DNA sensors to assess the effect of VKORC1 and CYP2C9 gene polymorphisms on warfarin dose requirement in Chinese patients with atrial fibrillation.

The optimal dose of warfarin depends on polymorphisms in the VKORC1 (the vitamin K epoxide reductase complex subunit (1) and CYP2C9 (cytochrome P450 2C9) genes. To minimize the risk of adverse reactions, warfarin dosages should be adjusted according to results from rapid and simple monitoring methods. However, there are few pharmacogenetic-guided warfarin dosing algorithms that are based on large cohorts from the Chinese population, especially patients with atrial fibrillation. This study aimed to validate a pharmacogenetic-guided warfarin dosing algorithm based on results from a new rapid electrochemical detection method used in a multicenter study. Three SNPs (CYP2C9 *2, *3 and VKORC1 c.-1639G > A) were genotyped by electrochemical detection using a sandwich-type format that included a 3' short thiol capture probe and a 5' ferrocene-labeled signal probe. A total of 1285 samples from four clinical hospitals were evaluated. Concordance rates between the results from the electrochemical DNA biosensor and the sequencing test were 99.8%. The results for gene distribution showed that most Chinese patients had higher warfarin susceptibility because mutant-type and heterozygotes were present in the majority of subjects (99.4%) at locus c.-1639G > A. When the International Warfarin Pharmacogenetics Consortium algorithm was used to estimate therapeutic dosages for 362 patients with AF and the values were compared with their actual dosages, the results revealed that 56.9% were similar to actual dosages (within the 20% range). A novel electrochemical detection method of CYP2C9 *2, *3and VKORC1 c.-1639G > A alleles was evaluated. The warfarin dosing algorithm based on data gathered from a large patient cohort can facilitate the reasonable and effective use of warfarin in Chinese patients with AF.

Changes in methylation patterns of multiple genes from peripheral blood leucocytes of Alzheimer's disease patients.

BACKGROUND: Efforts aiming at identifying biomarkers and corresponding methods for early diagnosis of Alzheimer's disease (AD) might be the most appropriate strategy to initiate promising new treatments and/or prevention of AD OBJECTIVE: The aim of our study is to assess the association of DNA methylation pattern of various leucocyte genes with AD pathogenesis in order to find potential biomarkers and corresponding methods for molecular diagnosis of AD. METHODS: DNA methylation level of various genes in AD patients and normal population were compared by bisulphite sequencing PCR and methylation-specific PCR (MSP). Furthermore, real-time PCR was used to explore the effects of DNA methylation on the expression of target genes. RESULTS: Results showed significant hypermethylation of mammalian orthologue of Sir2 (SIRT1) gene in AD patients compared with normal population. Meanwhile, changes in methylation level of SIRT1 gene between different severities of AD were also found. Specific primers were designed from the SIRT1 CpG islands to differentiate AD and control group by MSP method. Besides, significant demethylation of ß-amyloid precursor protein (APP) gene was observed in AD patients, whereas no difference was observed in other AD-related genes. Moreover, significant decrease in expression of SIRT1 gene and increase in expression of APP gene were also found in AD patients. In addition, the expression level of SIRT1/APP genes was associated with the severity, but not with the age or gender, of AD patients. CONCLUSION: SIRT1 and APP might be the interesting candidate biomarkers and valuable for clinical diagnosis or treatment of AD.

IL-1 polymorphisms in children with peptic symptoms in South China.

