Establishment Of Detection And Identification Methods Of Two Viruses?Two Bacteria And Enterocytozoon Hepatopenaei In Macrobrachium Rosenbergii

In this study,monoplex PCR and multiple PCR methods were established for detection WSSV,IHHNV and EHP of Macrobrachium rosenbergii.Aeromonas veronii and Aeromonas caviae of M.rosenbergii were also isolated and identified,and the pathogenetic and drug susceptibility of the strains were tested.The main results are as follows.1. Establishment of monoplex PCR methods for WSSV,IHHNV and EHP of M.rosenbergiiIn the experiment,the specific primers of monoplex PCR for WSSV and EHP have been designed and the IHHNV used the primer sequence recommended by OIE.And optimize the annealing temperature,initial denaturation conditions,denaturation conditions and cycle number to explore the best monoplex PCR reaction conditions,the sensitivity of the methods were also be tested,the results show that:The optimal reaction conditions of WSSV monoplex PCR reaction were:initial denaturation at 95?C for 5 min,followed by 35 cycles of denaturation at 95?C for 30s,annealing at 59.8?C for 30s and extension at 72?C for 50s and a final extension at 72?C for 8 min,200 fg is the detection limit for monoplex PCR of WSSV,containing 5.41x10~4copies of WSSV DNA;The optimal reaction conditions of IHHNV monoplex PCR reaction were:initial denaturation at 95?C for 5 min,followed by 35 cycles of denaturation at 95?C for 45s,annealing at57?C for 30s and extension at 72?C for 30s and a final extension at 72?C for 8 min,200 fg is the detection limit for monoplex PCR of IHHNV,containing 5.99x10~4copies of IHHNV DNA;The optimal reaction conditions of EHP monoplex PCR reaction were:initial denaturation at 95?C for 5 min,followed by 35 cycles of denaturation at 95?C for30s,annealing at 58?C for 30s and extension at 72?C for 20s and a final extension at 72?C for 8 min,2pg is the detection limit for monoplex PCR of EHP,containing6.36x10~5copies of EHP DNA.2. Establishment of multiplex PCR methods for EHP and IHHNV,EHP and WSSV,IHHNV and WSSV of M.rosenbergiiIn the experiment,multiplex PCR methods for EHP and IHHNV,EHP and WSSV,IHHNV and WSSV of M.rosenbergii were established,and optimize the annealing temperature,initial denaturation conditions,denaturation conditions and cycle number to explore the best multiplex PCR reaction conditions,and their specificity and sensitivity were also be tested,the results show that:The optimal reaction conditions of EHP and IHHNV multiplex PCR reaction were:initial denaturation at 95?C for 5 min,followed by36 cycles of denaturation at 95?C for 45s,annealing at 57?C for 30s and extension at72?C for 30s and a final extension at 72?C for 8 min,200 pg is the detection limit for multiplex PCR of EHP,containing 6.36x10~7copies of EHP DNA,20 pg is the detection limit for multiplex PCR of IHHNV,containing 5.99x10~6copies of IHHNV DNA;The optimal reaction conditions of EHP and WSSV multiplex PCR reaction were:initial denaturation at 95?C for 5 min,followed by 35 cycles of denaturation at 95?C for 30s,annealing at 58?C for 30s and extension at 72?C for 50s and a final extension at 72?C for8 min,200 pg is the detection limit for multiplex PCR of EHP,containing 6.36x10~7copies of EHP DNA,2 pg is the detection limit for multiplex PCR of WSSV,containing5.41x10~5copies of WSSV DNA;The optimal reaction conditions of IHHNV and WSSV multiplex PCR reaction were:initial denaturation at 95?C for 5 min,followed by 38cycles of denaturation at 95?C for 30s,annealing at 58.9?C for 30s and extension at 72?C for 50s and a final extension at 72?C for 8 min,20 pg is the detection limit for multiplex PCR of IHHNV,containing 5.99x10~6copies of IHHNV DNA,2 pg is the detection limit for multiplex PCR of WSSV,containing 5.41x10~5copies of WSSV DNA,and the specificity of three kinds of multiplex PCR was tested,the results show that,no PCR product was obtained from the DNA of SPF prawns and A.caviae,confirming that the three multiplex PCR methods established in this study had strong specificity.3.Isolation and identification of A.veronii and A.caviae from M.rosenbergiiIn the experiment,two strains with growth advantage were isolated from the dying M.rosenbergii hepatopancreas.Their hemolysis,gram staining,morphological characteristics,physiology and biochemistry were determined and the 16S r RNA gene of these isolate strains were sequenced and phylogenetic tree analyzed were constructed.The isolated strains were confirmed as A.veronii(Accession number MN733186)and A.caviae(Accession number MN736843)based on their biochemical characters and 16S r RNA sequence analysis.The artificial infection experiments were carried out with the isolated strains,the results revealed that,the infected M.rosenbergii showed similar symptoms to the diseased M.rosenbergii in natural waters,and died in a short time,which confirmed that the isolated strains were highly pathogenetic to M.rosenbergii.The results of drug susceptibility tests revealed that A.veroniiis highly sensitive to Ceftriaxone,Chloramphenicol,Florfenicol,Doxycycline,and Rifampicin;it was moderately sensitivity to Novobiocin and Compound Sulfamethoxazole and resistant to Ciprofloxacin,Norfloxacin,Erythromycin,Azithromycin,Streptomycin,Kanamycin,Tetracycline.The A.caviae is highly sensitive to Ceftriaxone,Chloramphenicol,Florfenicol,Tetracycline and Doxycycline;it was moderately sensitivity to Kanamycin and resistant to Ciprofloxacin,Norfloxacin,Erythromycin,Azithromycin,Novobiocin,Streptomycin,Compound Sulfamethoxazole and Rifampicin.This current study provides a reference and theoretical basis for the pathogen identification,drug diagnosis,and treatment of Aeromonas cavia in M.rosenbergii.