Identification of necrophagous fly species from 12 different cities in China using ISSR and SCAR markers

Objective:To identify necrophagous fly speies from different regions in China using inter simple sequenc repeat(ISSR) and sequence-characterized amplified region(SCAR) melocular markers and to analyze their gene difference and genetic relationship.Methods:Five carrion fly species were collected from 12 cities and regions in China,including Musca domestica(M.domestica), Lucilia sericata(L.sericata),Chrysomya megacephala(C.megacephala),Helicophagella melanura(H.melanura),Boethcherisca peregrina,and they were studied using ISSR and SCAR markers.Results:Eight ISSR primers were used for amplification of 121 samples.679 clear and stable bands were identified,of which 516 bands were polymorphic.Several species-specific ISSR fragment were cloned and sequenced as an initial effort to derive the SCAR markers.Using M.domestica SCAR specific primers,SCAR-PCR amplification was performed for 8 M.domestca population sample DNA from different regions in China as well as L sericata,C.megacephala, H.melanura and Lucillia cupirina.The result showed only M.domestica produced specificalty 600 bp fragment,but L sericata,C.megacephala,H.melanura and Lucillia cupirina did not produce the same specific fragment.Clustering analysis showed clustering of most flies of M. domestica,C.megacephala and L sericata.M.domestica samples from different regions in China yielded different banding patterns.Conclusions:Application of ISSR-PCR and SCAR markers to identify necrophagous fly species from 12 cities and regions in China is first reported.ISSR-PCR and SCAR markers provide a quick reliable molecular marker technique for the identification of different species of necrophagous fly.

Gold nanoparticle-based lateral flow immunoassay for the rapid and on-site detection of wheat allergen in milk

Wheat gliadin is one of the most important causes of gluten sensitivity.This study aimed to develop a sandwich enzyme-linked immunosorbent assay(ELISA)and gold nanoparticle-based lateral flow(LFIA)strips to detect wheat allergen in food.Through cell screening,ten monoclonal antibodies(mAbs)against gliadin and after optimization were obtained,a pair of mAbs suitable for its detection(capture antibody:mAb 7;detection antibody:HRP-labeled mAb 6)were identified.Based on two of these antibodies,a sandwich ELISA with limit of detection(LOD)of 60 ng/mL in negative milk,and more importantly,negligible cross-reactivity to other allergens was developed.The average recoveries for gliadin in negative milk were 99.16%-100.07%using the sandwich ELISA.We also developed LFIA strips for the rapid detection of gliadin with visual limit of detection(vLOD)of 25 ng/mL and calculated LOD value of 6.56 ng/mL in negative milk.The test results of positive samples obtained from the LFIA strips were highly consistent with those of the sandwich ELISA.Thus,the developed LFIA strip is an effective and reliable tool for the rapid and on-site detection of wheat allergen in milk.