Serum and urinary amino acid profiling analysis for urinary stone disease using GC-MS / 中华检验医学杂志

Objective:To analyze the serum and urinary amino acid (AA) profiles of urolithiasis patients to explore the potential biomarkers for clinical screening and early diagnosis.Methods:Case-control study. Serum and urine samples were collected from 74 urolithiasis patients (aged 20-82 years, 41 men, 33 female) in the department of urology of the First Affiliated Hospital of Fujian Medical University and 35 healthy controls (HC, aged 22-80 years old, 20 men, 15 female) from the health examination center from February 2015 to October 2017. Serum and urinary AA levels of patients and HC were analyzed using Gas Chromatography-Mass Spectrometry (GC-MS) based metabolomic strategy. The multivariate statistical analysis methods of principal component analysis (PCA) and orthogonal partial least squares discrimination analysis (OPLS-DA) were employed for modeling. The variable importance projection (VIP) value of OPLS-DA model>1 and P<0.05 of t test were selected to screen the differential amino acid metabolites. The diagnostic capabilities of potential markers were evaluated by receiver operating characteristic (ROC) curve and binary logistic regression analysis. Results:Five AA metabolites including serine, glutamate, aspartic acid, isoleucine and glycine were found, which had statistically significant differences between the patient group and the control group ( P<0.05) and were associated with seven metabolic pathways. Serum serine, glutamate, aspartic acid, isoleucine and urine glycine and aspartic acid were combined into an integrated marker panel whose AUC value was 0.890, the sensitivity was 78.0%, and the specificity was 96.4%. Conclusion:Five amino acids in serum and urine could be used as an integrated biomarker panel for the clinical screening and early diagnosis of urolithiasis, which could provide some experimental basis for molecular urolithiasis research.

Research progress in detection and clinical significance of three envelope proteins of hepatitis B virus / 中华检验医学杂志

The pre-S/S gene of hepatitis B virus (HBV) can encode for the production of large, medium and small surface protein. Different protein expression levels and their composition ratios have certain influences on the diagnosis, treatment and outcome of HBV infection. It is of great significance to clarify the functions of large, medium and small surface protein as serum markers and to explore their value in the diagnosis and treatment of HBV infection. In this paper, the expression status, detection methods and clinical significance of the three HBV proteins were reviewed.

Predictors of poor short-term outcome in patients with autoimmune encephalitis / 中华检验医学杂志

Objective:To analyze the risk factors of poor short-term outcome in patients with autoimmune encephalitis (AE).Methods:The data were collected retrospectively from patients hospitalized in the First Affiliated Hospital of Fujian Medical University from March 2016 to January 2021. According to the Modified Rankin Scale (mRS), eighty-one patients with AE were divided into the good prognosis group (mRS≤2, n=48) and the poor prognosis group (mRS>2, n=33). The clinical data, including the basic demographic data, main clinical manifestations, cranial MRI and electroencephalogram, as well as laboratory indicators from blood and cerebrospinal fluid (CSF), were compared between two groups, and the risk factors for poor outcome were analyzed by multivariate logistic regression. The receiver operating characteristic (ROC) curve was used to determine the cut-off value of these risk factors on predicting the poor short-term outcome for the AE patients. Results:The time of hospitalization was significantly longer in the poor prognosis group than in the good prognosis group ( P<0.05). Prevalence of severe disease course at admission, abnormal mental and behavior, δ wave and δ brush of abnormal EEG was significantly higher in the poor prognosis group than in the good prognosis group (all P<0.05). Serum leukocyte count, neutrophil count, lymphocyte count, mononuclear cell count, C-reactive protein, procalcitonin(PCT), alanine aminotransferase, aspartate transaminase, lactate dehydrogenase, creatine kinase, creatine kinase isoenzyme, apoA1/B, calcium, sodium, anion gap in serum, CSF oligoclonal bands, CSF-IgG index and antibody titer were significantly different between the two groups (all P<0.05). Severity of illness at admission ( OR=1.816, 95% CI 1.250-2.639, P=0.002), PCT ( OR=1.345, 95% CI 1.008-1.794, P=0.044), antibody titer in serum ( OR=1.422, 95% CI 1.071-1.888, P=0.015), CSF-IgG index ( OR=1.802, 95% CI 1.035-3.138, P=0.037) and anionic gap ( OR=1.640, 95% CI 1.191-2.259, P=0.002) were the independent risk factors for the poor short-term prognosis of patients with AE. The AUC value of combing the above 5 indexes to predict the poor short-term prognosis in patients with AE was 0.920 (95% CI 0.834-0.971), with 83.87% sensitivity and 88.37% specificity. Conclusion:Severity of illness at admission, PCT, antibody titer in serum, CSF-IgG index and anionic gap are the independent risk factors of poor short-term prognosis in patients with AE and the combination of these 5 indexes can sufficiently predict the poor short-term prognosis in patients with AE.

