Evaluating the efficiency of a TPIA method for the detection of two tobacco viruses

Field experiments are usually necessary for analyzing the efficiency of control methods, the resistance of varieties and many other virological studies. Such experiments generally include a large number of samples to be tested serologically. DAS-ELISA is a very accurate technique that has been widely used for identifying viral infections. For large numbers of plant samples, it takes a long time for the sample preparation, plate washing and other procedures. In this study, the efficiency of a simple and laborsaving TPIA (Tissue Print Immunoassay) method was evaluated for the identification of two important aphid-transmitted viruses (CMV and PVY) of tobacco fields in comparison with DAS-ELISA as the standard method. The leaf samples were collected from the fields of three commercial tobacco types (flue-cured, burley and oriental). Each sample was divided to two parts and each part was examined by one of the methods. DAS-ELISA was done based on the method described by Clark and Adams (1977). In TPIA, small parts of the leaf samples were rolled and then cut by a sterile sharp blade. The cut surface was gently printed on 1 cm2 blocks drawn on a nitrocellulose paper. Air dried paper was located first in 1% BSA for blocking the empty sites on paper, then in the buffer containing AP-conjugated polyclonal antibody for 3 h and finally in NBT-BCIP solution for color development. Between these stages, the paper was washed thoroughly (three times) by shaking in fresh washing buffer. The results of each sample were recorded and compared with those of DAS-ELISA. By considering DAS-ELISA as the reference method, the sensitivity of TPIA for the detection of PVY and CMV was 96.1% and 92.7%, respectively. The positive results by TPIA which were not detected positive by DAS-ELISA were regarded as false positive. These were 8 (out of 316 tested samples) for CMV and 6 (out of 204 samples) for PVY. Although the results of TPIA were not completely consistent with DAS-ELISA, it seems that this method can be used for some general studies. The most important advantages of this method were that it didn't need sample extraction and was done using only one antibody which was the conjugated antibody of each virus. This method gives more rapid results (within a day) in comparison with DAS-ELISA that needs more time.