The introduction of the pine wood nematode (PWN), Bursaphelenchus xylophilus, to new areas has impacted on the international economy. Therefore, accurate and reliable detection methods for PWN are essential for the control and management of this pest. A rapid and economic method for detecting PWN may be developed focusing on the PWN vector (Monochamus alternatus). This work standardized a loop-mediated isothermal amplification (LAMP) method using newly designed primer sequences based on the syg-2 gene, which encodes the synaptogenesis protein syg-2. Loopmediated isothermal amplification was conducted at 63°C for 60 min using six sets of primers. The result was confirmed by visual observation. A positive reaction was confirmed by SYBR Green I fluorescence dye under light thermal cycling. The lower limit of DNA detection was 51.4 pg/μl in both LAMP and 51.4 ng/μl in PCR. Therefore, the LAMP was 1,000 times more sensitive in DNA detection than PCR. The LAMP is a relatively new, highly accurate and rapid molecular technique that can rapidly detect infectious agents in the field without requiring sophisticated instruments, giving a visually readable result. The method greatly improves detection, without requiring professional knowledge and expensive, sophisticated equipment. Therefore, this system is suitable for quarantine and field detection. negative reaction (no target DNA template), the dye remained orange ( Figure 4). | DISCUSSIONBursaphelenchus xylophilus is an important pathogenic organism vectored between pine hosts by M. alternatus. Therefore, the number of M. alternatus is a crucial indicator of disease development. Unless B. xylophilus in M. alternatus is specifically treated in the primary phase of infection, PWD is fatal. The efficient, rapid detection of B. xylophilus from M. alternatus is necessary for monitoring and managing outbreaks, and in implementing specific therapeutics (Cardoso, Fonseca, & Abrantes, 2012; Wang et al., 2009). As the death of pines infected by PWD is very rapid, the novel PWD-detection technique must also be rapid.In the present work, we designed specific primers for rapid detection of B. xylophilus from M. alternatus using the LAMP. For this purpose, we targeted the conserved syg-2 gene reported by Gou (2014). The LAMP assay successfully detected all isolates of B. xylophilus, giving negative reactions in all other species tested. The high specificity of the LAMP is conferred by the six specially designed primers that recognize six regions on the syg-2 gene sequence, which are specific to B. xylophilus. The lower limit of DNA detection in the LAMP-based technique was 51.4 pg/μl of genomic DNA, compared with 51.4 ng/μl in conventional PCR. Therefore, the LAMP was 1,000-fold more sensitive than conventional PCR (Gou et al., 2015) and can rapidly detect B. xylophilus from M. alternatus.Given the high sensitivity of LAMP, post-amplification operations should be performed in a separate room, away from the reactions and reagents used in the PCR and LAMP, to reduce the chance of conta...

Aims:  Tuberculous pleurisy is an important cause of pleural effusions in areas with a high incidence of tuberculosis. In this study, we developed an IS1081‐based LAMP for the detection of Mycobacterium tuberculosis complex and investigated its usefulness in the diagnosis of tuberculous pleurisy. Methods and Results:  Investigation of pleural effusion samples from patients with tuberculous pleurisy, majority of them smear‐/culture‐negative, and control individuals with non‐TB diseases showed that the LAMP assay with incubation time of 60 min has much higher specificity and the LAMP assay with incubation time of 90 min has significantly higher sensitivity in the diagnosis of tuberculous pleurisy, as compared with fluorescent real‐time PCR. Conclusions:  The MTBC–LAMP is a useful assay for the diagnosis of tuberculous pleurisy, especially in pleural effusion smear‐/culture‐negative patients. Significance and Impact of the Study:  Tuberculous pleural effusion usually contains low number of mycobacteria, which leads to low diagnostic sensitivity of acid‐fast staining and mycobacterial culture methods. In this study, we developed a simple and sensitive LAMP assay for the diagnosis of tuberculous pleurisy. This assay should have broad application in resource‐limited settings.