ABSTRACT
To control and prevent the Echinococcus in the place ,we established a loop-mediated isothermal amplification (LAMP) assay for rapid detection of Echinococcus species specific DNA from dog faeces .Four primers which recognizing 6 dis-tinct regions on the NADH dehydrogenase subunit 2 (ND2) gene of Echinococcus granulosus were designed and used for LAMP assay .The specificity of LAMP assay was evaluated using DNA extracted from Echinococcus granulosus , Taenia saginata , and other dog intestinal parasites .In addition ,the sensitivity of LAMP assay was compared with that of conventional PCR using recombinant plasmid carrying Echinococcus granulosus ND2 gene fragment as standard template DNA after 10-fold serial dilution .Furthermore ,we extracted DNA from 46 canine fecal samples collected from endemic areas ,and tested the copro-DNA samples using LAMP and necropsy method .Results showed that E .g ND2 primer sets could differentiate Echinococcus granulosus from Echinococcus multilocularis without cross reaction among other parasites detected .Furthermore ,the LAMP assay with primer sets to the ND2 gene could detect 4 × 101 copies of target gene ,demonstrating 103 times higher sensitivity than that of conventional PCR methods .The LAMP assay with primer set to ND2 gene showed good sensitivity and specificity to detect copro-DNA samples extracted from fecal samples of 46 dogs tested in endemic areas .There was no statistically signifi-cant difference among LAMP and necropsy .In this study ,a sensitive ,specific and rapid copro-DNA detection LAMP assay was developed successfully for diagnosis of dogs infected with Echinococcus granulosus .Due to its rapidity ,simplicity ,speci-ficity and sensitivity ,the LAMP assay is a promising new tool for rapid detection of dogs infected with Echinococcus spp .dur-ing the field survey or in poor-equipped laboratories .
ABSTRACT
To isolate the specific genes in protoscoleses (PSC) of Echinococcus granulosus under oxidative stresses from the SSH library constructed in the previous study, the gene expression in PSC under oxidative stresses was studied by using real-time PCR. The previously amplified library was sequenced and analyzed in GenBank with Blast research. Sequence analysis indicated that all clones in the SSH library contained the coding sequences, of which some clones showed homology in the GenBank and others were unknown. Differential expression of 4 genes randomly selected and the TPx gene in this library were studied with real-time PCR. It was demonstrated that the gene expression of S88 and H32-1 in oxidative tissues was 2.0 and 2.3 times higher than the un-oxidative stresses respectively. The TPx gene was up-regulated when PSC was induced with H_2H_2 of more than 0.8 mmol/L. These results implies that the up-regulated expression of the above-mentioned genes may be related with the related functions of anti-oxidative process in PSC and they may be used as the candidate genes for the study of anti-oxidation of E.granulosus.