Eaves Deborah J, Liebana Ernesto, Woodward Martin J, Piddock Laura J V
Antimicrobial Agents Research Group, Division of Infection and Immunity, University of Birmingham, Birmingham B15 2TT, United Kingdom.
J Clin Microbiol. 2002 Nov;40(11):4121-5. doi: 10.1128/JCM.40.11.4121-4125.2002.
Denaturing high-performance liquid chromatography (DHPLC) was evaluated as a rapid screening and identification method for DNA sequence variation detection in the quinolone resistance-determining region of gyrA from Salmonella serovars. A total of 203 isolates of Salmonella were screened using this method. DHPLC analysis of 14 isolates representing each type of novel or multiple mutations and the wild type were compared with LightCycler-based PCR-gyrA hybridization mutation assay (GAMA) and single-strand conformational polymorphism (SSCP) analyses. The 14 isolates gave seven different SSCP patterns, and LightCycler detected four different mutations. DHPLC detected 11 DNA sequence variants at eight different codons, including those detected by LightCycler or SSCP. One of these mutations was silent. Five isolates contained multiple mutations, and four of these could be distinguished from the composite sequence variants by their DHPLC profile. Seven novel mutations were identified at five different loci not previously described in quinolone-resistant salmonella. DHPLC analysis proved advantageous for the detection of novel and multiple mutations. DHPLC also provides a rapid, high-throughput alternative to LightCycler and SSCP for screening frequently occurring mutations.
变性高效液相色谱法（DHPLC）被评估为一种用于检测沙门氏菌血清型gyrA喹诺酮耐药决定区DNA序列变异的快速筛选和鉴定方法。使用该方法对总共203株沙门氏菌分离株进行了筛选。将代表每种新型或多重突变类型以及野生型的14株分离株的DHPLC分析与基于LightCycler的PCR-gyrA杂交突变检测法（GAMA）和单链构象多态性（SSCP）分析进行了比较。这14株分离株呈现出七种不同的SSCP模式，LightCycler检测到四种不同的突变。DHPLC在八个不同密码子处检测到11个DNA序列变异，包括LightCycler或SSCP检测到的那些。其中一个突变是沉默突变。五株分离株含有多重突变，其中四株可以通过其DHPLC图谱与复合序列变异区分开来。在喹诺酮耐药沙门氏菌中，在五个先前未描述的不同位点鉴定出七个新突变。DHPLC分析被证明有利于检测新的和多重突变。DHPLC还为筛选常见突变提供了一种快速、高通量的替代LightCycler和SSCP的方法。
