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首页> 外文期刊>Journal of Clinical Microbiology >Loop-Mediated Isothermal Amplification Assay for Detection of Haemophilus influenzae Type b in Cerebrospinal Fluid
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Loop-Mediated Isothermal Amplification Assay for Detection of Haemophilus influenzae Type b in Cerebrospinal Fluid

机译：循环介导的等温扩增检测脑脊液中的b型流感嗜血杆菌

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Haemophilus influenzae type b (Hib) is one of the leading causes of meningitis in developing countries. To establish and evaluate a novel loop-mediated isothermal amplification (LAMP) assay for Hib, we designed a LAMP primer set targeting the Hib-specific capsulation locus. LAMP detected 10 copies of purified DNA in a 60-min reaction. This indicated that the detection limit of LAMP was >100-fold lower than the detection limits of both a PCR for the detection of bexA and a nested PCR for Hib (Hib PCR). No H. influenzae, other than Hib or control bacteria, was detected. Linear determination ranged from 10 to 1,000,000 microorganisms per reaction mixture using real-time turbidimetry. We evaluated the Hib LAMP assay using a set of 52 randomly selected cerebrospinal fluid (CSF) specimens obtained from children with suspected meningitis. For comparison, the CSF specimens were tested using a conventional Hib PCR assay. Hib was detected in 30 samples using LAMP and in 22 samples using the Hib PCR assay. The Hib PCR showed a clinical sensitivity of 73.3% and a clinical specificity of 100% relative to the Hib LAMP assay. These results suggest that further development and evaluation of the Hib LAMP will enhance the global diagnostic capability for Hib detection.
机译：b型流感嗜血杆菌（Hib）是发展中国家脑膜炎的主要原因之一。为了建立和评估针对Hib的新型环介导的等温扩增（LAMP）分析，我们设计了针对Hib特异封装位点的LAMP引物组。 LAMP在60分钟的反应中检测到10个拷贝的纯化DNA。这表明，LAMP的检测限比用于检测 bexA 的PCR和嵌套式PCR的Hib（Hib PCR）的检测限低100倍以上。除Hib或对照细菌外，未检测到流感嗜血杆菌。使用实时比浊法，线性测定的范围是每个反应混合物10到1,000,000个微生物。我们使用一组52个随机选择的脑脊液（CSF）标本评估了Hib LAMP分析，这些标本是从怀疑患有脑膜炎的儿童那里获得的。为了比较，使用常规Hib PCR测定法测试了CSF样品。使用LAMP在30个样品中检测到Hib，使用Hib PCR分析在22个样品中检测到Hib。相对于Hib LAMP分析，Hib PCR的临床敏感性为73.3％，临床特异性为100％。这些结果表明，Hib LAMP的进一步开发和评估将增强Hib检测的整体诊断能力。

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