Establishment And Application Of Visual LAMP And Its Nucleic Acid Dipstick Assay For Detection Of Two Pathogens From Fish

Plesiomonas shigelloides has been isolated from a variety of environmental sources,primarily aquatic,and is distributed worldwide,which is an enteric pathogen of human,animal and various aquatic animals.It can cause human infectious diarrhea and food poisoning.Klebsiella pneumoniae is a common conditional pathogen,which can also cause diseases to fish and pose a great threat to aquaculture.Most of the detection methods of P.shigelloides and K.pneumoniae have been established,but depending on expensive equipment and professional technicians,which can not meet the requirements of on-site rapid diagnosis.Therefore,evelopmenting a simple,sensitive and rapid methods is significance for the detection,prevention and control of disease.In this study,the reaction conditions of visual Loop-Mediated Isothermal Amplification?LAMP?rapid detection method,which was combined the LAMP with dye fluoresent calcein and nucleic acid dipstick assay?NADA?,was optimized for P.shigelloides and K.pneumoniae detection.The main contents and results of present study were as follows:1.Two strains of pathogens isolated from diseased hybrid snakehead and snakehead fish?Channa argus?,named FS170522 and ZS685683 respectively.Strain FS170522 was identified as P.shigelloides and strain ZS685683 was confirmed as K.pneumoniae by physiological and biochemical characteristics,and the sequence analysis of 16S rDNA.The P.shigelloides strain was re-isolated from the artificial infected hybrid snakehead,and the half lethal dose(LD₅₀)was about 7.5×10⁴ CFU/g.The symptoms of strain ZS685683infected shakehead fish were similar with natural infected fish.The LD₅₀0 of strain ZS685683to shakehead fish was 1.4×10⁵ CFU/g per fish.Results of drug sensitive tests showed that strain FS170522 was resistant to ampicillin and cephradine,etc.Strain ZS685683 was resistant to cotrimoxazole,clindamycin,madamecin,piperacillin,etc.2.Based on the conserved sequence of P.shigelloides hugA gene and K.pneumoniae FimK gene,a set of LAMP primers were designed using Primer Explorer V4 and Larsergene7.0 Primer Select online.The constructed plasmid of hugA and FimK gene were used as the standard template in LAMP and following optimization test.In this study,the reaction conditions of visual LAMP rapid detection method,which was combined the LAMP with calcein,was optimized for P.shigelloides and K.pneumoniae detection.At the same time,the LAMP-NADA?nucleic acid dipstick assay?method to detect P.shigelloides and K.pneumoniae.was developed.Results showed that the detection methods established were specific and completed in50 min at 64?,with the limit detection of 20 copies/reaction for the target gene,which sensitivity was 100 times higher than conventional PCR.The result of detecting clinical samples showed the visual LAMP and LAMP-NADA could detect more positive samples.This rapid and accurate visual LAMP as well as LAMP-NADA methods could be used in the on-site diagnosis for effective prevention.