Laboratory diagnosis of HBV infection: present and future / 中华检验医学杂志

At present, the situation of prevention and treatment of hepatitis B(HBV) infection in China is still serious. Factors affecting the course and outcome of HBV infection mainly include virus and hosts. While exploring the new clinical significance of HBV infection classic diagnostic indicators such as hepatitis B sunface antigen, hepatitis B core antibody and HBV DNA, more attention should be paid to the application value and prospect of new indicators such as HBV RNA and hepatitis B core-related antigen. With the proposing of the omics concept and the development of technology, people begin to focus on HBV infection with the concept of omics and integrate genomics, metabolomics, proteomics, etc., to achieve the goal of accurate diagnosis.

The application of serum HBV RNA detection in anti-HBV treatment / 中华检验医学杂志

The persistence of covalently closed circular DNA (cccDNA) in the nucleus of liver cells is a key factor that hinders the cure of chronic hepatitis B. However,it is difficult to eliminate cccDNA with existing anti-HBV therapy. Recent studies have found that serum HBV RNA may be a new indicator reflecting the activity of cccDNA in hepatocytes and evaluating the clinical efficacy of CHB patients . This article reviews recent advances in the properties,detection methods,and clinical significance of HBV RNA, particularly the application of antiviral therapy in CHB patients.

The genetic polymorphism of CYP7A1 in Fujian Han HBV infected patients / 中华检验医学杂志

Objective The study aims to investigate the associationbetweencholesterol 7α-hydroxylase (CYP7A1) gene polymorphism and different clinical outcomes after Hepatitis B virus (HBV)infection in Fujian Han population and lay a foundation for understanding the mechanisms of genesis anddevelopment of HBV-related diseases.Methods Case-control study was conducted.586 patients of HBVpersistent infection without antiviral therapy and 225 HBV rehabilitation patients (35-55 years old) werecollected from May 2015 to June 2016 in the Liverish Center of First Clinical College of Fujian MedicalUniversity.The group of HBV persistent infection without antiviral therapy included 246 patients with chronichepatitis B, 177 patients with hepatitis B-related cirrhosis, and 163 patients with hepatitis B-related liver cancer.The rs3824260, rs4738687and rs8192871 loci of CYP7A1 gene were detected by improved multipleligase detection reaction (iMLDR).Logistic regression analysis and chi-square test were used to analyze thegenotyping results.Results Three SNPs ( single nucleotide polymorphisms ) of CYP7A1 gene wereselected and compared between HBV persistent infection group and HBV rehabilitation group and betweenchronic hepatitis B subgroup, liver cirrhosis subgroup and liver cancer subgroup.After adjustment for factorsincluding age andgender, there was no significant difference in the distribution of rs3824260 genotype amongthe groups(χ2 =1.565,P =0.459), however,the frequency of allele C in HBV rehabilitation group wassignificantly higher than in HBV persistent in fectiongroup for men (χ2 =4.365,P =0.037), whereas thefrequency of rs3824260 CC and CT was more likely to be observed in liver cancer group than in non -livercancer group (chronic hepatitis B subgroup and liver cirrhosis subgroup ) for women (χ2 =5.768,P =0.012;χ2 =10.130,P =0.001).The frequency of rs4738687 GG genotype was more likely to be observed innon-liver cancer group than in liver cancer group (χ2 =4.403,P =0.041;χ2 =6.940,P =0.009).Theresults of gender stratification showed that there were significant differences in the distribution of rs 4738687among the HBV persistent infection groups for men (χ2 =10.697,P =0.030), however, there was nosignificant difference in the distribution of rs4738687 among the HBV persistent infection groups for women(χ2 =4.627,P =0.329), and there was no significant difference in the distribution of genotype frequencyand allele frequency among all groups(χ2 =0.489,P =0.792).There was no significant difference after sexstratification either (χ2 =1.282, P =0.526;χ2 =1.565,P =0.465) .Conclusions These findingssuggested that CYP7A1 gene polymorphism was related todifferent clinical outcomes in Fujian Hanpopulation.The rs3824260 mutation had a certain gender preference and the mutation allele was detected ina higher proportion in male patients.Male HBV patients with rs3824260 C allele had more chance ofswitching to rehabilitation.The rs4738687 was likely to be related to the occurrence of liver cancer in FujianHan population, and GG genotype may delay the occurrence and development of liver cancer especially in themale group.The rs8192871 was not found to be related to the different clinical outcomes of HBV infection.