BACKGROUND: Polymorphisms of IL-1 gene cluster are reported to be associated with histological changes and IL-1ß expression in the gastric mucosa in adults, especially in Helicobacter pylori-infected subjects. As H. pylori infecting adults and children own different virulence genotypes, the aim of this study was to investigate whether IL-1 polymorphisms are risk factors in young children in South China. MATERIALS AND METHODS: A total of 128 children with peptic symptoms were enrolled in this study. Polymorphisms of IL-1B-511 and IL-1B-31 were identified by dual fluorescence PCR. Variable number of tandem repeat region in IL-1RN was detected by conventional PCR and IL-1ß mRNA expression by real-time PCR ddCT assay. RESULTS: IL-1B-31T and IL-1B-511C were completely linked in this study. Significant differences of IL-1B-511/-31 genotypes were observed among different clinical outcomes (p = .001). The IL-1B-511TT/-31CC was mostly found in the moderate gastritis and the above (severe gastritis or gastric ulcer) groups, with percentage of 60.7%. While no association was observed between IL-1RN genotypes and the gastric mucosal histological changes (p = .128). Also no relationships were found between IL-1 polymorphisms and H. pylori infection or gastric mucosal IL-1ß mRNA expression level. CONCLUSION: Children with IL-1B-511TT/-31CC may have a risk to develop relatively severe gastric mucosal histological changes in South China.

Non-invasive prenatal diagnosis of trisomy 21 by reverse transcriptase multiplex ligation-dependent probe amplification.

BACKGROUND: Obtaining fetal DNA or RNA by either chorionic villus sampling (CVS) or amniocentesis is currently, the gold standard prenatal diagnosis. However, these invasive procedures carry risk of miscarriage. A reliable method for non-invasive prenatal diagnosis (NIPD) has long been sought to reduce the risk of miscarriage. METHODS: Cell-free fetal RNA was extracted from the plasma of peripheral blood from 121 women 9-20 weeks of pregnancy. Five single nucleotide polymorphism (SNP) loci in PLAC4 gene were analyzed by reverse transcriptase multiplex ligation-dependent probe amplification (RT-MLPA), followed by capillary electrophoresis. Karyotype analysis was used for confirmation of prenatal diagnosis of trisomy 21. RESULTS: Of 121 samples, 23 were diagnosed with trisomy 21, 87 with normal ploidy, nine had all five SNP loci homozygous and two had one heterozygous SNP locus. Comparing with karyotype analysis, the diagnostic sensitivity and specificity of RT-MLPA were 92% and 100%, respectively. CONCLUSIONS: RT-MLPA is a convenient and reliable method for the diagnosis of trisomy 21. We have shown that this method has good specificity, high sensitivity, and high throughput, making this technique applicable for NIPD in clinical practice.

Development of a multiplex MethyLight assay for the detection of multigene methylation in human colorectal cancer.

In peripheral blood, cell-free methylated DNA has been reported to be a useful biomarker of noninvasive blood screening for the detection of colorectal cancer (CRC), including the genes ALX homeobox 4 (ALX4), septin 9 (SEPT9), or transmembrane protein with EGF-like, and two follistatin-like domains 2 (TMEFF2). Here we report a multiplex MethyLight polymerase chain reaction (PCR) assay that simultaneously detected the methylation status of ALX4, SEPT9, and TMEFF2, as well as quantifying methylation level of these genes in a total of 127 fresh tissue samples and 182 peripheral blood samples from CRC patients. Using the multiplex MethyLight assay, methylated ALX4, SEPT9, and TMEFF2 occurred in 56, 78, and 75% of CRC tissue samples and in 48, 75, and 71% of peripheral blood samples from CRC patients. The sensitivities of the combined study using the three genes as biomarkers for the detection of CRC in primary tissues and peripheral blood samples were 84 and 81%, with specificities of 87 and 90%, respectively. Combining the specificity of real-time PCR, the high throughput of multiplex PCR, and the high sensitivity of multigene detection, this multiplex MethyLight PCR assay may allow for future screening programs with large-scale noninvasive blood testing for early-stage CRC.

Development of primer-special TaqMan PCR: a novel SNP detection method to detect CYP2C9 3 in South Chinese.

