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首页> 美国卫生研究院文献>Journal of Clinical Microbiology >New Real-Time PCR-Based Method for Kingella kingae DNA Detection: Application to Samples Collected from 89 Children with Acute Arthritis
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New Real-Time PCR-Based Method for Kingella kingae DNA Detection: Application to Samples Collected from 89 Children with Acute Arthritis

机译：基于实时荧光定量PCR的金黄色小金藻DNA检测新方法：应用于从89例急性关节炎儿童中收集的样品

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Inoculation of blood culture vials with joint fluid samples has revealed the important pathogenic role of Kingella kingae in pediatric arthritis. However, recent studies based on broad-range 16S ribosomal DNA PCR and real-time PCR without a probe suggest that conventional methods remain suboptimal. We developed a new real-time PCR method with a probe that is highly specific for K. kingae and applied it to joint fluid samples collected from 89 children with suspected arthritis admitted to our institution during a 2-year period. Real-time PCR was also applied to blood samples obtained before surgery and to joint drainage fluid samples obtained during several days after surgery. Thirty-six (40%) of the 89 cases of suspected septic arthritis had positive culture. Staphylococcus aureus was the main isolate (n = 19/36, 53%), followed by K. kingae (n = 7/36, 19%). Specific real-time PCR identified K. kingae in 24 of the 53 culture-negative cases. Thus, K. kingae was present in 31 (52%) of the 60 documented cases, making it the leading pathogen. Real-time PCR on all 15 blood DNA extracts from patients with K. kingae infection was negative, demonstrating that joint fluid positivity did not result from DNA circulating in blood. Real-time PCR amplification of drainage fluid samples showed that the pathogen could be detected for up to 6 days after antibiotic initiation. K. kingae real-time PCR applied to DNA extracted from joint fluid samples, but not from blood samples, markedly improved the etiological diagnosis of septic arthritis in children. Retrospective diagnosis is feasible for up to 6 days after treatment initiation.
机译：用关节液样本接种血液培养小瓶表明，金黄色小Kingella在小儿关节炎中具有重要的致病作用。但是，最近基于宽范围16S核糖体DNA PCR和实时PCR的研究（没有探针）表明，常规方法仍然欠佳。我们开发了一种新的实时PCR方法，该方法具有对金黄色K.菌高度特异的探针，并将其应用于从两年内入院的89例疑似关节炎儿童收集的关节液样本中。实时PCR还应用于手术前获得的血液样本以及手术后几天内获得的关节引流液样本。在89例疑为化脓性关节炎的病例中，有36例（40％）的培养呈阳性。金黄色葡萄球菌是主要的分离株（n = 19 / 36，53％），其次是金黄色葡萄球菌（n = 7 / 36，19％）。在53例培养阴性病例中，有24例通过实时荧光定量PCR鉴定了金黄色葡萄球菌。因此，在记录的60个病例中有31个（占52％）存在金缕梅，使其成为主要病原体。对K. kingae感染患者的所有15种血液DNA提取物进行的实时PCR均为阴性，这表明血液中循环的DNA并非导致关节液阳性。引流液样品的实时PCR扩增表明，在抗生素启动后长达6天可以检测到病原体。 K. kingae实时PCR应用于从关节液样本而非血液样本中提取的DNA，显着改善了儿童败血性关节炎的病因学诊断。在开始治疗后最多6天进行回顾性诊断是可行的。

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