Epidemiological and molecular virological characteristics of HBV infected patients of hospital with simultaneously positive HBsAg and anti-HBs / 临床检验杂志

Objective To investigate the epidemiological and molecular virological characteristics in HBV-infected patients with copositive HBsAg and anti-HBs.Methods HBV serological markers were analyzed in 52 070 specimens.The epidemiological characteristics of HBsAg and anti-HBs simultaneously positive patients (the experimental group) and HBsAg positive and auti-HBs negative patients (the control group) were compared.The S protein of HBV coding region was amplified by semi-nested PCR and sequenced.The statistical differences between the two groups were compared in different gene regions,genotypes and different clinical diagnosis.Results HBsAg was positive in 20.40％ (10 621/52 070) of all specimens.In the patients with positive HBsAg,2.48％ (263/10 621) was positive anti-HBs.The prevalence of co-positive HBsAg and auti-HBs was higher in aged 0 to 9 years and greater than or equal to 80 years than that in other age,and the prevalence of positive HBsAg and negative anti-HBs was completely opposite.The mutation rate of S protein in the experimental group was significantly higher than that in the control group (1.52％ vs 0.81％,P ＜0.01) with the mutation in the major hydrophilic region (MHR) (1.68％ vs 0.57％,P ＜0.01).The mutation rates of S protein of HBV carriers,chronic hepatitis B (CHB) patients and patients with liver cirrhosis (LC) in the experimental group were significantly higher than those in the control group (1.47％ vs 0.65％,1.28％ vs 0.84％,2.21％ vs 0.44％,P ＜0.05,respectively),except for the patients with hepatocellular carcinoma (HCC) (1.97％ vs 2.21％,P ＞ 0.05).Conclusion Co-positive HBsAg and anti-HBs in HBV-infected patients was more common in HBsAg positive patients aged 0 to 9 years and greater than or equal to 80 years than the others.Coexistence of HBsAg and anti-HBs in HBV-infected patients may relate to immune escape caused by mutation of S protein (mainly MHR).The mutation rates of S protein in the two groups of patients,co-positive HBsAg and anti-HBs and the positive HBsAg combined with negative anti-HBs,were associated with the stage of liver disease.

Exploration and practice of student' clinical thinking ability in the courses of Medical Laboratory Technology / 中华医学教育探索杂志

This study was conducted to explore a proper training model of interns' clinical thinking ability under the construction of a new four-year system of medical laboratory technology courses, combined with the establishment of innovative standard whole process practice mode. Multi-teaching methods of clinical thinking, such as explanation of laboratory sheet, interactive teaching based on micro digital system, interdisciplinary multiple information system, combined PBL teaching and intern report, were applied and evaluated in the laboratory. Integrated application of these methods remarkably improved the intern's com-prehensive professional quality and their practice performance. All methods received high evaluation from both the interns and teachers.