BACKGROUND: CYP2C9 3 (1075A/C) is an inherited single nuclear polymorphism (SNP) of cytochrome P450 (CYP) 2C9, which affects the activity of the enzyme. In vitro studies with several drugs have indicated that the CYP2C9 3 variant has an impaired capacity for drug metabolism. Therefore an efficient detection assay for this mutation may be important for clinical dose adjustment. OBJECTIVE: The aim of this work was to develop an appropriate tool for detection of the CYP2C9 3 polymorphism in the clinical laboratory. STUDY DESIGN: The previously described TaqMan mismatch amplification mutation assay (TaqMAMA) was modified to a primer-special (PS)-TaqMan PCR to satisfy the high-throughput requirements of a clinical laboratory. 404 genomic DNA samples from South Chinese individuals were genotyped to test the detection system. The results were checked by bi-directional sequencing. RESULTS: PS-TaqMan PCR could correctly genotype the CYP2C9 allele from a genomic template at a concentration of 1 x 104 to 1 x 1011 copies/PCR. Among the 404 genomic DNA samples, 24 heterozygotes and 380 wild-type homozygotes were detected and confirmed by bi-directional sequencing. CONCLUSION: PS-TaqMan PCR was successfully developed for CYP2C9 3 detection. This efficient, reliable, high-throughput tool could satisfy the requirements of a clinical laboratory test.

The manual Polybrene test has limited sensitivities for detecting the Kidd blood group system.

BACKGROUND AND OBJECTIVES: The Kidd system antibodies, if undetected, can cause immediate or delayed hemolytic transfusion reactions as well as hemolytic disease of the newborn. There have been anecdotal reports about the inefficiency of the manual Polybrene test in detecting these antibodies. Here, we sought to determine the sensitivity of the manual Polybrene test in detecting anti-Jk(a) and anti-Jk(b) antibodies and Jk(a) and Jk(b) antigens. MATERIALS AND METHODS: Ten archived anti-Jk(a)/Jk(b) antibody positive human sera were examined by both the manual Polybrene test and the indirect antiglobulin test using polyspecific antibodies, monospecific anti-IgG antibodies and anti-C3 antibodies. Furthermore, 40 randomly selected donor blood samples were collected and phenotyped for the frequencies of Jk(a) and Jk(b) antigens using the manual Polybrene test and the indirect antiglobulin test. The results from these tests were further confirmed by saline tube tests. RESULTS: The manual Polybrene test displayed an overall sensitivity of 60% in detecting anti-Jk(a) and anti-Jk(b) antibody. Specifically, it had a sensitivity of 57.14% for anti-Jk(a) antibody and a sensitivity of 66.7% for anti-Jk(b) antibody. Furthermore, the manual Polybrene test exhibited a sensitivity of 46.15% for Jk(a) antigen and a sensitivity of 77.42% for Jk(b) antigen. CONCLUSION: The manual Polybrene test has a very low sensitivity in detecting anti-Jk(a) and anti-Jk(b) antibody, especially anti-Jk(a) antibody. It is also a very insensitive test for detecting Jk(a) antigen.

Distinctiveness of the cagA genotype in children and adults with peptic symptoms in South China.

BACKGROUND: Helicobacter pylori infection is different between children and adults, not only in infection rate but also in virulence genotypes. However, the 3' region of CagA, important in stomach carcinogenesis, still remains unclear in children. The present study aims to compare the frequency of cagA and the distribution of its subtypes between children and adults in South China. MATERIALS AND METHODS: One hundred and twenty-eight children and 99 adults with peptic symptoms were enrolled in our research. Histology, rapid urease test, and real-time polymerase chain reaction (PCR) assay were used to diagnose H. pylori infection. vacA s1 was detected by real-time PCR, and EPIYA motifs in the 3' region of CagA by conventional PCR and DNA sequencing. RESULTS: H. pylori infection was diagnosed in 53 children and 62 adults. vacA s1 was identified in 90.6% and 91.9% of infected children and adults, respectively. Furthermore, cagA was identified in 73.6% and 82.3% of infected children and adults, respectively. No patient with multiple cagA subtypes was observed. A higher prevalence of more virulent cagA genotype was found in children compared to adults (p < .05). Thirty-eight of 39 (97.4%) cagA-positive children were found to have EPIYA-ABD and only one (2.6%) with EPIYA-ABC. In adults, four types of EPIYA motifs--ABC (29.4%), ABD (64.7%), ABAB (2%), and AAD (3.9%)--were identified, and the ABD type was found more commonly in severe diseases, such as atrophic gastritis (53.3%) and gastric cancer (71.4%). CONCLUSION: cagA genotypes in children and in adults are different, and EPIYA-ABD may have potential clinical implication in the development of gastric cancer in South China.

