Journal of Jiangsu University Medicine Edition basic core promoter BCP 1762 /1764 BCP. TaqMan MGB % 33 /80

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1 Journal of Jiangsu UniversityMedicine Edition Vol. 21 No. 6 Nov BCP 1762 / basic core promoter BCP1762 /1764 BCP A1762T /G1764A TaqMan MGB PCRPSRT - PCR % 33 / % 36 / % 34 / % 39 /50 P < HBV DNA 10 6 /ml % 25 / % 27 /43 HBV DNA < 10 6 /ml % 8 / % 9 /37 P < BCP R446. 5R A Detection and significance of double mutation in basic core promoter in hepatitis B virus in chronic hepatitis B and hepatocellular carcinoma SHEN Yue-ping XU JunMAN Xiao-boZHANG Yuan-hai 1. Department of Infectious Disease the Third People's Hospital of ZhenjiangZhenjiang Jiangsu Department of OutpatientEastern Hepatobiliary Surgery Hospital of Second Military Medical UniversityShanghai China AbstractObjectiveTo explore the feasibility of quantitative detection of double mutation in basic core promoter BCP1762 /1764 sites in hepatitis B virus HBVin chronic hepatitis B CHBand hepatocellular carcinoma HCCand the relationship between the mutation and HCC and its clinical significance. MethodsThe mutant was accurately detected using probe-specific real-time quantitative polymerase chain reaction PSRT-PCRwhich adopted TaqMan MGB probe the designing was depended on the double mutation plasmids which were constructed based on the wild recombinant replication competent plasmids of HBV. Their different effluence was compared between CHB and HCC. ResultsAfter detecting the serum and hepatic tissue in 80 CHB patients and 50 HCC patients all patients never accepted antiviral treatmentthe HCC group expressed a higher mutant rate % vs % P < in the serum as well as in the hepatic tissue % vs % P < respectively. The difference of mutation rate between the serum and the hepatic tissue was not significant either in CHB group and HCC group. In 80 CHB patients compared with low viral load group HBV DNA < 10 6 copies /ml 37 caseshigh viral load group HBV DNA 10 6 copies /ml 43 casesexpressed a higher mutant rate not only in the serum % vs % P < but also in the hepatic tissue % vs % P < BK zhangyuanhai1962@ 163. com

2 6. BCP 1762 / ConclusionThe higher rate of double mutation in patients with HCC hinted that the double mutation was related to the occurrence of HCC which can be forecasted by detecting the double mutation. Based on the satisfactory consistency in detecting serum and hepatic tissueto detect the patients'serums who could not accept drawing the tissue from their liver can accurately reflect their states of double mutation tooand it can easily be wide spreaded in clinic. High viral load hinted high mutation rate in hepatitis B patients without HCC so furthermore early antiviral treatment can help to decrease the mutation rate. Key wordshepatitis B virusbasic core promotermutationhepatitis Bhepatocellular carcinoma hepatitis B virus HBV 52 1 HBV ~ ± ~ ± HBV phbv1. 5 C HBV DH5α 5-7 HBV HBV BCP 1762 / base core promoterbcp A - T/1764G - A 15 BCP PMD18T 10 BCP 1762 /1764 PCR icycler PCR BioRad BCP BCP Primer Premier BCP 1762 / HBV 100 μl 2% BCP 1762 / μl min PCR r /min 15 min BCP DNA NaCl-EDTA min 10 min 95% hepatitis B chronic r /min 15 min 80% PCR PCR TaKaRa DNA Na r /min 10 min 60 SDS 30 min min r / CHB 80 hepatocellular carcinomahcc PMD-HBV1. 5-AG PMD-HBV1. 5-TA

3 μg PCR PSRT- TaqMan MGB PCR PSRT-PCR HBV DNA 10 PSRT-PCR F 5'-GTAACTCCACAGAAGCTCCAAAT-3' BCP R5'-GTTGGGCACTTTGCCACAGGAAC-3' A1762T /G1764A HBV DNA GTAMRA > PSRT-PCR BCP TGGCCTATGG < NFQ M' 100% FAM > GACTTATAAC < NFQ TaqMan MGB PSRT-PCR % % χ 2 = P = % % χ 2 = P = HBV DNA 10 6 / ml 43 HBV DNA < 10 6 /ml 37 P = P = % Tab 1 Comparison of double mutant on hepatitis B and liver cancer serum and liver tissue n χ > > χ P Fig 1 1 Curve of plasmid standard amplification 2 % DNA Tab 2 Comparison of double mutant in different PSRT-PCR 95 3 min 60 viral load groups of hepatitis B 4 min 72 5 min s n s72 30 s % HBV χ DNA P 3 3 BCP 1762 / BCP 1762 /1764 BCP P

