Manchanda V, Rai S, Gupta S, Rautela R S, Chopra R, Rawat D S, Verma N, Singh N P, Kaur I R, Bhalla P
Clinical Microbiology and Infectious Diseases, Chacha Nehru Bal Chikitsalaya, (Affiliated to Maulana Azad Medical College), Govt of NCT of Delhi, Geeta Colony, Delhi - 110 031, India.
Indian J Med Microbiol. 2011 Jul-Sep;29(3):249-53. doi: 10.4103/0255-0857.83907.
The newly emerging form of the so-called New Delhi Metallo-beta-lactamases (NDM-1) has been reported recently from patients worldwide and broadly thought as a potential source for the major global health problem. Thus, it is important to study the epidemiology of the so-called NDM-1 harbouring bacteria to prevent its further spread and to place effective control measures. The present study describes the use of the real-time polymerase chain reaction (PCR) assay for the detection of the bla NDM-1 gene using TaqMan probes among clinical isolates.
Clinical isolates of Escherichia coli (11 strains), Klebsiella pneumoniae (17 strains) and Acinetobacter baumannii (six strains) that were resistant to either of the carbapenems (meropenem or imipenem) were included in the study. The presence of carbapenemases in such strains was confirmed using the modified Hodge test. A real-time PCR assay was optimized for the detection of NDM-1 using a cloned synthetic gene fragment followed by testing of the clinical isolates. The findings were further confirmed using PCR and gene sequencing.
TaqMan probe assay displayed a good detection limit with analytical sensitivity of the assay up to 10 copies of bla NDM-1 gene per reaction. The isolates of E. coli and K. pneumoniae revealed narrow range crossing point values (Cp values) between (12-17) cycles (mean Cp value 14), indicating number of bla NDM-1 gene copies of 106-108. The wider range of Cp values (15-34) cycles with a higher mean Cp value (23.6) was observed in A. baumannii with number of bla NDM-1 gene copies of 103-108.
The study demonstrates that real-time PCR assay based on TaqMan chemistry is a useful technique for the detection of bla NDM-1 harbouring clinical isolates of E. coli, K. pneumoniae and A. baumannii. The assay has great precision in measuring the number of bla NDM-1 gene copies per specimen of DNA.
近期在全球范围内的患者中报道了所谓的新德里金属β-内酰胺酶（NDM-1）的新出现形式，普遍认为它是全球主要健康问题的一个潜在源头。因此，研究携带NDM-1的细菌的流行病学情况对于防止其进一步传播并采取有效的控制措施很重要。本研究描述了在临床分离株中使用基于TaqMan探针的实时聚合酶链反应（PCR）检测bla NDM-1基因。
本研究纳入了对碳青霉烯类药物（美罗培南或亚胺培南）耐药的大肠埃希菌临床分离株（11株）、肺炎克雷伯菌临床分离株（17株）和鲍曼不动杆菌临床分离株（6株）。使用改良的 Hodge试验确认此类菌株中碳青霉烯酶的存在。使用克隆的合成基因片段对实时PCR检测NDM-1进行优化，随后对临床分离株进行检测。研究结果通过PCR和基因测序进一步确认。
TaqMan探针检测法显示出良好的检测限，每次反应对bla NDM-1基因的分析灵敏度高达10个拷贝。大肠埃希菌和肺炎克雷伯菌的分离株显示出较窄范围的交叉点值（Cp值），介于（12 - 17）个循环之间（平均Cp值为14），表明bla NDM-1基因拷贝数为10⁶ - 10⁸。在鲍曼不动杆菌中观察到更宽范围的Cp值（15 - 34）个循环，平均Cp值更高（23.6），bla NDM-1基因拷贝数为10³ - 10⁸。
该研究表明基于TaqMan化学的实时PCR检测法是检测携带bla NDM-1的大肠埃希菌、肺炎克雷伯菌和鲍曼不动杆菌临床分离株的有用技术。该检测法在测量每份DNA样本中bla NDM-1基因拷贝数方面具有很高的精确度。
