Graduate Thesis Or Dissertation
 

Serological and molecular approaches for distinguishing bean common mosaic and bean common mosaic necrosis potyviruses and their respective pathogroups

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  • Polyclonal antisera were raised against isolates of bean common mosaic virus (BCMV) and bean common mosaic necrosis virus (BCMNV) using conventional serological methods. Infected tissues containing, respectively, 22 recognized BCMV and BCMNV isolates were tested against the two antisera by antigen-coated plate (ACP) ELISA and double antibody sandwich (DAS) ELISA. Results indicated that each immunoglobulin was virus-specific by DAS-ELISA, providing clear distinction between BCMV and BCMNV. A reverse transcription, polymerase chain reaction (RT-PCR)-based assay in combination with restriction endonuclease analyses, was developed for molecular detection of BCMV, BCMNV and their pathogroups. Specific detection of the two viruses was accomplished by constructing two virus-specific primer pairs that amplified a PCR product specific for each virus. Distinction of two BCMNV pathogroups (PG-III and PG-VI) was achieved by restriction enzyme XbaI digestion of BCMNV PCR products. However, none of the tested restriction enzymes clearly differentiated the five recognized BCMV pathogroups. A primer pair Dts/Uny15 specific for BCMV pathogroup V was also developed. By its RT-PCR application, four BCMV-PG-V isolates were differentiated from the other known variants of BCMV pathogroup I, II, IV and VII. Thus, by a combination of RT-PCR and restriction enzyme analyses, it was possible to differentiate both viruses, and two pathogroups of BCMNV, and one pathogroup of BCMV.
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