4 6. BCP 1762 / BCP PCR-SSCP PCR BCP 1762 / SOMA pyrosequence BCP 1762 / B CT BCP 1762 /1764 PSRT-PCR HBV BCP A1762T /G1764A PCR 2 h BCP PCR BCP PCR 1762 /1764 BCP PCR TaqMan MGB Non-Fluorescent Quencher MGB Minor Groove Binder Tm 10 tion of hepatitis B virus over 25 years J. J Virol 1 Osiowy CGiles ETanaka Yet al. Molecular evolu Tm MGB TaqMan 2 Tharayil VSRoberts LR. Molecular targets for therapy TaqMan MGB of hepatitis B virus-induced hepatocellular carcinoma A /T J. Minerva Gastroenterol Dietol Marschenz SEndres ASBrinckmann Aet al. Functional analysis of complex hepatitis B virus variants asso- ciated with development of liver cirrhosis J. Gastroenterology BCP 21 BCP AT 4Wands JR. Prevention of hepatocellular carcinoma J. N BCP C Engl J Med RNA HBeAg BCP 5 Palumbo E. Hepatitis B genotypes and response to antiviral therapya review J. Am J Ther RNA 6Szmaragd CFoster GRManica Aet al. Genome-wide

5 characterisation of hepatitis B mutations involved in clinical outcome J. Heredity Echevarria JMAvellon A. Hepatitis B virus genetic diversity J. J Med Virol Suppl Yuan HJLee WM. Molecular mechanisms of resistance to antiviral therapy in patients with chronic hepatitis B J. Curr Mol Med Waltz TLMarras SRochford Get al. Development of a molecular-beacon assay to detect the G1896A precore mutation in hepatitis B virus-infected individuals J. J Clin Microbiol Kramvis A Kew MC. The core promoter of hepatitis B virus J. J Viral Hepat Jardi RRodriguez FButi Met al. Mutations in the basic core promoter region of hepatitis B virus. Relationship with precore variants and HBV genotypes in a Spanish population of HBV carriers J. J Hepatol Yotsuyanagi HHino KTomita Eet al. Precore and core promoter mutationshepatitis B virus DNA levels and progressive liver injury in chronic hepatitis B J. J Hepatol Erhardt AReineke UBlondin Det al. Mutations of the core promoter and response to interferon treatment in chronic replicative hepatitis B J. Hepatology Buckwold VEXu ZChen Met al. Effects of a naturally occurring mutation in the hepatitis B virus basal core promoter on precore gene expression and viral replication J. J Virol Song BCCui XJKim HUet al. Sequential accumulation of the basal core promoter and the precore mutations in the progression of hepatitis B virus-related chronic liver disease J. Intervirology Baptista MKramvis AKew MC. High prevalence of 1762T1764Amutations in the basic core promoter of hepatitis B virus isolated from black Africans with hepatocellular carcinoma compared with asymptomatic carriers J. Hepatology Blackberg JKidd-Ljunggren K. Mutations within the hepatitis B virus genome among chronic hepatitis B patients with hepatocellular carcinomaj. J Med Virol A1762T /G1764A J Zhang MGong YOsiowy Cet al. Rapid detection of hepatitis B virus mutations using real-time PCR and melting curve analysis J. Hepatology Chen CHLee CMLu SNet al. Clinical significance of hepatitis B virus HBVgenotypes and precore and core promoter mutations affecting HBV e antigen expression in TaiwanJ. J Clin Microbiol Du HLi TZhang HYet al. Correlation of hepatitis B virus HBVgenotypes and mutations in basal core promoter / precore with clinical features of chronic HBV infection J. Liver Int Liu CJChen BFChen PJet al. Role of hepatitis B virus precore / core promoter mutations and serum viral load on noncirrhotic hepatocellular carcinomaa casecontrol studyj. J Infect Dis Gandhe SSChadha MSWalimbe AMet al. Hepatitis B virusprevalence of precore /core promoter mutants in different clinical categories of Indian patients J. J Viral Hepat

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