Survey And Identification Of Gnathostoma In Eel And Loach And Detection By LAMP Method

Gnathostomiasis is the result of accident human infection with the larval nematode species found in raw or undercooked freshwater fish and other animals. Larval and immature Gnathostoma sp. adults cause migratory, often transitory, subcutaneous swellings in humans. The worms occasionally enter the internal organs such as lung, eye,liver, kidney and central nervous system, causing severe symptoms, and can lead to death.Gnathostomiasis is a kind of emerging diseases, increasing badly. As a result of the population flow, culture and international trade globalization, gnathostomiasis presents the trend of globalization. Gnathostomosis has been reported in travelers who acquired the infection in one of these endemic countries and developed the disease when they returned to the home country, or in persons who acquired the infection consuming raw fish imported from endemic countries. At present, the United Nations food and agriculture organization(FAO) has put it as an important pathogenic factor. 13 species have been recognized by morphology in the genus Gnathostoma. This study conducted an investigation of domestic and imported swamp eels and loach, and established reliable detection methods of Gnathostoma by the LAMP and PCR, providing effective method for Gnathostoma identified, the Gnathostoma epidemiological investigation, pathogenic diagnosis, as well as the prevention and control.1. Gnathostoma epidemiology investigation From November2015 to December 2015, 13 batch of swamp eels were detected as Gnathostoma positively within 19 batch swamp eels imported from 2 countries. The positive rate was 68.4%. From September 2015 to December 2015, swamp eels and loach were investigated for larvae of Gnathostoma. Although no infection were detected in swamp eels and loach from Guangzhou, Sichuan, Chongqing, Hubei, Heilongjiang, a Gnathostomawas found in swamp eels from Fujian, showing the existence of the Gnathostoma.2. Species identification To identify the species of Gnathostoma in the present study, we observed the Gnathostoma under light microscope, then measured and analyzed data of morphological characteristics. A set of specificity PCR primer was designed from COX1 gene as well.After PCR amplification of DNA of parasite, PCR product was connected to the T-vector.DNA sequencing was performed by Invitogen Company. As a result, the COX1 sequences were determined for Gnathostoma. Spinigerum. Themethod is suitable for specifically detecting Gnathostoma in general laboratory.3. The establishment of the Gnathostoma LAMP detection method The method was based on LAMP reaction. Three sets of specificity and sensitivity of primers were designed from 28 S gene. The specificity, sensitivity and stability were tested.Specific amplification products were obtained with Gnathostoma, while no amplification products were detected with DNA of related parasites, demonstrating the specificity of the assay. The capable detection of plasmid DNA in the method was 1 fg/?L. The LAMP assay was proved to be 100 times more sensitive than a conventional polymerase chain reaction for detection of Gnathostoma. 51 samples from South East Asian and South Asian testing by the LAMP method and PCR method showed that, the result of the LAMP method was the same with PCR method. The established LAMP method is suitable for specifically detecting Gnathostoma.