CpG island methylation status in the EpCAM promoter region and gene expression.

The epithelial cell adhesion molecule (EpCAM) gene has been found to be highly expressed in carcinomas of various origins; however, the molecular mechanisms underlying the transcriptional regulation of EpCAM remain poorly understood. The purpose of this experiment was to study the relationship between EpCAM gene overexpression and CpG island methylation status in the promoter region in cell cultures and normal colon epithelial and colorectal cancer tissues. Real-time quantitative PCR and bisulfite sequencing PCR were employed to detect EpCAM gene expression and to analyze the methylation status of the CpG islands. In addition, EpCAM gene expression and methylation status of CpG islands were studied in promoter methylation cell lines treated with a DNA methyltransferase inhibitor. It was found that most CpG dinucleotides were unmethylated in cell lines and cancer tissues where the EpCAM gene was highly expressed whereas most CpG dinucleotides were methylated in EpCAM gene unexpressed cell lines and normal colon tissues. When cells were treated with demethylating agent, CpG islands were demethylated, although EpCAM gene expression did not increase suggesting that unmethylation of the EpCAM promoter region was not responsible for EpCAM overexpression. The results of the study described herein suggest that unmethylation of the EpCAM promoter region was associated with EpCAM overexpression; however, it was not responsible for EpCAM overexpression by itself. Further research is required to determine which factors are responsible for EpCAM gene expression.

Critical effect of VEGF in the process of endothelial cell apoptosis induced by high glucose.

The underlying molecular mechanism whereby hyperglycemia causes endothelial cell apoptosis is not well understood. This study aims to elucidate the role of survival factor VEGF involved in the apoptosis of endothelial cells induced by elevated glucose. The present study confirmed that high concentration of glucose (25 mmol/l) significantly increased the apoptotic cell number in cultured primary human umbilical vein endothelial cells (HUVEC). Up-regulation of Bax/Bcl-2 ratio and activation of caspase-3 induced by high glucose suggested that mitochondria apoptosis pathway was involved. High glucose significantly reduced VEGF expression in HUVEC both at mRNA and protein levels. p42/44 MAPK phosphorylation was transitory attenuated when exposed to high glucose and preceded VEGF reduction, thus suggesting down-regulation of VEGF through inhibition of p42/44 MAPK. Addition of VEGF prevented HUVEC apoptosis from high glucose exposure. Moreover, elevated reactive oxygen species (ROS) generation, calcium overload, Bax/Bcl-2 ratio, caspase-3 activation in HUVEC induced by high glucose were reversed by pre-challenge with VEGF. This may represent a mechanism for the anti-apoptotic effect of VEGF. These results suggest that down-regulation of VEGF plays a critical role in apoptosis of endothelial cells induced by high glucose and restoration of VEGF might have benefits in the early stage of diabetic endothelial dysfunction.

Simultaneous detection of different respiratory virus by a multiplex reverse transcription polymerase chain reaction combined with flow-through reverse dot blotting assay.

Cell culture and immunofluorescence (IF) assays have been traditionally used for the laboratory diagnosis of respiratory viral infections, but these assays have a low sensitivity and are time consuming. We developed a multiplex reverse transcription polymerase chain reaction combined with flow-through reverse dot blotting (mRT-PCR-FT-RDB) assay for the simultaneous detection of influenza virus type A including H5 subtype and H9 subtype, influenza virus type B, parainfluenza virus types 1 and 3, respiratory syncytial virus, human rhinovirus, and human coxsackievirus. In comparison with viral culture and IF assay as the gold standard method, the mRT-PCR-FT-RDB assay gave a sensitivity and a specificity of 100% and 98%. The high sensitivity and specificity, the rapid result turnaround time, and the reduced expense of the mRT-PCR-FT-RDB assay compared with viral culture and IF assay suggest that this assay would be a significant improvement over traditional ones for the detection of respiratory viruses in a clinical laboratory.

