Development And Application Of Recombinase Polymerase Amplification Assays For Detection Of Cryptosporidium Spp.and Toxoplasma Gondii

Cryptosporidium spp.and Toxoplasma gondii,two kinds of worldwide parasitic protozoan,infect allmost all the warm-blood vertebrates,resulting in a great threat to human health and livestock industry.Dairy cattle infected with Cryptosporidium spp.,can shed faeces with huge number of oocysts into environment.With the development of dairy cattle farming,the dairy cattle faeces become an important source of Cryptosporidium infection.T.gondii oocysts are shed worldwide into environment by felids.The sporulated oocysts that have capability to infect definitive and intermediate hosts are very resistant to environmental conditions.Therefore,development of efficient and rapid detection method for Cryptosporidium spp.oocysts in dairy cattle feces and T.gondii oocysts in the environment is critical for control of cryptosporidiosis and toxoplasmosis spread.Recombinase polymerase amplification(RPA)is a newly developed nucleic acid amplification technology that is more efficient and more suitable in filed detection than Polymerase Chain Reaction method(PCR).Unlike PCR,heat denaturation of the template strand is not needed in the RPA technology.Besides,RPA is proved to be highly specific,sensitive,fast,and easy to be operated,therefore it is more suitable for quick field diagnosis than other technologies in the early diagnosis of animal diseases,rapid quarantine of import and export.The amplification product can be visually displayed on lateral flow(LF)strip by adding the specific probe into RPA reaction solution.This complex assay form LF-RPA with higher efficiency is more suitable in filed detection than PCR.In this study,we established the LF-RPA assay for the detection of Cryptosporidium spp.in dairy cattle feces and T.gondii oocysts in the environment,respectively.The results are as follows:1.Cryptosporidium oocyst DNA could be extracted in hot water with 0.1% N-lauroylsarcosine sodium salt(LSS).The lowest detection threshold of the LF-RPA assay was 0.5 oocysts/reaction,which was more sensitive than the nest PCR method.The assay can work well in a range of temperature from 30? to 45? and the whole process can be completed within 30 min.No cross-reaction happened in negative control and T.gondii,G.lamblia,Ei.Cylindrica,E.bieneusi.Four out of the 12 clinical samples were tested positive by the nest PCR,while 6 of the 12 samples were positive by the LF-RPA.2.In order to extract the DNA of T.gondii oocusts,oocysts of T.gondii were repeatedly freezen and grinded by liquid nitrogen due to the hard shell of T.gondii oocysts.The LF-RPA primers and probe were designed based on the B1 repeat gene of T.gondii,and was proved to be with a limit of detection threshold of 0.1 T.gondii oocyst per reaction,which is tenfold sensitivity over the nest PCR method.This method was also highly specific for the detection of T.gondii.Five out of 35 samples were detected T.gondii oocysts-positive by using both LF-RPA and nest PCR.