Rapid detection of blaKPC carbapenemase genes by real-time PCR
Rapid detection of blaKPC carbapenemase genes by real-time PCR
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DOI：
10.1128/jcm.00661-08
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发表时间：
2008-09-01
影响因子：
9.4
通讯作者：
Keller, Nathan
中科院分区：
文献类型：
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作者：
Hindiyeh, Musa;Smollen, Gill;Keller, Nathan
肠杆菌科细菌对碳青霉烯类抗生素的耐药性是一个全球性的问题。肺炎克雷伯菌碳青霉烯酶（bla（KPC））是所述最常见的β-内酰胺酶。在这项研究中，我们报告的发展和验证的实时PCR（q-PCR）检测bla（KPC）基因使用TaqMan化学。bla（KPC）DNA的q-PCR扩增在7个对数稀释度内呈线性关系（r（2）= 0.999;斜率为3.54），扩增效率为91.6%。q-PCR检测限为1 CFU，与多种多重耐药菌DNA无交叉反应。从Sheba Medical Center外科重症监护室收治的128例患者中一式两份采集肛周/直肠拭子（n = 187），通过在麦康凯琼脂加碳青霉烯纸片上培养评价碳青霉烯类耐药菌的存在，并通过q-PCR评价bla（KPC）基因。碳青霉烯类耐药菌，均为K.从收集的187个样品中的47个（25.1%）分离出肺炎链球菌，而在通过NucliSENS easyMAG系统提取的54个（28.9%）患者样品中检测到bla（KPC）基因。其中，7份样本通过q-PCR检测bla（KPC）基因呈阳性，但通过培养检测碳青霉烯类耐药性呈阴性，而所有通过培养未检测到碳青霉烯类耐药性细菌的样本通过q-PCR检测也呈阴性。因此，通过NucliSENS easyMAG系统提取后q-PCR测定的灵敏度和特异性分别为100%和95%。在通过Roche MagNA Pure LC仪器提取DNA后获得了相似的值：97.9%的灵敏度和96.4%的特异性。总体而言，bla（KPC）q-PCR检测似乎具有高度灵敏度和特异性。q-PCR的使用将blaKPC检测时间从24小时缩短至4小时，并将有助于快速分离定殖或感染患者并将其分配至队列。
Carbapenem resistance among Enterobacteriaceae is an emerging problem worldwide. Klebsiella pneumoniae carbapenemase (bla(KPC)) enzymes are among the most common beta-lactamases described. In this study, we report the development and validation of a real-time PCR (q-PCR) assay for the detection of bla(KPC) genes using TaqMan chemistry. The q-PCR amplification of bla(KPC) DNA was linear over 7 log dilutions (r(2) = 0.999; slope, 3.54), and the amplification efficiency was 91.6%. The q-PCR detection limit was 1 CFU, and there was no cross-reaction with DNA extracted from several multidrug-resistant bacteria. Perianal/rectal swabs (n = 187) collected in duplicate from 128 patients admitted to Sheba Medical Center surgical intensive care units were evaluated for the presence of carbapenem-resistant bacteria by culturing on MacConkey agar-plus-carbapenem disks and for bla(KPC) genes by q-PCR. Carbapenem-resistant organisms, all K. pneumoniae, were isolated from 47 (25.1%) of the 187 samples collected, while bla(KPC) genes were detected in 54 (28.9%) of the patient samples extracted by the NucliSENS easyMAG system. Of these, seven samples were positive for bla(KPC) genes by q-PCR but negative for carbapenem resistance by culture, while all samples in which no carbapenem-resistant bacteria were detected by culture also tested negative by q-PCR. Thus, the sensitivity and specificity of the q-PCR assay after extraction by the NucliSENS easyMAG system were 100% and 95%, respectively. Similar values were obtained after DNA extraction by the Roche MagNA Pure LC instrument: 97.9% sensitivity and 96.4% specificity. Overall, the bla(KPC) q-PCR assay appears to be highly sensitive and specific. The utilization of q-PCR will shorten the time to blaKPC detection from 24 h to 4 h and will help in rapidly isolating colonized or infected patients and assigning them to cohorts.