Melatonin Ameliorates Tau Hyperphosphorylation of Mice with Exposure to Lead(Pb)at Early Development Stage by Mediating ER Stress in Hippocampus / 华中科技大学学报(医学版)

Objective To investigate the effect of melatonin(MT)on tau hyperphosphorylation in hippocampus of mice with exposure to lead(Pb)at early development stage and its possible mechanism.Methods Healthy C57BL/6 mice were randomly divided into four groups:control group(received normal water),lead exposure group(exposed to 0.2% Pb acetate from postnatal day 1(PND 1)to PND 21 through drinking water),MT group(received 50 mg/mL MT through drinking water since 12-month-old for 3 months)and MT combined with lead exposure group(exposed to 0.2% Pb acetate from PND 1 to PND 21 and then given 50 mg/mL MT since 12-month-old for 3 months through drinking water).The Pb levels in the blood and hippocampus were determined by graphite furnace atomic absorption spectrometry.Morris water maze was used to detect the spatial learning and memory.The phosphorylation level of tau and the protein level of GRP78 and CHOP in hippocampus were detected by Western blotting.Results Compared with the control group,the Pb levels in the blood and hippocampus were significantly increased in lead exposure group(P<0.05),while MT treatment did not affect the Pb levels both in the blood and hippocampus.In the lead exposure group,the phosphorylation level of tau in the hippocampus was significantly increased compared with control group(P<0.01),and after treatment with MT,the phosphorylation level of tau in MT combined with lead exposure group was significantly decreased compared with the lead exposure group(P<0.05).Furthermore,the expression of GRP78 and CHOP in the hippocampus was significantly higher in lead exposure group than in control group(P<0.05),and after treatment with MT,the expression levels of GRP78 and CHOP were lower than those of the control group.Conclusion MT can ameliorate the phosphorylation level of tau in the hippocampus and defects of learning and memory in C57BL/6 mice with exposure to lead at early development stage,and this may be related with the modulation of endoplasmic reticulum stress.

The regulative function of mitochondria in innate immune response to chronic HBV infection / 中华检验医学杂志

Innate immunity initially resists the infection of pathogenic microorganism in host immune response.Recent researches confirmed that mitochondria participated in a wide range of innate immune pathways,mainly including contributing to innate immune activation,regulating antiviral signaling pathways and antibacterial immunity.Therefore,further studies on the relationship among mitochondria,hepatitis B virus(HBV)infection and innate immune response might contribute to elucidate the mechanism of chronic HBV infection and explore the mechanism of host immune to clear HBV.Here,mitochondria playing a vital role in regulations of innate immune response,HBV infection tending to chronicity by suppressing innate immune response and chronic HBV infection by regulating the innate immune response through injuring mitochondria,were reviewed.

Advances in research of HBV quasispecies / 中华检验医学杂志

Hepatitis B virus exists in the form of quasispecies.Quasispecies are an important basis for the pathogenesis and drug resistance of hepatitis B virus.Researches for quasispecies could contribute to evaluating the progress of disease, predicting drug resistance, adjusting the therapeutic regimen and performing individual treatment.

The relationship between HBV P region genetic muation and serum markers in hepatitis B patients in Fuzhou region / 中华检验医学杂志

