21 6 2011 11 Journal of Jiangsu UniversityMedicine Edition Vol. 21 No. 6 Nov. 2011 BCP 1762 /1764 1 1 2 1 1. 2120212. 210438 basic core promoter BCP1762 /1764 BCP A1762T /G1764A TaqMan MGB PCRPSRT - PCR 80 50 41. 25% 33 /80 45. 00% 36 /80 68. 00% 34 /50 78. 00% 39 /50 P < 0. 005 HBV DNA 10 6 /ml 43 58. 14% 25 /43 62. 79% 27 /43 HBV DNA < 10 6 /ml 37 21. 62% 8 /37 24. 32% 9 /37 P < 0. 01 BCP R446. 5R512. 61 A 1671-7783201106 - 0514-05 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 2120212. Department of OutpatientEastern Hepatobiliary Surgery Hospital of Second Military Medical UniversityShanghai 210438 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 68. 00% vs 41. 25% P < 0. 005in the serum as well as in the hepatic tissue 78. 00% vs 45. 00% P < 0. 005respectively. 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 58. 14% vs 21. 62% P < 0. 005 but also in the hepatic tissue 62. 79% vs 24. 32% P < 0. 005. BK2006083 1966 E-mailzhangyuanhai1962@ 163. com
6. BCP 1762 /1764 515 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 28 30 ~ 56 42. 4 ± 10. 62 41 9 45 ~ 67 48 2-4 ± 8. 36 1. 2 1. 2. 1 1. 5 HBV phbv1. 5 C HBV DH5α 5-7 HBV HBV BCP 1762 /1764 8 1896 9 18 base core promoterbcp1762 10 - A - T/1764G - A 15 BCP PMD18T 10 BCP 1762 /1764 PCR icycler 11-15 PCR BioRad BCP 1. 2. 3 BCP Primer Premier 5. 0 16-17 BCP 1762 /1764 1. 3 1. 5 HBV 100 μl 2% BCP 1762 /1764 18 50 μl 100 15 min PCR 18 000 r /min 15 min BCP 1. 3. 2 DNA NaCl-EDTA- 1 1. 1 2005 1 2009 1 min 10 min 95% 4 000 hepatitis B chronic r /min 15 min 80% 1. 2. 2 PCR PCR TaKaRa 1. 3. 1 DNA Na 2 3 000 r /min 10 min 60 SDS 30 min - - 20 min 4 000 r / CHB 80 hepatocellular carcinomahcc50 1. 3. 3 2005 PMD-HBV1. 5-AG PMD-HBV1. 5-TA
516 21 1 μg 3. 31 10 11 2 10 7 10 6 10 5 10 4 10 3 1. 3. 4 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 80 50 TaqMan MGB PSRT-PCR 68. 00% 1 41. 25% χ 2 = 8. 815 P = 0. 003 78. 00% 45. 00% χ 2 = 13. 728 P = 0. 000 1 HBV DNA 10 6 / ml 43 HBV DNA < 10 6 /ml 37 P = 0. 001 P = 0. 001 2 1 % Tab 1 Comparison of double mutant on hepatitis B and liver cancer serum and liver tissue n χ 2 80 3341. 25 3645. 00 0. 229 > 0. 05 50 3468. 00 3978. 00 1. 268 > 0. 05 χ 2 8. 815 13. 728 P 0. 003 0. 000 Fig 1 1 Curve of plasmid standard amplification 2 % 1. 3. 5 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 95 30 s n 60 30 s72 30 s 50 72 10% HBV 43 37 2558. 14 821. 62 2762. 79 924. 32 χ DNA P 3 3 BCP 1762 /1764 10. 94 0. 001 11. 90 0. 001 10 BCP 1762 /1764 BCP P
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