Development of a multiplex quantitative fluorescent PCR assay for identification of rearrangements in the AZFb and AZFc regions.

The azoospermia factor b (AZFb) and azoospermia factor c (AZFc) regions in the human Y chromosome consist of five palindromes constructed from six distinct families of amplicons and are prone to rearrangement. Partial deletion and duplication in the region can cause azoospermia or oligozoospermia and male infertility. The aim of the study was to establish a quantitative fluorescent PCR (QF-PCR) assay to classify AZFb and AZFc rearrangements. A single pair of fluorescent primers was designed to amplify simultaneously the amplicon in AZFc and the length-variant homologous sequences outside of the region as control. Since the copy number of the control sequences is fixed in the human genome, dosage of the target could be easily obtained through comparing the height of the fluorescent peaks between the target and the control after amplification with limited PCR cycles. Most types of rearrangements in AZFb and AZFc regions could be classified with QF-PCR containing four such primer pairs. Eleven types of rearrangement in AZFb and AZFc regions were well discriminated with QF-PCR. In conclusion, QF-PCR is a simple and reliable method to detect rearrangements in AZFb and AZFc.

Development of a multiplex real-time polymerase chain reaction for the detection of influenza virus type A including H5 and H9 subtypes.

Avian influenza viruses (AIVs) are endemic in wild birds and, if transmitted to poultry, can cause serious economic losses. The aim of this study was to develop a multiplex real-time reverse transcriptase polymerase chain reaction (RT-PCR) for rapid detection of influenza virus type A, including H5 and H9 subtypes. The selected primers and various labeled TaqMan reporter probes corresponding to matrix, H5, and H9 genes were used in a multiplex real-time RT-PCR to simultaneously detect triple fluorescent signals. The results showed that the multiplex real-time RT-PCR assay can be applied to detect RNA of influenza virus type A including H5 and H9 subtypes with a high specificity and a sensitivity of 10 copies per reaction. As a result of its short turnaround times and a high specificity and sensitivity, the assay is very suitable for large-scale screening during AIV outbreaks.

Determination of hepatitis B virus genotype by flow-through reverse dot blot.

BACKGROUND: The clinical outcome and response to therapy of hepatitis B virus infection differ depending upon viral genotype. Most methods of determining the viral genotype are relatively time-consuming and costly. Moreover, the results of some methods are influenced by single nucleotide mutations. OBJECTIVES: To develop a novel HBV genotyping process insensitive to single nucleotide mutations using an improved reverse dot blot method employing the principle of "flow-through hybridization". STUDY DESIGN: The flow through reverse dot blot (FT-RDB) method was developed using DNA from different HBV genotypes. HBV sequences from Genebank were used to design primers and probes. Specificity and sensitivity of the method were evaluated with clinical samples in which the HBV viral load was quantified by real-time PCR. Results were compared to those of multiplex PCR and sequencing. Another 59 clinical samples were used to test the clinical applicability of the method. RESULTS: We showed that FT-RDB could be made insensitive to single nucleotide mismatch by adjusting the hybridization temperature. All HBV-negative samples showed no signals in the assay. The detection sensitivity of the method was found to be between 10(3) and 10(4) DNA copies/ml. The results of FT-RDB were 84% concordant with those of multiplex PCR, and 96% concordant with sequencing results in 101 cases. The genotype all 59 clinical samples was accurately identified. CONCLUSIONS: We demonstrated that the FT-RDB method was rapid, reliable, accurate and inexpensive. It appears to be useful for routine clinical HBV genotyping even in non-specialized hospital laboratories.

