Development Of Rapid Detection Techniques For Two Marine Pathogenic Microorganisms

Vibrio parahaemolyticus?VP?,a kind of gram-negative pathogenic bacteria can cause disease for human and aquacutured animals.Outbreaks of V.parahaemolyticus associated with gastroenteritis are usually attributed to the consumption of eating raw or undercooked seafood or pickled food.With the improvement of seafood consumption level,the disease caused by V.parahaemolyticus has showed a rise trend and it has become the first foodborne disease.Scylla serrata reovirus?SsRV?is one of the most prevalent viral pathogens of the scylla serrata discovered in recent years.It has a serious impact on the development of scylla serrata breeding industry due to the higher fatality rate and the rapid spread of SsRV.Currently,several methods have been developed for detection of V.parahaemolyticus,including various molecular biology and immunology methods.For SsRV,the detection techniques mainly include microscopic observation,traditional tissue section,ultrathin sections technique,PCR and its derivatives.However,these procedures were disadvantages due to a long assay time,low sensitivity and some expensive equipments and skilled persons are needed.Therefore,they are difficult to be applied in many quarantine institutes.Cross primer amplification?CPA?is a class of isothermal amplification reactions that is carried out by a strand displacement DNA polymerase and does not require an initial denaturation step or the addition of a nicking enzyme.CPA can achieve efficiently amplification for the target fragment under constant temperature condition.Colloidal gold is a kind of commonly marking technology,with colloidal gold as tracer marker applied to a new kind of antigen antibody immune markers,has its unique advantages.In this study,we developed and evaluated the specificity and sensitivity both of a CPA combined with immuno-blotting analysis for rapid detection of V.parahaemolyticus and immuno gold staining for the detection of SsRV respectively.The main results are as followings:?1?The establishment of CPA-nucleic acid strip method for rapid detection of V.Parahaelyticus.Based on the conservative and species specific gene“tlh”in V.parahaemolyticus,we established CPA-nucleic acid strip method for the detection of V.parahaemolyticus by designing two pairs of sequence-specific primers and one pair prodes.The CPA system parameters,including the concentration of Mg²⁺,dNTPs,betaine,Bst DNA polymerase,reaction time and temperature,were optimized.The optimum reaction system included:0.1?mol/mL each of VPOF and VPOR,0.6?mol/mL each of VPIF and VPIR;0.3?mol/mL each of VPDF and VPDR;4.0 mmol/mL Mg ²⁺;0.8 mol/L betaine;0.4 mmol/mL dNTPs;2?l 10×ThermoPol Reaction Buffer;1?l templates and sterilized double-distilled water in 20?l volumes.The mixture was incubated at 63?for 1 hour.By comparing traditional PCR method and CPA method for V.parahaemolyticus detection sensitivity,it indicated that the detection limit of CPA for pure cultures and genome were 5.8×10²cfu/mL and 11.4 pg/mL respectively.It was 10 times of traditional PCR.This method only took 75 minutes and provided an effective technical support for the quick detection of V.parahaemolyticus.?2?Construction of a prokaryotic expression system for SsRV protein coat VP12:The VP12gene following a PCR-amplification from PMD18-T-VP12 was cloned to an expression vector pET-28a to abtain pET-28a-VP12.The VP12 recombinant protein was expressed in Escherichia coli BL21 as a fusion protein of 32.7 kDa.The recombinant prokaryotic expression system was successfully established with high expression efficiency for target fusion protein 83%.By Ni²⁺affinity chromatography method,the recombinant protein was purified.?3?Preparation of SsRV coat protein VP12 polyclonal antibody:VP12 recombinant protein was used as antigen to immunize the New Zealand white rabbit for polyclonal antibody preparation.Immunoblot analysis revealed that the antibody reacted strongly with recombinant protein VP12.The mean titer of survey sera was 1:51200 by indirect ELSIA detection.?4?Preparation of monoclonal antibody against reovirus coat protein VP12:VP12recombinant protein was used as antigen to prepare monoclonal antibodies by outine immunization technology and the fusion technology of spleen and hybridoma cell.Female BALB/c mice were immunized with recombinant protein VP12 and yielded 5 hybridomas secreting McAb.Dot blotting analysis showed that cell strains 1M9 and 2N5 had a higher affinity,so they were selected as the capture antibody and detector antibody labeled with colloidal gold for the gold immnochromatographic assay based on the principle of double antibody.Then,ascitic fluids were obtained and purified by ammonium sulphateprecipitation and protein G affinity chromatography method.The concentration of purified monoclonal antibody were 4.004mg/mL and 4.078 mg/mL,respectively.The purified McAbs were identified by SDS-PAGE.It indicated that the McAb protein was pure and contained little other protein.Analyzed by western blotting,two kinds of McAb only reacted specifically with protein VP12.The titer of McAb1M9 and 2N5 were determined to be 1:102400,1:51200 by indirect ELISA.?5?Preparation of colloidal gold immunochromatography strip for detection of SsRV.Colloidal gold with about 20 nm particle diameter were prepared by sodium citrate reduction method.The colloidal gold was homogeneous,without fragments and agglutination characterized by visual observation,ultraviolet-visible absorption spectroscopy and transmission electron microscopy.McAb 1M9 was conjugated with collidal gold.Then the colloidal gold-antibody conjugation was dispensed on a porous glass fiber to form the gold conjugated released pad.Purified McAb 2N5 and goat anti-mouse IgG were coated on the nitrocellulose membrane as the test line and the control line,respectively.Through a series of optimization,the monoclonal antibody 1M9 was labelled with colloidal gold of 20 nm particle diameter at a rate of 37.5?g monoclonal antibody per 1 mL colloidal gold.Optimum coating concentrations of McAb 2N5and goat anti-mouse IgG were determined as 0.75 mg/mL and 1.0 mg/mL,respectively.Sensitivity test showed that the colloidal gold immunochromatography strip could detect 3.8?g/mL purified protein VP12 and 20.4?g/mL purified virus.Specificity tests showed that the strip is positive only for SsRV.The current study demonstrated that the assay method of CPA combined with immuno-blotting analysis and colloidal gold immunochromatography strip were specific and sensitive detection for the rapid detection of V.parahaemolyticus and SsRV respectivelly.It has provided more practical detection tools for rapid diagnosis,early prevention,monitoring and control of V.parahaemolyticus and Scylla serrata reovirus.