用于脑脊液中脑膜炎奈瑟菌快速检测的环介导等温扩增（LAMP）测定法的临床评估
Clinical evaluation of a loop-mediated isothermal amplification (LAMP) assay for rapid detection of Neisseria meningitidis in cerebrospinal fluid.
作者信息
Lee DoKyung, Kim Eun Jin, Kilgore Paul E, Kim Soon Ae, Takahashi Hideyuki, Ohnishi Makoto, Anh Dang Duc, Dong Bai Qing, Kim Jung Soo, Tomono Jun, Miyamoto Shigehiko, Notomi Tsugunori, Kim Dong Wook, Seki Mitsuko
机构信息
Department of Pharmacy, College of Pharmacy, Hanyang University, Ansan, Republic of Korea; Institute of Pharmacological Research, Hanyang University, Ansan, Republic of Korea.
Wayne State University, Eugene Applebaum College of Pharmacy & Health Sciences, Department of Pharmacy Practice, Detroit, Michigan, United States of America.
出版信息
PLoS One. 2015 Apr 8;10(4):e0122922. doi: 10.1371/journal.pone.0122922. eCollection 2015.
BACKGROUND
Neisseria meningitidis (Nm) is a leading causative agent of bacterial meningitis in humans. Traditionally, meningococcal meningitis has been diagnosed by bacterial culture. However, isolation of bacteria from patients' cerebrospinal fluid (CSF) is time consuming and sometimes yields negative results. Recently, polymerase chain reaction (PCR)-based diagnostic methods of detecting Nm have been considered the gold standard because of their superior sensitivity and specificity compared with culture. In this study, we developed a loop-mediated isothermal amplification (LAMP) method and evaluated its ability to detect Nm in cerebrospinal fluid (CSF).
METHODOLOGY/PRINCIPAL FINDINGS: We developed a meningococcal LAMP assay (Nm LAMP) that targets the ctrA gene. The primer specificity was validated using 16 strains of N. meningitidis (serogroup A, B, C, D, 29-E, W-135, X, Y, and Z) and 19 non-N. meningitidis species. Within 60 min, the Nm LAMP detected down to ten copies per reaction with sensitivity 1000-fold more than that of conventional PCR. The LAMP assays were evaluated using a set of 1574 randomly selected CSF specimens from children with suspected meningitis collected between 1998 and 2002 in Vietnam, China, and Korea. The LAMP method was shown to be more sensitive than PCR methods for CSF samples (31 CSF samples were positive by LAMP vs. 25 by PCR). The detection rate of the LAMP method was substantially higher than that of the PCR method. In a comparative analysis of the PCR and LAMP assays, the clinical sensitivity, specificity, positive predictive value, and negative predictive value of the LAMP assay were 100%, 99.6%, 80.6%, and 100%, respectively.
CONCLUSIONS/SIGNIFICANCE: Compared to PCR, LAMP detected Nm with higher analytical and clinical sensitivity. This sensitive and specific LAMP method offers significant advantages for screening patients on a population basis and for diagnosis in clinical settings.
背景
脑膜炎奈瑟菌（Nm）是人类细菌性脑膜炎的主要致病原。传统上，脑膜炎球菌性脑膜炎通过细菌培养进行诊断。然而，从患者脑脊液（CSF）中分离细菌耗时且有时会得出阴性结果。近来，基于聚合酶链反应（PCR）的检测Nm的诊断方法因其与培养法相比具有更高的敏感性和特异性而被视为金标准。在本研究中，我们开发了一种环介导等温扩增（LAMP）方法，并评估了其检测脑脊液（CSF）中Nm的能力。
方法/主要发现：我们开发了一种针对ctrA基因的脑膜炎球菌LAMP检测法（Nm LAMP）。使用16株脑膜炎奈瑟菌（A、B、C、D、29-E、W-135、X、Y和Z血清群）和19种非脑膜炎奈瑟菌验证了引物特异性。在60分钟内，Nm LAMP检测到每个反应低至10个拷贝，敏感性比传统PCR高1000倍。使用1998年至2002年期间在中国、越南和韩国收集的一组1574份疑似脑膜炎儿童的随机选择的脑脊液标本对LAMP检测法进行评估。结果表明，对于脑脊液样本，LAMP方法比PCR方法更敏感（LAMP检测出31份脑脊液样本呈阳性，而PCR检测出25份）。LAMP方法的检测率显著高于PCR方法。在PCR和LAMP检测法的比较分析中，LAMP检测法的临床敏感性、特异性、阳性预测值和阴性预测值分别为100%、99.6%、80.6%和100%。
结论/意义：与PCR相比，LAMP检测Nm具有更高的分析和临床敏感性。这种灵敏且特异的LAMP方法在基于人群的患者筛查和临床诊断方面具有显著优势。
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