Abstract
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.
Highlights
Mycoplasma pneumoniae (MP) is one of the leading causes of community acquired pneumonia (CAP) of all ages, especially in school-age children (Marston et al 1997)
By lateral flow biosensor (LFB), two red lines were shown in the positive reactions, whereas only one red line (CL) was exhibited in the negative and blank controls (Fig. 2b). These results demonstrated that the MP-Loopmediated isothermal amplification method (LAMP) primer set designed here was able to amplify the target sequence and can be used as a good candidate for establishment of LAMP-LFB assay for MP detection
Current available techniques for MP detection, such as culture-based method, serological test and realtime PCR assay are time-consuming, sophisticated and not suitable for clinical application. To overcome these shortcomings posed by facilities illustrated above, we developed a simple, fast, and reliable assay, LAMP-LFB assay, for MP detection
Summary
Mycoplasma pneumoniae (MP) is one of the leading causes of community acquired pneumonia (CAP) of all ages, especially in school-age children (Marston et al 1997). MP was responsible for 40% of cases of CAP in children, and as many as 18% of patients requiring. Comparing with traditional culture-based methods, nucleic acid amplification techniques, such as conventional PCR and real-time PCR, which are fast, sensitive and specific, have been widely used for MP detection (Law et al 2015). Loopmediated isothermal amplification method (LAMP), a simple isothermal amplification test with high sensitivity and specificity developed by (Notomi et al 2000), which has been successfully applied to MP identification (Zhao et al 2013; Ratliff et al 2014; Petrone et al 2015; Yuan et al 2018). The interpretation of LAMP result depends on complicated instruments (real-time turbidimeter), laborious process (agarose gel electrophoresis) and special reagents (colorimetric indicator), which were subjective and limited its application for routine diagnosis
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