Detection of gyrA mutations in quinolone-resistant Salmonella enterica by denaturing high-performance liquid chromatography
Detection of gyrA mutations in quinolone-resistant Salmonella enterica by denaturing high-performance liquid chromatography
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DOI：
10.1128/jcm.40.11.4121-4125.2002
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发表时间：
2002-11-01
影响因子：
9.4
通讯作者：
Piddock, LJV
中科院分区：
文献类型：
--
作者：
Eaves, DJ;Liebana, E;Piddock, LJV
变性高效液相色谱法（DHPLC）可用于沙门氏菌血清型gyrA基因喹诺酮类耐药决定区DNA序列变异的快速筛查和鉴定。共203株沙门氏菌使用该方法进行了筛选。DHPLC分析代表每种类型的新的或多个突变和野生型的14个分离株进行了比较，基于LightCycler的PCR-gyrA杂交突变分析（GAMA）和单链构象多态性（SSCP）分析。14个分离株给出了7种不同的SSCP模式，LightCycler检测到4种不同的突变。DHPLC检测到8个不同密码子的11个DNA序列变异，包括LightCycler或SSCP检测到的变异。其中一个突变是沉默的。5株分离株含有多个突变，其中4株可通过DHPLC图谱与复合序列变体区分开来。在5个不同的位点上发现了7个新的突变，这些突变以前在喹诺酮类耐药的沙门氏菌中没有描述过。DHPLC分析证明有利于检测新的和多个突变。DHPLC还提供了一种快速，高通量的替代LightCycler和SSCP筛选频繁发生的突变。
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.