Objective To investigate the correlation of hepatitis B patients in Fuzhou between resistance patterns in HBV P region and genotype,HBeAg,the hepatitis B process.Methods This was a retrospective study.The serum and clinic data of 1 115 hepatitis B patients were collected from the inpatient and outpatient Center for Liver Diseases in First Affiliated Hospital of Fujian Medical University between October 2011 and January 2015.HBV DNA was extracted and sequenced using the Sanger method to detect HBV genotype and resistance mutations in P region,HBeAg and HBeAb concentration were detected by chemiluminescent assay.The relationship between P region resistance mutations pattern,HBV genotype,serum HBeAg and the hepatitis B process was analyzed.The x2-test was used to compare the resistance rate and positive rate.Results There were significant differences between 14 kinds of resistance loci and the genotype distribution(x2 =30.788,P =0.004),the C/B genotype ratio of three common resistance loci (rtM204V/I,rtL180M,rtA181T/V) were 85/82,49/25 and 27/9,respectively,which in genotype C was higher than genotype B.The resistance ratio of hepatocellular carcinoma,liver cirrhosis,chronic hepatitis B,hepatitis B carriers was 31.4％ (11/35),37.6％ (65/173),27.3％ (146/535) and 21.8％ (43/197),respectively,which showed significant difference between the four clinical diagnosis (x2 =11.858,P =0.008).The highest percentage of resistance was liver cirrhosis,followed by hepatocellular carcinoma and chronic hepatitis B.There was significant difference in the distribution of HBV genotype between HBeAg (+) group and HBeAg (-) group (x2 =11.093,P =0.001),the HBeAg positive rate in genotype C [37.53％ (295/786)] was higher than in genotype B[35.62％ (280/786)].However,the total resistance rate between HBeAg (+) group and HBeAg(-) group was not significantly different[23.7％ (136/573) and 24.6％ (52/211),respectively,x2 =0.07,P =0.791].Conclusions HBV genotype was related to the resistance rates,HBeAg levels and the progress of hepatitis B.The resistance rate and HBeAg positive rate of genotype C were higher than those of genotype B,and clinical outcomes were worse in genotype C.HBV resistance rates and HBeAg levels were related to the progress of hepatitis B,the higher the resistance rates,the worse clinical outcomes.

Advances in detection technology for HBV phenotypic resistance / 中华检验医学杂志

Long-term use of nucleoside ( nucleotide ) analogues for anti-HBV treatment care easily lead to drug resistance. As compared to genotypic resistance detection, the detection technology of phenotypic resistance is a method used to analysis the sensitivity of antiviral drugs in vitro cell model, the result can directly reflect the relationship between drug-resistance mutations and drug susceptibility, thus perform a better prediction of clinical resistance.Among those technologies, some analysis strategies like transient transfection assay, stable cell lines and enzymatic assay, have been developed for early diagnoses as well as dynamic monitoring of drug resistance, and contribute greatly to proper selection of therapeutic regimen, reduction of resistance rate and implementation of individual treatment.

Clinical distribution and drug resistance of 179 isolates of Streptococcus agalactia / 国际检验医学杂志

Objective To investigated the clinical distributions and antimicrobial susceptibility of Streptococcus agalactia strains isolated from the patients .Methods The identification and susceptibility of the strains were mainly measured by automatic VITEK‐Ⅱ system ,the K‐B disc diffusion tests were used for the resistance test of erythromycin ,meropenem ,and D‐test .Results The iso‐lates were mainly from urine (63 .1% ) ,genital tract(7 .8% ) and wound secretion(6 .7% ) .They were obtained from patients in dif‐ferent situations ,including 110 patients who were older than 50 years old (61 .5% ) ,113 female patients (63 .1% ) ,12 gravidas (6 .7% ) ,3 vertical transmitted newborns(1 .7% ) ,and 82 patients with cancer ,undergoing chemo radiotherapy ,with diabetes ,tuber‐culosis or after operations(45 .8% ) .The resistant rates of the isolated Streptococcus agalactia to erythromycin and clindamycin were 42 .9% -93 .3% and 41 .9% -80 .0% respectively .The positive rate of D‐test was 4 .1% .The strains were highly resistant to tet‐racycline(>80% ) ,while the resistance to penicillin was below 10% except in 2008 .All isolates were susceptible to vancomycin and meropenem .Only one strain was resistant to Quinupristin‐dalfopristin .Conclusion Streptococcus agalactia infection in adults most‐ly cause genitourinary tract ,skin and soft tissue infections .There were more females than males with Streptococcus agalactia infec‐tion .Penicillin andβ‐lactams are still the first choice for the treatment .Erythromycin ,clindamycin and tetracycline should be used with caution under the guidance of laboratory susceptibility test results .

