DNA detection of Paragonimus westermani: Diagnostic validity of a new assay based on loop-mediated isothermal amplification (LAMP) combined with a lateral flow dipstick.

Paragonimus westermani (P. westermani) is widely spread in Asian countries and is one of the most important causative agents for lung fluke diseases. The prevention and control of Paragonimiaisis mainly depends on the accurate diagnosis and effective treatment. In this study, we developed a loop-mediated isothermal amplification (LAMP) assay targeted to a portion of the Ty3/gypsy-like LTR retrotransposon (Rn1) sequence coupled with a lateral flow dipstick (LFD) for the rapid detection of P. westermani-specific amplicons. The positive LAMP products were biotin-labeled and hybridized with a fluorescein isothiocyanate-labeled probe which could be visually detected by LFD. No cross-reaction were observed with other parasitic pathogens including Trichinella spiralis, Anisakis simplex, Schistosoma japonicum and Gnathostoma spinigerum, but this LAMP assay could not distinguish P. westermani with Paragonimus skrjabini and Paragonimus heterotremus. The detection limit of the LAMP assay for P. westermani was 2.7 fg/µL, while that of PCR method was 27 fg/µL. LAMP method was applied to detect P. westermani genomic DNA in blood samples form experimental infected dogs, and results showed the parasite was detectable as early as week 2. LAMP-LFD assay applicability was successfully tested in dog blood samples collected from five cities (Wenzhou, Hangzhou, Huzhou, Jiaxing and Shaoxing) in Zhejiang province. In summary, the established LAMP-LFD assay targeted to the Rn1 sequence is a rapid and convenient method for specific detection of P. westermani.

Cloning and expression of gene encoding myophilin-like protein of Schistosoma japonicum and study on the antigenicity of recombinant protein / 中国血吸虫病防治杂志

Objective To clone and express the gene encoding Schistosoma japonicum myophilin-like protein (SjcMLP) and to study the antigenicity of the recombinant protein. Methods The SjcMLP gene was amplified by PCR. The PCR product was cloned into T vector, and then subcloned into expression vector pQE30. The recombinant plasmid of pQE30-SjcMLP was transformed into E.coli M15, and induced with IPTG for expression. The bacterial lysis was conducted by ultrasonication and the supernatant was analysed by SDS-PAGE. The recombinant protein (reSjcMLP)was purified with the Ni-NTA resin, and analysed with SDS-PAGE and Western blot. The titers of sera from C57BL/6 mice immunized subcutaneously with reSjcMLP were detected by ELISA. Results The results of SDS-PAGE and Western blot showed that the molecular weight of expressed fusion protein was around 24.8 kDa and was recognized by the sera from the mice infected with Schistosoma japonicum. The purified protein of reSjcMLP was coated for ELISA test and the IgG titers in the sera from the mice immunized with reSjcMLP were as high as 1∶12 800 reacted with. However, no significant difference was found in worm reduction rates between the immunized mice and control mice. Conclusions The fused recombinant protein of reSjcMLP is successfully ex-pressed and purified. The recombinant protein in this experiment fails to induce significant protection against the challenge infection in C57BL/6 mice.