Development And Application Of LAMP Method For The Identification Of Echinococcus Spp. In Dog Faeces

Echincococcosis, which is caused by the infection of the larval stages of Echinococcus (E.) spp.parasitizing in the liver, lungs and other organs of intermediate host, is a zoonotic disease with a globaldistribution. Echincococcosis is regarded as an emerging and re-emerging infectious disease, and isbecoming serious public health concerns. Carnivores infected with the Echinococcus tapeworms are themain sources of infection for echinococcosis of other animals including humans and livestock. So thediagnosis and epidemiological survey of carnivores with Echinococcus species plays a key role indeveloping guidelines for the prevention and control of this disease. Echincococcosis is not onlyharmful to human health but also hinders the development of livestock industry seriously.The necropsy method is the main diagnosis for detection and epidemiological survey of definitivehosts with Echinococcus infections, and other methods including immunological and molecularbiological techniques are also increasingly being developed and improved. In present study, fourprimers were designed according to the nad5gene of mtDNAs of Echinococcus tapeworms including E.granulosus and E. multilocularis, to establish the method of loop-mediated isothermal amplification(LAMP) for determing whether definitive hosts are infected with Echinococcus spp. Our results showedthat the LAMP method had no cross-reaction with other species including E. shiquicus, Taenia (T.)hydatigena, T. pisiformis, T. taeniaeformis, T. multiceps, Dipylidium (D.) caninum, which are oftenfound in the intestinal of carnivores, indicating that it is highly specific. And its good reproducibilitywas also confirmed.The g-DNA of larval E. multilocularis was diluted to10ng/μl and then diluted in10-fold seriallyand was detected using LAMP and PCR assays. Both PCR and LAMP had the same sensitivity with10-3ng. However, positive occurred at the twelfth day post infection using the LAMP assay whereas positiveat the seventeenth day by use of the traditional PCR approach when detecting the f-DNA of differentday post infection, suggesting that LAMP assay was more sensitive than PCR and had the earlydetection value. Furthermore, at least four eggs per gram of faece were sufficiently to be detectable withLAMP assay when the smallest eggs were determined.The g-DNA was diluted to contain10ng/μl and then serially diluted10-fold. The result showedthat LAMP was102times more sensitive than the conventional PCR assay. Similar outcome wasobtained when diluted faecal DNA of E. granulosus was used to determine the sensitivity of LAMP. Theresult also demonstrated that the LAMP assay was102times more sensitive than a conventional PCRassay. Besides, the LAMP method was able to identify the pathogen DNA from dog’s faeces at the22ndday post-infection; while, the PCR positive occurred until the26th day. However, eggs existing in faecalsamples were been observed using microscopy on the69th day post infection and the result with theELISA assay was positive on the25th day post-infection according to the kit’s criterion, revealing thatLAMP method was more sensitive than other assays. All these results indicated that LAMP assay wasmore sensitive than PCR and had the early detection value. Furthermore, at least five eggs per gram of faece were sufficiently to be tested with LAMP assay when minimum eggs were assayed.Additionally, field sample collected from Tianzu county of Gansu province (n=30) and Zhiduocounty (n=48), Jiuzhi county (n=9) and Dari county (n=132) of Qinghai province were detected usingthe two LAMP methods. No positive samples were characterized from Tianzu county of Gansuprovince. The infection rate of E. multilocularis and E. granulosus (G1strain) were16.40%and13.76%in Qinghai province, respectively.The LAMP methods developed in present study for the identification of the definitive hosts with E.granulosus and E. multilocularis have highly specificity and sencitivity with the early detection value.In addition, epidemiological investigations have been performed using these LAMP methods in Gansuand Qinghai provinces, which play a crucial role in formulating the prevention and treatment measuresand provide the foundation for supporting the development of rapid diagnostic kits for the eximinationof definitive hosts with Echinococcus infections.