Correlation of maternal plasma total cell-free DNA and fetal DNA levels with short term outcome of first-trimester vaginal bleeding.

BACKGROUND: The current methods using sonographic parameters and/or maternal serum beta-HCG levels to predict spontaneous abortion are not satisfactory. The aim of this study was to determine whether maternal plasma fetal DNA and total DNA levels could be used to predict spontaneous abortion. METHODS: We prospectively studied pregnant women who presented with vaginal bleeding in the first trimester of pregnancy, and those who had no vaginal bleeding (controls). DYS14 and the beta-globin gene were used to measure the maternal plasma levels of fetal and total DNA, respectively, by real-time PCR. RESULTS: A total of 1114 women were studied. Both maternal plasma fetal and total DNA concentrations increased with gestation from 6 to 11.6 weeks in the controls. The multiple of medians (MoMs) of fetal and total DNA concentration in those who miscarried were significantly greater (P < 0.001) than in the normal controls by about 5- and 4-fold respectively. Using a cut-off value of 1.6 MoMs for total DNA to predict spontaneous abortion, the sensitivity was 98.2% and false positive rate was 4.7%. However, using a cut-off value of 1.8 MoMs for fetal DNA, the corresponding figures were 97% and 44.3%, respectively. CONCLUSIONS: Both maternal plasma fetal and total DNA concentrations increased throughout the first trimester. Significantly high levels of fetal and total DNA were found in those who miscarried.

Relationship between apolipoprotein E, D10S1225 polymorphisms and late-onset Alzheimer's disease.

BACKGROUND: There were some papers published in the Jonrnal of Science, December 2000 suggesting that one or more important susceptibility genes for late onset Alzheimer's disease (LOAD) were located on the long arm of chromosome 10. Linkage analysis showed maximum lod score close to D10S1225 loci, which indicated the loci might contribute to the etiology of Alzheimer's disease (AD). METHODS: Fifty-nine LOAD patients and 107 controls were recruited. Apolipoprotein E (ApoE) genotypes were determined by reverse dot blotting hybridization assay. The D10S1225 was genotyped by 12% nondenaturing polyacrylamide gels electrophoresis and analyzed by silver staining. Statistical analysis was used to compare genotype and allele distributions between LOAD group and control group for ApoE and D10S1225 polymorphisms. RESULTS: ApoE epsilon 4 was significantly higher in LOAD group in comparison with the control group (chi(2) = 6.530, P = 0.011). Seven different alleles of D10S1225 have been identified. The length of these gene fragments were 178 bp, 181 bp, 184 bp, 187 bp, 190 bp, 193 bp, and 196 bp, respectively. A total of 21 different genotypes were observed. There was no relationship between D10S1225 polymorphism and LOAD (chi(2) = 4.488, P > 0.05). Conclusion This study suggests that ApoE epsilon 4 is a risk factor for LOAD, however, the results indicated that there is not any possible linkage for disequilibria with a nearby AD risk gene near D10S1225.

5' CpG island methylation analysis identifies the MAGE-A1 and MAGE-A3 genes as potential markers of HCC.

OBJECTIVES: The change in DNA methylation patterns can be used to distinguish between normal and cancer cells. The aim of the present study was to examine the 5' CpG island methylation patterns of the cancer-testis antigen (CT antigen) gene family, MAGE-As, in hepatocellular carcinoma (HCC), and to develop the DNA demethylation pattern as a novel tumor biomarker. METHODS: We used bisulfite-sequencing PCR (BSP) to map the methylation status of the CpG site among the promoter of the MAGE-A gene family in several HCC cell lines including Hep G2, BEL7402, BEL7404, and BEL7407, and normal peripheral blood white blood cells (WBCs). According to differences of the methylation pattern between HCC cell lines and the control, methylation-special PCRs (MSP) have been developed. The developed MSPs were used to detect the paraffin-embedded slices that were pathologically diagnosed as HCC, hepatocirrhosis, hepatitis, and healthy. RESULTS: We found that several CpG sites among the MAGE-A1 and MAGE-A3 promoters have different methylation patterns in the HCC cell lines as compared to those in normal WBCs. Two sets of MSP primers were designed to distinguish the HCC genomic DNA and normal control cell genomic DNA as novel tumor biomarkers, and the biomarkers were validated on the archived paraffin sections of liver primary tissue. In the detection of 34 HCCs and 17 tumor-free liver tissues, the clinical sensitivity and specificity were 91.2% and 100%, respectively. CONCLUSION: Detection of aberrant methylation patterns of MAGEs CpG islands using MSP may be useful for diagnosis of HCC.

