Development of diagnostic kit for the detection of Chrysanthemum virus B

Abstract

The present invention provides a method for detection of Chrysanthemum virus B in plants using desined primers of Sequence ID 1:Upstream primer TGCCTCCCAAACCGGCACCAGGTGAT Sequence ID 2: Downstream primer:TTTATAATGTCTTATTATTCGCAT It also relates to a diagnostic kit useful for detection of coat protein of Chrysanthemum virus B in plants comprising: polyclonal antibodies against Chrysanthemum virus B coat protein in plants; conjugate labeled with alkaline phosphatase; coating buffer; extraction buffer; ECI buffer; PNP buffer.

1 . Primers useful for detection of Chrysanthemum virus B (CVB) in plants, comprising the following sequence:  
       
         
           
                 
                 
                 
                 
               
                     
                     
                 
                     
                   Sequence ID 1: 
                     
                     
                 
                     
                   Upstream primer 
                   ATGCCTCCCAAACCGGCACCAGGTGAT 
                 
                     
                     
                 
                     
                   Sequence ID 2: 
                 
                     
                   Downstream primer 
                   TTTATAATGTCTTATTATTCGCAT 
                 
                     
                     
                 
             
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         2 . A method for detection of Chrysanthemum virus B (CVB) in plants, wherein the said method comprising the steps of: 
 a) providing a purified coat protein of CVB by using designed primers of                                          Sequence ID 1:                 Upstream primer   ATGCCTCCCAAACCGGCACCAGGTGAT                   Sequence ID 2:         Downstream primer   TTTATAATGTCTTATTATTCGCAT                                 b) preparing polyclonal antibodies against CVB coat protein obtained from step (a);    c) performing direct antibody sandwich enzyme linked immunosorbent assay (DAS ELISA) for detection of CVB.    
     
     
         3 . A method as claimed in claim  2 (a), wherein the complete coat protein of CVB is amplified from rose using designed primers having a  
       
         
           
                 
                 
                 
                 
               
                     
                     
                 
                     
                   Sequence ID 1: 
                     
                     
                 
                     
                   Upstream primer 
                   ATGCCTCCCAAACCGGCACCAGGTGAT 
                 
                     
                     
                 
                     
                   Sequence ID 2: 
                 
                     
                   Downstream primer 
                   TTTATAATGTCTTATTATTCGCAT 
                 
                     
                     
                 
             
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         4 . A method as claimed in claim  2 (a), wherein the complete coat protein of CVB comprising sequence ID having No. CAI51623: MPPKPAPGDNEGNASGSTPTPSPPHPARTAEEARLRLAEMEREREQEQSLEE MNSNTPDDDARNISRLTQLAALLRREQTNVHVTNMALEIGRPALQPPPNMR GDPTNMYSQVSTDFLWKIKPQRISNNMATSEDMVKIQVALEGLGVPTESVKE VIIRLVLNCANTSSSVYQDPKGVIEWDGGAIIADDVVGVINEHSTLRKVCRLYA AVAWNYMHLQQTPPSDWSAMGFHPNVKYAAFDFFDYVENGAAIGPSGGIV PKPTRAEYVAYNTYKMLALNKANNNDTFGNFDSAITGGRQGPAIHNNLNN ANNKTL is cloned in pGEX-2TK followed by transformation using  E - coli  strain BL 21.  
     
     
         5 . A method as claimed in claim  2 (a), wherein the optimal expression of CVB coat protein is checked with 0.25-1 mM IPTG concentration at about temperature 25 degree C. for 3.54 h.  
     
     
         6 . A method as claimed in claim  2 (a), wherein the purification of CVB coat protein is carried out by the known method.  
     
     
         7 . A method as claimed in claim  2 (b), wherein the immunization in rabbits are carried out four times with purified coat protein of PNRSV obtained from step  1 (a) and Freund's complete adjuvant in the ratio of 1:1 at weekly intervals.  
     
     
         8 . A method as claimed in claim  2 (b), wherein the route for immunization may be intramuscularly, subcutaneously or intravenously.  
     
     
         9 . A method as claimed in claim  2 (b), wherein the rabbits are bled after 14 to 15 days to obtain polyclonal antibodies against CVB coat protein.  
     
     
         10 . A method as claimed in claim  2 (b), wherein the polyclonal antibodies against CVB coat protein are purified from the serum by known methods.  
     
     
         11 . A method as claimed in claim  2 (c), wherein the microtiter plates are coated with polyclonal antibodies diluting in a coating buffer in a ratio ranges from 1:500-1:1000 followed by 4-5 times washing with PBS-T.  
     
     
         12 . A method as claimed in claim  2 (c), wherein the test samples are prepared in microtiter plates by macerating infected leaf tissue from plant with extraction buffer followed by dilution from 1×-1/150× of the original antigen.  
     
     
         13 . A method as claimed in claim  2 (c), wherein the microtiter plate is incubated overnight at about 37° C. followed by washing to allow coating of antigen in the wells.  
     
     
         14 . A method as claimed in claim  2 (c), wherein antibody conjugate obtained from step 2(b) in ECI buffer is added in the ratio ranges between 1:500 to 1:1000 for a period of about 2 hrs at about 37° C. followed by washing with PBS-T.  
     
     
         15 . A method as claimed in claim  2 (c), wherein about 100 μl of about 1 mg/ml p-nitrophenyl phosphate solution in PNP buffer is added in the mix as claimed in  claim 14 .  
     
     
         16 . A method as claimed in claim  2 (c), wherein the reaction is terminated by adding about 50 μl of about 3M NaOH after 15-20 min to obtain yellow color product.  
     
     
         17 . A method as claimed in claim  1 (c), wherein the color product is antigen and antibody conjugate.  
     
     
         18 . A method as claimed in claim  2 (c), wherein absorbance of colored product is measured at 405 nm for detection of Chrysanthemum virus B (CVB).  
     
     
         19 . A diagnostic kit useful for detection of coat protein of Chrysanthemum virus B (CVB) comprising: 
 a) polyclonal antibodies against Chrysanthemum virus B (CVB) coat protein in plants as claimed in  claim 2;     b) conjugate labeled with alkaline phosphatase;    c) coating buffer;    d) extraction buffer;    e) ECI buffer;    f) PNP buffer;    g) Instruction manual.