISA),cross reaction test and competitive inhibition assay.The identified positive clones were analyzed by DNA sequencing.The decoded amino acids sequences,which displayed on the surface of phage,were aligned with the hemagglutinin(HA)gene of influenza virus by homology comparison for definition of the mimotopes of influenza A(H1N1)virus.Results After enriching the specific antibody-binding phages from phage displaying peptide library,12 positive clones were chosen from 32 randomly selected clones.DNA sequencing and homology comparison showed that one epitope PLHARLP was confirmed as mimotope of swine influenza A(H1N1)virus antigen,which was composed of the 52nd,53rd,59th,60th,61st,83rd and 271st amino acid.Conclusions Swine influenza A(H1N1)mimotope has been obtained by cyclic 7-mer peptide phage library screening.This result provides a basis for developing new influenza virus vaccine from virus antigen mimotopes.

Techniques and clinical significance of determination of drug resistance of hepatitis B virus / 解放军医学杂志

Nucleoside analogs (NA) are currently the major anti-hepatitis B virus (HBV) agents in clinic. However,long-term use of NA may initiate a state of drug resistance due to HBV mutation. The genotyping and phenotyping are two basic methods for analysis of HBV drug resistance. Genotyping is done mainly by DNA sequencing and reverse hybridization line probe to detect the well-known drug-resistant mutations. Phenotyping is an essential tool used to identify "novel" and complex resistant mutations,and it is based on the comparison of HBV replication capacities of variants and wild-type counterparts by cell-transfer of individual viral genomes followed by quantitation of HBV replication intermediates in the presence of antivirals in different concentrations. Clinical choice of HBV resistance detection assays relies on their sensitivity,specificity and cost-effectiveness. In our recent studies,some novel HBV resistant features have been found,including multidrug-resistant strain with mutations due to the use of both entecavir and adefovir,association of HBV genotype/subgenotype with the resistant mutational patterns,transmission of the resistant HBV to cause acute hepatitis,replication compensation of rt229 variants,etc. These findings are important for better understanding the clinical features of HBV drug-resistant mutations and strengthening the management of HBV drug resistance in clinic.

Virological characteristics and the clinical significance of hepatitis B virus / 解放军医学杂志

Hepatitis B virus(HBV) is a double-strand hepadnavirus.HBV genomics have unique properties:(1) Viral polymerase/reverse transcriptase(RT) is lacking in proofreading activity,leading to higher mutation occurrence than other DNA viruses.(2) Overlapping open reading frames in viral genomics may produce the possibility of mutation of two viral proteins mutation as a result of one-site genetic change.(3) A stable covalently closed circular DNA(cccDNA) form exists as an important intermediate in the life cycle.(4) Viral gene fragments may be integrated into host cellular genomic DNA.(5) Viral proteins could be a trans-regulator for certain host cellular protein expression.The virological characteristics of HBV are closely related with the chronicity and disease course of hepatitis B.Multi-site detection of HBV RT-gene mutations is very important for monitoring drug resistance and rational administration of anti-HBV therapy in clinic.Analysis of viral genomic variation and detection of cccDNA are highly valuable in better understanding virological pathogenesis and evaluate therapeutic efficacy.Methodological innovation and improvement is the basis for the study of virological characteristics of HBV.

The comparison of YMDD mutation results detected by direct sequencing and real-time PCR / 解放军医学杂志

Objective To compare the efficiency of detecting YMDD mutations by direct DNA sequencing and real time fluerescence PCR, and to explore the significance of the mutations of drug-resistance gene other than YMDD mutation. Methods 92 serum samples from 89 patients with chronic hepatitis B were collected and all the samples were detected by real-time PCR for YMDD mutation. HBV DNA was extracted from serum and was amplified by nest PCR to achieve HBV P gene RT region sequence. The PCR products were sequenced at both directions, and the sequencing results were contrasted through NTI program. The other 11 known drug-resistance mutation sites at the HBV RT region were also analyzed. Results Among the 37 samples with no YMDD mutation detected by real-time PCR, 33 samples were with M204M (without mutation), 1 sample with M204I and 3 samples with M204V by direct sequencing. Mutation and wild-type standard sequences were all coexisted in the 4 positive samples. There were 7 samples detected with ADV resistant mutation, accounting for 18.9% (7/37). Among the 55 samples with YMDD mutation detected by real-time PCR, 52 samples were detected by DNA sequencing, the accordance rate was 94.5% (52/55); 5 samples with ADV or ETV resistant mutation were detected, accounting for 9.1% (5/55). Conclusions Direct DNA sequencing is a high sensitive, repeatable method to detect drug-resistance mutation at RT region of HBV P gene. The result is well consistent with that attained by real-time PCR. Direct DNA sequencing can also detect various drug-resistance mutations as well as YMDD mutation, which is helpful to generally understand the nucleoside analogue resistant mutation and adopt more reasonable therapy projects against HBV.