|
|
|
| Establishment of an assay for identifying mutation ofclarithromycin-resistance gene of Helicobacter pylori by real timePCR with a novel fluorescence quencher |
| CHEN Zhi-yong, LUO Yun,HUANG Chen, YE Ju-lian, YING Shen, LI Hui, JIN Da-zhi
|
| Fenghua People's Hospital,Ningbo,Zhejiang,315500,China |
|
|
|
|
Abstract Objective To establish a real time PCR assay with a novel fluorescence quencher for identification of mutation of clarithromycin-resistance gene of Helicobacter pylori.Methods Two mutations of 23S rDNA gene in Helicobacter pylori, No. 2142 and 2143, were chosen as targets for detection, and then the primers and the probe with a novel fluorescence quencher were designed. The genome DNA of Helicobacter pylori was extracted, and then detected by real time PCR reported here. Meanwhile, the specificity, reproducibility and sensitivity of the assay were evaluated. Finally, the real time PCR described here, the real time PCR based on TaqMan, and a sequencing assay were applied to detect 55 Helicobacter pylori strains isolated from clinical specimens, respectively. The results from three assays were compared with each other in order to further evaluate the applicability of this assay in clinic. Results It indicated that the mutation points related to clarithromycin-resistance, A2142G and A2143G, were identified by real time PCR with a novel fluorescence quencher rapidly and accurately. Moreover the coefficient of variation was less than 5%. The limit of detection was 100 copies/reaction. While this assay was applied directly to detect 55 Helicobacter pylori strains, the results were in accordance with those obtained from a TaqMan real time PCR and a sequencing assay, respectively. Conclusion The real time PCR described here was a simple, reliable and accurate approach and substituted for the TaqMan real time PCR for identification of two mutation points of clarithromycin-resistance, A2142G and A2143G in Helicobacter pylori. Thus, a novel tool for diagnosis of gene mutation was provided and the results might be regarded as a substantial evidence for clinical individual therapy.
|
|
Published: 25 December 2017
|
|
|
|
|
|
[1]MALATY H M,EVANS DJ Jr,ABRAMOVITCH K, et al. Helicobacter pylori infection in dental workers: a sero-epidemiology study [J]. Am J Gastroentero, 1992, 87 (12) : 1728-1731.
[2]MISIEWICZ J J. Working Party Reports. World Congresses Gastroenterology [M]. Melbourne: Blackwell, 1990: 1210.
[3]GERRITS M M, VAN VLIET A H, KUIPERS E J, et al. Helicobacter pylori and antimicrobial resistance: molecular mechanisms and clinical implications[J]. Lancet Infect Dis, 2006, 6(11): 699-709.
[4]郝庆,李岩,高红,等. 幽门螺杆菌对克拉霉素耐药的分子基础 [J]. 世界华人消化杂志,2003,11(10):1485-1487.
[5]刘永军,梅浙川. 分子生物学技术在幽门螺杆菌耐药检测中的应用[J]. 现代医药卫生,2004,20(24):2663-2665.
[6]RIBEIRO M L, VITIELLO L, MIRANDA M C, et al. Mutations in the 23S rRNA gene are associated with clarithromycin resistance in Helicobacter pylori isolates in Brazil[J] . Ann Clin Microbiol Antimicrob, 2003, 2(1):11.
[7]王小红,王升启. 荧光定量PCR技术研究进展[J]. 国外医学分子生物学分册,2001,23(3): 42-45.
[8]SAMBROOK J, RUSSELL D W. Molecular Cloning [M]. 黄培堂,王恒樑,周晓巍,等译.分子克隆实验指南(精编版).北京:化学工业出版社,2008:29-449.
[9]OLEASTRO M, MENARD A, SANTOS A, et al. Real-time PCR assay for rapid and accurate detection point mutations conferring resistance to clarithomycin in Helicobacter pylori[J]. J Clin Microbial, 2003, 41(1):397-402.
[10]MARSHALL B J, WARREN J R. Unidentified curved bacilli in stomach patients with gastritis and peptic ulceration [J]. Lancet, 1984, 323(8390):1311-1315.
[11]PARSONNET J. Helicobacter pylori and gastric cancer [J]. Gastroenterol Clin North Am, 1993,22(1): 89-104.
[12]PEREZ A L, KATO M, NAKAGAWA S, et al.The relationship between consumption of antimicrobial agents and the Prevalence of Primary Helicobaeter Pytori resistance[J].Helicobaeter, 2002,7(5):306-309.
[13]史彤,刘文忠,萧树东. 上海地区幽门螺杆菌对抗生素耐药率的变迁[J].中华内科杂志,2000,39(8): 576.
[14]成虹,胡伏莲,王蔚虹. 108株幽门螺杆菌(HP)菌株的耐药分析及其对HP根除的影响[J]. 中国临床药理学杂志,2001,17(6): 415-418.
[15]黄衍强,黄赞松,何勇强,等. 桂西地区幽门螺杆菌对克拉霉素耐药的调查[J]. 现代医药卫生,2008,24(20):3029-3030.
[16]王艳,周惠琴,唐文. 苏州市胃肠病患者幽门螺旋杆菌耐药分析[J]. 中国公共卫生,2010,26(1):122-123.
[17]秦基取,钱福初,何建方,等. 浙江省湖州地区幽门螺杆菌感染及耐药研究[J]. 疾病监测,2010,25(10):795-798.
[18]夏成静,陆学东. 分子生物学技术在病原菌耐药检测中的应用进展[J]. 中华医院感染学杂志,2008,18(2):297-300.
[19]FANG W J, JIN D Z, LUO Y, et al. A DNA minor groove binder shows high effectiveness as a quencher for FRET probes [J]. Bioorganic and Medicinal Chemistry Letters, 2014, 24(16): 3956-3960.
|
|
|
|
