OBJECTIVE: To study the role of hepatitis B virus with A1762T/G1764A double mutation in liver cirrhosis and hepatocellular carcinoma, and create a sensitive, fast, accurate assay for detection of A1762T/G1764A double mutation.METHODS: We developed an accurate and fast real-time amplification refractory mutation system to detect A1762T/G1764A double mutation. Cloned hepatitis B virus genome was used as a control. Assay sensitivity was determined by serial dilution and mixed template experiments. Specificity was determined by cross experiments with wild and mutant hepatitis B virus. Fifty clinical samples were tested by the real-time amplification refractory mutation system and the results were compared with sequencing.RESULTS: The real-time amplification refractory mutation system had a sensitivity of 100 copies of virus with these mutations, and 0.1% weak population virus with double mutation could be found in mixtures. A total of 50 randomly collected clinical samples were detected by real-time amplification refractory mutation system, and the results were consistent with those by DNA sequencing. Hepatitis B virus genotype C was more prevalent in 39 of 50 samples than genotype B (11 samples), and about 75% of genotype C carried a double mutation compared to 45% of genotype B. However, the percentage of A1762T/G1764A double mutation in hepatitis B e antigen-negative (58.3%) samples was almost the same as in hepatitis B e antigen-positive (61%) samples.CONCLUSION: The real-time amplification refractory mutation system is sensitive and specific for detection of hepatitis B virus double mutation.

ObjectiveTo study the effect of ischemia preconditioning (IP) on renal ischemia/reperfusion (I/R)-associated functional injury and expression of renal adhesion molecules in rats.Materials and MethodsThe ischemia preconditioning plan adopted in this experiment involved renal warm ischemia for 6 min. and blood flow for 4 min., repeated four times. The Wistar rat kidneys used for warm ischemia preconditioning were subjected to 60 min of renal warm ischemia followed by reperfusion. The rat kidneys with ischemia/reperfusion were compared with the ischemia preconditioning group to observe rat renal function and changes in the expression of renal adhesion molecules ICAM-1, P--Selectin, and E-Selectin.ResultsThe expression of rat renal adhesion molecules (ICAM-1, P-Selectin, and E-Selectin) with ischemia preconditioning was significantly lower than that of the ischemia/reperfusion group. Serum creatinine was significantly lower than that in the ischemia/reperfusion group after 48 hours.ConclusionsIschemia preconditioning has a protective effect on renal function. Reduced expression of renal adhesion molecules is likely a mechanism involved in the observed protection.

OBJECTIVES: Detection of mutations associated to nucleos(t)ide analogs and hepatitis B virus (HBV) genotyping are essential for monitoring treatment of HBV infection. We developed a multiplex polymerase chain reaction-ligase detection reaction (PCR-LDR) assay for the rapid detection of HBV genotypes and mutations associated with lamivudine, adefovir, and telbivudine resistance in HBV-infected patients. METHODS: HBV templates were amplified by PCR, followed by LDR and electrophoresis on a sequencer. The assay was evaluated using plasmids that contained wild-type or mutant HBV sequences and 216 clinical samples. RESULTS: The PCR-LDR assay and sequencing gave comparable results for 158 of the 216 samples (73.1%) with respect to mutation detection and genotyping. Complete agreement between the two methods was observed for all the samples (100%) at codon 180 and codon 204. Concordant results were observed for 99.4% of the 158 samples at codon 181 and 98.7% at codon 236. The genotyping results were completely concordant between the PCR-LDR assay and sequencing. The PCR-LDR assay could detect a proportion of 1% mutant plasmid in a background of wild-type plasmid. CONCLUSION: The PCR-LDR assay is sensitive and specific for detection of HBV genotypes and drug resistance mutations, and could be helpful for decision making in the treatment of HBV infection.