Lee W K, Cho M J, Choi H J, Rhee K H
Department of Microbiology, Gyeongsang National University College of Medicine, Chinju, Kyung-Nam, Korea.
J Korean Med Sci. 1991 Dec;6(4):338-47. doi: 10.3346/jkms.1991.6.4.338.
The polymerase chain reaction was used to develop a method for the detection of Helicobacter pylori, a causative agent of gastritis, as well as for the elucidation of its mode of transmission. A genomic library of Helicobacter pylori DNA in Escherichia coli JM109 was constructed by cloning Hind III-digested DNA fragments into plasmid vector pUC18. The nucleotide sequences from seven recombinant clones were determined and five sets of oligonucleotide primers were synthesized on the basis of the sequences from five clones (B4, B9, B10, C15 and I22). The PCR amplifications with these primers were performed using DNA samples from five strains of Helicobacter pylori, two Campylobacter spp. and eleven species of enteric bacteria. Amplifications of the target DNA fragments in all of 5 strains of Helicobacter pylori were observed from the PCR with primers derived from clone B4, B9, C15 and I22. When the specificity was checked with the DNA samples from 13 other bacteria as template DNA for the PCR, specific amplification that produced the correct size of the target DNA of Helicobacter pylori was shown only in the PCR with primers derived from clone B9 and C15. The detection limit in the PCR amplification, determined by the heat-lysis method, was 500 cells of Helicobacter pylori.
聚合酶链反应被用于开发一种检测幽门螺杆菌（胃炎的病原体）的方法，以及阐明其传播方式。通过将经Hind III消化的DNA片段克隆到质粒载体pUC18中，构建了幽门螺杆菌DNA在大肠杆菌JM109中的基因组文库。测定了七个重组克隆的核苷酸序列，并根据五个克隆（B4、B9、B10、C15和I22）的序列合成了五组寡核苷酸引物。使用来自五株幽门螺杆菌、两株弯曲菌属细菌和十一种肠道细菌的DNA样本，用这些引物进行聚合酶链反应扩增。用来自克隆B4、B9、C15和I22的引物进行聚合酶链反应时，在所有五株幽门螺杆菌中均观察到目标DNA片段的扩增。当用来自其他13种细菌的DNA样本作为聚合酶链反应的模板DNA检查特异性时，只有在用来自克隆B9和C15的引物进行的聚合酶链反应中显示出产生正确大小的幽门螺杆菌目标DNA的特异性扩增。通过热裂解法测定，聚合酶链反应扩增的检测限为500个幽门螺杆菌细胞。