Studies on the association of single nucleotide polymorphisms of HLA-DP and DQ genes with the outcome of chronic hepatitis B virus infection / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the association of single nucleotide polymorphisms in the HLA-DP and DQ genes with the outcome of chronic hepatitis B virus infection.</p><p><b>METHODS</b>Two hundred and four healthy subjects, 255 clearance subjects, 204 asymptomatic HBV carriers (AsC), 136 chronic hepatitis B (CHB), 68 liver cirrhosis (LC) and hepatocellular carcinoma (HCC) were enrolled. Genotypes of rs3077, rs9277535 and rs2647050 were determined by sequence specific primers-PCR (PCR-SSP).</p><p><b>RESULTS</b>By using healthy subjects and clearance subjects as the control groups, rs3077 and rs9277535 were significantly associated with chronic HBV infection under additive and dominant models (P< 0.05). Meanwhile, haplotypes GGA, AGA, AAA appeared to be protective factors against chronic HBV infection (P < 0.05). By using AsC as the control group, comparison with the CHB, LC and HCC groups showed no association of the 3 SNPs or haplotypes with the clinical outcome (P > 0.05).</p><p><b>CONCLUSION</b>HLA-DP gene polymorphisms are strongly associated with chronic HBV infection. The presence of A allele at rs3077 and rs9277535 of the HLA-DP gene may decreased the risk for chronic HBV infection.</p>

Establishment and application of cubic and conductive teaching pattern in clinical microbiolog-ical analysis / 中华医学教育探索杂志

Cubic and conductive teaching pattern is based on standardized operation process of microbiological specimens. It uses multi-dimensional teaching methods including tests, question guid-ance, discussion, lectures, measurements and scientific researches to lead the undergraduate students to master the standardized inspection process and to train talents with good gifts and abilities in prac-tical application. The teaching pattern helps the trainees complete training in basic experiment skills, analysis and problem solving skills, scientific thinking and working methods. Then the corresponding practical quantification evaluation criteria has been established according to the standardized inspec-tion process to assess the interns' level of grasping various vocational skills and comprehensive appli-cation in the inspection process. This pattern reflects the teaching philosophy of modern laboratory medicine and can scientifically and objectively assess the talent cultivation quality and teaching quality. Since its implementation, these trainees have improved their overall vocational abilities, and made a good preparation for their practical work after school. Besides, it has enhanced the teaching level of microbiology laboratory practice and has been highly valued by interns. It is hoped that this practical experience will be a useful attempt to promote medical education development of modern laboratory medicine.

An innovative HBV genotyping and drug-resistant mutation detection platform / 中华检验医学杂志

Despite decades of the prevention and control.China is still one of the countries with high prevalence of hepatitis B virus (HBV).It is imperative for us to innovate.Lab diagnostic platforms for drug-resistant mutation and HBV genotyping to enhance the management level in patients with CHB.Specifically,the following three strategies should be included:firstly,choose an appropriate platform for drug-resistant mutation detection according to the actual situation in laboratory.Secondly,establish a practical and simple genotyping platform for real-time monitoring of HBV genotypes.Thirdly,combined with clinical,explore the correlation among genotypic resistance,phenotypic resistance and clinical resistance when different medications are adopted,confirm the percentage or threshold of resistant strains causing clinical resistance,and ascertain the influence of HBV genome polymorphism (genotype) on clinical prognosis.These strategies above mentioned will lay a foundation for CHB personalized treatment and prognosis.

Status quo and reflection of clinical immunology in laboratory medicine / 中华检验医学杂志

With the rapid advancement of immunology and molecular biology,many new revelations and ideas were drew for clinical immunology in laboratory medicine.In the current circumstances of application of clinical pathway management and individual medical care,how to accurately grasp these changes to build perfect quality system for clinical immunology laboratory,clarify the immunology laboratory pathway and integrate that into clinical pathway management,make solid progress for the promotion and application of individual diagnosis and evaluate the diagnostic value of new technique and item for clinical immunology laboratory accurately might be worth pondering for every people working in clinical immunology laboratory.In this paper,the status quo and reflection of clinical immunology is reviewed.

