AIM： To screen for metronidazole （MTZ）-resistance associated gene fragments of H pylori by suppression subtractive hybridization （SSH）. METHODS： Five MTZ-resistant （tester, T） and 1 MTZ- susceptible （driver, ...AIM： To screen for metronidazole （MTZ）-resistance associated gene fragments of H pylori by suppression subtractive hybridization （SSH）. METHODS： Five MTZ-resistant （tester, T） and 1 MTZ- susceptible （driver, D） clinical H pylori isolates were selected. Genomic DNAs were prepared and submitted to Rsa I digestion. Then two different adaptors were ligated respectively to the 5＇-end of two aliquots of the tester DNA fragments and SSH was made between the tester and driver DNAs. The specific inserts of tester strains were screened and MTZ-resistance related gene fragments were identified by dot blotting. RESULTS： Among the randomly selected 120 subtractive colonies, 37 DNA fragments had a different number of DNA copies （≥ 2 times） in resistant and susceptible strains and 17 of them had a significantly different number of DNA copies （≥ 3 times）. Among the sequences obtained from the 17 DNA fragments, new sequences were found in 10 DNA fragments and duplicated sequences in 7 DNA fragments, representing respectively the sequences of depeptide ABC transporter periplasmic dipeptide-binding protein （dppA）, permease protein （dppB）, ribosomal protein S4 （rps4）, ribonuclease Ⅲ （rnc）, protease （pqqE）, diaminopimelate epimerase （dapF）, acetatekinase （ackA）, H pylori plasmid pHP51 and Hpylori gene 1334. CONCLUSION： Gene fragments specific to MTZ-resistant H pylori strains can be screened by SSH and may be associated with MTZ-resistant Hpylori.展开更多
基金Supported by the Natural Science Foundation of ZhejiangProvince, No. 29801
文摘AIM： To screen for metronidazole （MTZ）-resistance associated gene fragments of H pylori by suppression subtractive hybridization （SSH）. METHODS： Five MTZ-resistant （tester, T） and 1 MTZ- susceptible （driver, D） clinical H pylori isolates were selected. Genomic DNAs were prepared and submitted to Rsa I digestion. Then two different adaptors were ligated respectively to the 5＇-end of two aliquots of the tester DNA fragments and SSH was made between the tester and driver DNAs. The specific inserts of tester strains were screened and MTZ-resistance related gene fragments were identified by dot blotting. RESULTS： Among the randomly selected 120 subtractive colonies, 37 DNA fragments had a different number of DNA copies （≥ 2 times） in resistant and susceptible strains and 17 of them had a significantly different number of DNA copies （≥ 3 times）. Among the sequences obtained from the 17 DNA fragments, new sequences were found in 10 DNA fragments and duplicated sequences in 7 DNA fragments, representing respectively the sequences of depeptide ABC transporter periplasmic dipeptide-binding protein （dppA）, permease protein （dppB）, ribosomal protein S4 （rps4）, ribonuclease Ⅲ （rnc）, protease （pqqE）, diaminopimelate epimerase （dapF）, acetatekinase （ackA）, H pylori plasmid pHP51 and Hpylori gene 1334. CONCLUSION： Gene fragments specific to MTZ-resistant H pylori strains can be screened by SSH and may be associated with MTZ-resistant Hpylori.
