Mycoplasma pneumoniae (MP) is one of the most common pathogens causing respiratory tract infection, especially for community-acquired pneumonia (CAP) in school-age children. There was considerable amount of studies on loop-mediated isothermal amplification (LAMP) assay for MP detection. However, the result interpretation of these developed LAMP assays was sophisticated and subjective. Therefore, we developed and evaluated a LAMP coupled with nanoparticle-based lateral flow biosensor (LFB) assay (LAMP-LFB) for simple, reliable, and objective identification of MP (MP-LAMP-LFB). Six primers specific to P1 gene of MP were designed, and the preferred temperature for this assay was confirmed to be 65 °C. The amplification products could be visually interpreted by LFB within 2 min. The MP-LAMP-LFB assay specifically identified DNA templates of MP, and no cross-reactivity with other pathogens was obtained. The limit of the detection for this assay was 600 fg of DNA templates in pure cultures, which was in complete accordance with colorimetric indicator detection and agarose gel electrophoresis analysis. This assay was applied to 209 oropharyngeal swab specimens collected from children with acute respiratory tract infection for clinical evaluation, and compared to real-time PCR detection. Using the LAMP-LFB and real-time PCR assay, the positive rates of MP were 47.8% and 31.6%, respectively. Results suggested that the LAMP-LFB assay displayed high sensitivity compared to real-time PCR method. In summary, LAMP-LFB assay established here was a simple, objective, and sensitive assay for MP detection, which can be widely applied in clinical settings, especially in rural areas.

中文翻译：

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循环介导的等温扩增与基于纳米颗粒的侧向流生物传感器检测技术相结合，用于肺炎支原体检测的开发。

肺炎支原体（MP）是导致呼吸道感染的最常见病原体之一，尤其是对于学龄儿童的社区获得性肺炎（CAP）。关于用于MP检测的环路介导的等温扩增（LAMP）分析，已有大量研究。但是，这些发达的LAMP分析的结果解释是复杂而主观的。因此，我们开发并评估了LAMP与基于纳米颗粒的侧向流生物传感器（LFB）分析（LAMP-LFB）结合使用，以简单，可靠和客观地鉴定MP（MP-LAMP-LFB）。设计了六种对MP的P1基因具有特异性的引物，确定该测定的优选温度为65°C。LFB可以在2分钟内从视觉上解释扩增产物。MP-LAMP-LFB检测可特异性鉴定MP的DNA模板，并没有与其他病原体发生交叉反应。该测定的检测极限是纯培养物中600 fg DNA模板，这完全符合比色指示剂检测和琼脂糖凝胶电泳分析。该测定方法应用于从急性呼吸道感染儿童中收集的209口咽拭子标本进行临床评估，并与实时PCR检测进行了比较。使用LAMP-LFB和实时PCR检测，MP的阳性率分别为47.8％和31.6％。结果表明，与实时PCR方法相比，LAMP-LFB测定法显示出高灵敏度。总之，此处建立的LAMP-LFB检测是一种简单，客观，灵敏的MP检测方法，可广泛应用于临床，尤其是农村地区。