Advances in HBV drug-resistance mutations and detection technologies / 中华检验医学杂志

Drug-resistance mutation is inevitable during long-term monotherapy with nucleus(t)ide analogues in hepatitis B patients.Early detection the drug-resistance mutations could contribute to adjusting the therapeutic regimen,reducing resistance rate,improving therapeutic efficacy and performing individual treatment.

Quantification and genotyping of hepatitis B virus in a single reaction based on dual molecular beacon real-time PCR / 中华检验医学杂志

Objective To develop a new method for simultaneous quantifying and genotyping of HBV in a single reaction based on dual molecular beacon real-time PCR.Methods Genotype B and C recombinant plasmids were constructed as the standards and genotype-specific primers and molecular beacons were designed for each genotype.The molecular beacons of genotype B and C were labeled with FAM and Hex respectively.In this way,a simultaneous qualification and genotyping method for HBV DNA in a single real-time PCR reaction system was developed.Firstly,10-fold gradient dilution of genotype B and C standard plasmids (103-1011 kIU/L) were utilized to evaluate the linear ranges and sensitivity of this approach.The clinical specificity was tested with twenty different serum specimens (5 cases with hepatitis C virus,5 cases with herpes simplex virus and 5 cases with human papilloma virus as well as 5 healthy volunteers) ; the reproducibility was assessed by intra-assay and inter-assay coefficient of variation (CV) of cycle threshold (Ct) value through 10 repeated detections within a batch and between batches of the B,C standard plasmids (108,106 and 104 kIU/L).Then the accuracy of qualifying and genotyping of the self-built method was evaluated by a parallel examination with 132 HBV infected patients by use of two commercial kits as the references.Finally,these HBV-positive patients were divided into 4 groups:asymptomatic carrier (n =21),chronic hepatitis (n =77),liver cirrhosis (n =25) and hepatocellular carcinoma (n =9) to investigate the relationship of genotypes,stages of disease progression and HBV DNA load.Results A simultaneous qualification and genotyping assay was successfully built and its genotyping sensitivity was 103 kIU/L and the linear range was 103-1011 kIU/L.The intra-assay CV of B genotyping was 1.51％ to 1.80％ and the interassay CV was 2.11％ to 3.03％,while the intra-assay CV of C genotyping was 1.79％ to 1.95％ and the inter-assay CV was 2.53％ to 2.91％.The results of non HBV infected cases and healthy volunteers showed negative.In the test of 132 HBV infected patients,the general coincident rate of genotyping results comparing our assay and HBV DNA genotyping kit was 90.9％ (120/132,Kappa =0.832,P ＜ 0.05).The HBV DNA quatitive results between the assay[5.07 (3.89-6.33)] and HBV DNA quatitive kit [5.19 (4.15-6.32) lg kIU/L] were well correlative (R2 =0.8477,P ＜ 0.05).69 genotype B cases,51 genotype C cases and 12 B/C mixed-genotype cases were detected by dual molecular beacon real-time PCR method and their HBV DNA load were 4.54 (3.83-6.17),5.53 (4.02-6.55),4.58 (3.68-4.98) lg kIU/L respectively.Where the patients with genotype C had higher DNA load than the patients with other two genotypes (Z =-2.195and-2.162,P ＜ 0.05).The HBV DNA load of asymptomatic group,chronic hepatitis group,liver cirrhosis group and hepatocellular carcinoma group were 7.02 (6.35-7.84),4.94 (4.16-6.25),4.37(3.50-5.17) and 3.45 (3.25-4.92) lg kIU/L,respectively.Among them,the asymptomatic group was significantly higher than those of other three groups (Z =-4.244,-4.568 and-3.489,P ＜0.001) and DNA load comparing with the chronic hepatitis group,liver cirrhosis group and hepatocellular carcinoma group also showed statistically different (Z =-2.894 and-2.413,P ＜ 0.05).However,compared with the liver cirrhosis group and hepatocellular carcinoma group there was no significant difference (Z =-0.995,P =0.335).Conclusion A dual molecular beacon real-time PCR assay which can simultaneously quantifying and genotyping HBV DNA with highly accuracy,sensitive and specificity is successfully developed.(Chin J Lab Med,2013,36:333-338)