Gürtler Volker, Barrie Helen D, Mayall Barrie C
Department of Microbiology, Austin & Repatriation Medical Centre (Austin Campus), Heidelberg, Victoria, Australia.
Electrophoresis. 2002 Sep;23(19):3310-20. doi: 10.1002/1522-2683(200210)23:19<3310::AID-ELPS3310>3.0.CO;2-O.
To obviate the need for multilocus sequencing, a method using denaturing gradient gel electrophoresis (DGGE) was developed for the multilocus sequence typing (MLST) of Staphylococcus aureus isolates. Sequence types (STs) were obtained on the basis of sequences of polymerase chain reaction (PCR) products from seven housekeeping genes and compared to the reference MLST database. The melt curves, sequences and DGGE profiles were compared for 100 STs (i) to determine PCR conditions with 40-mer GC-clamps attached to the forward and reverse primers; (ii) to choose single restriction enzyme sites for digestion of PCR products into two fragments each with a GC-clamp attached and (iii) to optimize DGGE conditions. When the DGGE types (DT) were analyzed, the majority of DTs (76/100) were accurately classified into one ST (95% of nucleotide changes were detected), 10 DTs were classified into one of two STs corresponding to a single nucleotide ambiguity and 14 DTs were classified into 3 or 4 STs corresponding to 4 or 5 nucleotide ambiguities. A combination of STs and DTs were used to obtain septuplet sets of STs (7-ST) for 25 S. aureus isolates. When compared to the reference MLST database, one methicillin-resistant S. aureus (MRSA) isolate had the same genotype as the first MRSA clone. The DGGE-MLST method can be used as a rapid, accurate and 20-fold less expensive method than DNA sequencing for the detection of all sequence types. This combined laboratory and in silico approach could have wide applicability not only to MLST methods for other bacteria but to the screening of multilocus nucleotide differences deposited in other mutation databases.
为避免进行多位点测序，开发了一种使用变性梯度凝胶电泳（DGGE）的方法，用于金黄色葡萄球菌分离株的多位点序列分型（MLST）。根据来自七个管家基因的聚合酶链反应（PCR）产物的序列获得序列类型（STs），并与参考MLST数据库进行比较。对100个STs的熔解曲线、序列和DGGE图谱进行比较，以：（i）确定正向和反向引物连接40个碱基的GC夹的PCR条件；（ii）选择单一限制性酶切位点，将PCR产物消化成两个片段，每个片段都连接一个GC夹；以及（iii）优化DGGE条件。分析DGGE类型（DTs）时，大多数DTs（76/100）被准确分类为一种ST（检测到95%的核苷酸变化），10个DTs被分类为与单核苷酸歧义相对应的两种STs之一，14个DTs被分类为与4或5个核苷酸歧义相对应的3或4种STs。使用STs和DTs的组合，为25株金黄色葡萄球菌分离株获得了七重STs集（7-ST）。与参考MLST数据库相比，一株耐甲氧西林金黄色葡萄球菌（MRSA）分离株与第一个MRSA克隆具有相同的基因型。DGGE-MLST方法可作为一种快速、准确且成本比DNA测序低20倍的方法，用于检测所有序列类型。这种实验室和计算机相结合的方法不仅可广泛应用于其他细菌的MLST方法，还可用于筛选其他突变数据库中存储的多位点核苷酸差异。
