Studies Ofcross-priming Isothermal Amplification Methods For Two Fish Pathogens,vibrio Harveyi And Amyloodinium Ocellatum

Vibrio harveyi is a common opportunist pathogen of aquaculture animals in seawater.Isolate ML01 of V.harveyi causes the skin ulcer diseasein juvenile hybrid groupers,Epinephelus fuscoguttatus × Epinephelus lanceolatus with high mortality and leading to mass economic losses.In this work,the primers specifically targeting V.harveyi were designed and a rapid detecting method,cross-priming isothermal amplification coupled with a lateral flow dipstick?CPA-LFD?,for V.harveyi was established and optimized.The optimized method included two steps.First,the CPA reaction tube was settled in a metal water bath and incubated at 64°C for 60 min,then incubated at 80 °C for 5 min to terminate the reaction.Second,the amplified product of the CPA was visualized by an LFD nucleic acid detection device.The result showed that the sensitivity of this method was 100 copies/?L,103 times higher than conventional PCR and similar with real-time PCR.This CPA-LFD method was specifically for the V.harveyi and had no cross-reactions with tested common fish pathogens,such as Megalocytivirus,TRBIV,LCDV,V.anguillarum,V.parahaemolyticus,V.alginolyticusand V.campbellii.This method is simple and rapid with high sensitivity and good specificity and can be widely applied for rapid detection of V.harveyi on the spot.Recently,a pathogenic dinoflagellate?the Bohai-1407 isolate?was isolated from diseased Spotted Knifejaw?Oplegnathus puncatus?.In order to perform the molecular identification and phylogenetic analysis,four ribosomal DNA?rDNA?fragments of the Bohai-1407 isolate were amplified,cloned,sequenced,assembled and analyzed by BLAST.The result showed that the sequenced rDNA operon was 6530 bp in length.It was composed of 74 bp of partial external transcribed spacer?ETS?,1813 bp of 18S?also called small subunit,SSU?,352 bp of internal transcribed spacers 1?ITS1?,159 bp of 5.8S,698 bp of internal transcribed spacers 2?ITS2?and 3388 bp of 23S?also called large subunit,LSU?,accompanied by a 46 bp of partial non-transcribed spacer?NTS?in the 3' end.The sequence identity between the Baohai-1407 isolate and the Ningde1412 strain of A.ocellatum was as high as 99.5%.Based on the SSU sequence of 10 dinoflagellates in family Blastodiniidae,a phylogenetic tree was constructed and the Bohai-1407 isolate clustered with isolates of A.ocellatum.Two phylogenetic trees based on the ITS1 and ITS2 sequences of 19 isolates/clones in species A.ocellatum were also constructed.The results showed that the Bohai-1407 isolate always clustered with the Gulf of Mexico isolate FL₂1?DQ490260.1?.Based on the sequenced SSU gene of Bohai-1407 isolate,the CPA-LFD detecting method of A.ocellatumwas established and optimized.The optimized method included two steps.First,the CPA reaction tubes containing specific primers of A.ocellatum was settled in a metal water bath and incubated at 64°C for 60 min,then incubated at 80 °C for 5 min to terminate the reaction.Second,the amplified product of the CPA was visualized by an LFD nucleic acid detection device.The result showed that the sensitivity of this method was 100 copies/?L,103 times higher than conventional PCR.This CPA-LFD method was specifically for A.ocellatum and had no cross-reactions with tested common fish pathogens,such as Megalocytivirus,TRBIV,LCDV,V.harveyi,V.anguillarum,V.parahaemolyticus,V.alginolyticus and V.campbellii.This method is simple and rapid with high sensitivity and good specificity and can be widely applied for rapid detection of A.ocellatum on the spot.