Diagnostic Systems Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland, USA.
Mol Cell Probes. 2010 Jun;24(3):154-60. doi: 10.1016/j.mcp.2010.01.003. Epub 2010 Jan 25.
The potential for genetic modification of biological warfare agents makes rapid identification of antibiotic resistant strains critical for the implementation of suitable infection control measures. The fluorinated quinolone, ciprofloxacin, is an antibiotic effective for treating bacterial infections by inhibiting the enzyme activity of the DNA type II topoisomerases DNA gyrase and topoisomerase IV. The genes that encode the subunits of DNA gyrase (gyrA and gyrB) and topo IV (par C and parE) contain hotspots within an area known as the quinolone resistance-determining region (QRDR). Base pair changes within this region give rise to mutations that cause resistance to the antibiotic by altering amino acids within the enzymes. Ciprofloxacin-resistant (cipro(r)) strains of Bacillus anthracis, Yersinia pestis, and Francisella tularensis with one or more known mutations within the QRDR of gyrA, gyrB, parC, and parE genes were tested with SimpleProbe and High Resolution Melt (HRM) dye chemistries and Pyrosequencing genetic analysis to evaluate the ability to rapidly detect ciprofloxacin-induced mutations. While SimpleProbe and Pyrosequencing successfully identified all known mutants, the HRM assay identified all but those resulting from G<-->C or A<-->T substitutions.
生物战剂的遗传修饰潜力使得快速鉴定抗生素耐药菌株对于实施适当的感染控制措施至关重要。氟喹诺酮类药物环丙沙星是一种有效的抗生素，通过抑制 DNA 拓扑异构酶 II 型（gyrase 和拓扑异构酶 IV）的酶活性来治疗细菌感染。编码 DNA 拓扑异构酶（gyrA 和 gyrB）和拓扑异构酶 IV（parC 和 parE）亚基的基因在称为喹诺酮类药物耐药决定区（QRDR）的区域内存在热点。该区域内的碱基对变化导致抗生素耐药性的出现，从而改变酶内的氨基酸。用 SimpleProbe 和高分辨率熔解（HRM）染料化学法和焦磷酸测序遗传分析对炭疽芽胞杆菌、鼠疫耶尔森菌和土拉弗朗西斯菌的环丙沙星耐药（cipro(r)）菌株进行了测试，这些菌株在 gyrA、gyrB、parC 和 parE 基因的 QRDR 内有一个或多个已知突变，以评估快速检测环丙沙星诱导突变的能力。虽然 SimpleProbe 和焦磷酸测序成功地鉴定了所有已知的突变体，但 HRM 检测法除了那些由 G<-->C 或 A<-->T 取代引起的突变体外，都能识别出来。
