CSIR-Institute of Genomics and Integrative Biology, Mall Road, Delhi, 110007 India.
Department of Zoology, M. D. University, Rohtak, 124001 Haryana India.
Indian J Microbiol. 2014 Jun;54(2):170-7. doi: 10.1007/s12088-013-0435-7. Epub 2013 Oct 27.
Human brain bacterial meningitis is a life-threatening disease mainly caused by Neisseria meningitidis, lead to several complications including damage of brain or even death. The present available methods for diagnosis of meningitis have one or more limitations. A rmpM gene based genosensor was fabricated by immobilizing 5'-amino modified 19-mer single stranded DNA probe onto carbon-mercaptooctadecane/carboxylated multi-walled carbon nanotubes composite electrode and hybridized with 2.5-40 ng/6 μL of single stranded genomic DNA (ssG-DNA) of N. meningitidis from cerebrospinal fluid (CSF) of the suspected meningitis patients. The electrochemical response was measured by using cyclic voltammetry and differential pulse voltammetry (DPV) using 1 mM methylene blue as redox indicator in 30 min (including a response time of 1 min) at 25 °C. The sensitivity of the genosensor was 3.762 (μA/cm(2))/ng and limit of detection was 2 ng of ssG-DNA of N. meningitidis with DPV. The genosensor has specificity only to N. meningitidis and does not hybridize with the genomic DNA of any other possible pathogen in human CSF. The immobilization of the probe and hybridization of the ssG-DNA were characterized by using electrochemical impedance in presence of 5 mM potassium ferricyanide and scanning electron microscopy. The genosensor loses only 12 % of its original DPV current on storage at 4 °C for 6 months. Carbon composite based electrochemical array can be constructed to detect multiple bacterial meningitis suspected patient CSF samples during an outbreak of the disease.
人类细菌性脑膜炎是一种危及生命的疾病,主要由脑膜炎奈瑟菌引起,可导致多种并发症,包括脑损伤甚至死亡。目前用于脑膜炎诊断的方法存在一种或多种局限性。本研究通过将 5'-氨基修饰的 19 个碱基单链 DNA 探针固定在碳-巯基十八烷/羧基多壁碳纳米管复合电极上,制备了一种基于 rmpM 基因的基因传感器,并与来自疑似脑膜炎患者脑脊液的脑膜炎奈瑟菌单链基因组 DNA(ssG-DNA)2.5-40ng/6μL杂交。在 25°C 下,使用 1mM 亚甲基蓝作为氧化还原指示剂,通过循环伏安法和差分脉冲伏安法(DPV)在 30 分钟内(包括 1 分钟的响应时间)测量电化学响应。该基因传感器的灵敏度为 3.762(μA/cm(2))/ng,DPV 检测限为 2ng 的脑膜炎奈瑟菌 ssG-DNA。该基因传感器仅对脑膜炎奈瑟菌具有特异性,不会与人类脑脊液中任何其他可能病原体的基因组 DNA 杂交。通过电化学阻抗法在存在 5mM 铁氰化钾的情况下,对探针的固定和 ssG-DNA 的杂交进行了表征,并采用扫描电子显微镜进行了分析。基因传感器在 4°C 下储存 6 个月后,其 DPV 电流仅损失 12%。在疾病爆发期间,可以构建基于碳复合材料的电化学阵列来检测多个疑似细菌性脑膜炎患者的脑脊液样本。