Population genetic history of hepatitis C virus 1b infection in China.

Subtype 1b is the most common strain of Hepatitis C virus (HCV) in China. Here, the molecular epidemiology and epidemic history of this strain were investigated by conducting phylogenetic and population genetic analyses of E1 and NS5B gene sequences sampled from nine Chinese cities. The phylogenetic analysis indicated the presence of two clusters of Chinese strains that did not include reference strains from other countries, suggesting that these clusters represent two independent chains of HCV transmission within China. The remaining Chinese isolates were more closely related to reference strains from other countries. The date of origin and past population dynamics of the two groups were investigated using a new population genetic method, the Bayesian skyline plot. The estimated dates of origin of both groups coincide with the period of the Chinese 'Cultural Revolution' during the years 1966-1976. Both groups grew at a rapid exponential rate between approximately 1970 and approximately 1990, after which transmission slowed considerably. Possible explanations for the groups' fast spread and subsequent slowdown are discussed, including parenteral transmission by unsafe injection, iatrogenic transmission by infected blood or blood products and improvements in blood safety since 1990. These results shed light on HCV transmission in China and may help to predict the future burden of HCV-related disease in the country.

Rapid and accurate genotyping of YMDD motif variants in the hepatitis B virus genome by an improved reverse dot blot method.

By using the "flow-through hybridization" principle, we developed a new, rapid and accurate reverse dot blot (RDB) method to detect lamivudine resistance-associated YMDD motif variants in hepatitis B virus (HBV) genome. The improved RDB method was very fast at simultaneously detecting HBV YMDD wild-type and mutant motifs. In a blind analysis, 100 samples previously genotyped by DNA clonal sequencing analysis were used to evaluate the sensitivity and specificity of this assay. Conventional restriction fragment length polymorphism (RFLP) data were also used to test our method. In blind experiments, our improved RDB method had an accuracy and specificity of 100%, which was much higher than RFLP, which had an accuracy and specificity of only 83.0%. In clinical detection practice, 49 patients highly suspected of lamivudine resistance were successfully diagnosed by this method. Our improved RDB assay is a simple, rapid, cheap, semiautomatic, accurate, sensitive, and contamination-proof method of detecting lamivudine resistance-associated mutants in the human hepatitis B virus genome.

Hepatitis C virus genotype distribution in China: predominance of closely related subtype 1b isolates and existence of new genotype 6 variants.

To determine hepatitis C virus (HCV) genotype distribution in China, a total of 148 HCV RNA positive serum samples were collected from nine geographic areas and subjected to RT-PCR followed by direct DNA sequencing and phylogenetic analysis of the core, E1, and NS5B regions. HCV was genotyped in 139 (93.9%) samples. Among them subtype 1b was the most predominant [66% (92/139)] followed by 2a [14% (19/139)]. Of 92 subtype 1b isolates, 35 (38%) and 30 (33%) formed two clusters, designated groups A and B. Group A was prevalent throughout China, while group B was predominant in the central and southern regions. In three cities in the Pearl River Delta, subtype 6a replaced 2a as the second most predominant subtype, and in Kunming (southwest) multiple HCV genotypes/subtypes were present. New variants of HCV genotype 6 were discovered in three samples from Kunming and one in Guangzhou in the Pearl River Delta.