Three pairs of primers were designed and synthesized from nucleotide sequences of garlic latent virus （GLV）, onion yellow dwarf virus （OYDV）, and leek yellow stripe virus （LYSV） by using PCR primer design softwa...Three pairs of primers were designed and synthesized from nucleotide sequences of garlic latent virus （GLV）, onion yellow dwarf virus （OYDV）, and leek yellow stripe virus （LYSV） by using PCR primer design software. The expected fragments about 170 bp, 287 bp, and 191 bp were amplified by RT-PCR for GLV, OYDV, and LYSV, respectively in disease-infected plants of potato onion （Allium cepa L., Aggregatum group）, but such fragments were not obtained from healthy-looking plants and virus-free seedlings of shoot-tips. The amplified products ofGLV, OYDV and LYSV were cloned into pGEM-T vectors, and transformed into Escherichia coli. JM109. The recombinant plasmids were obtained and sequenced. The nucleotide sequences were compared with corresponding viral nucleotide sequences reported in GenBank by performing a NCBI BLAST. The analysis showed that their homology attained 75% to 90%,89.5% to 96.1%,and 91.6% to 96.3% in GLV, OYDV, and LYSV, respectively. The total RNA of 6.34 ug·uL^-1 from infected plants was diluted to a series of 10^-1 to 10^5 and the detection sensitivity of RT-PCR was 10^4 （about 4 ng）. Thus, a method of identification and detection by RT-PCR of GLV, OYDV, and SLYV was established.展开更多
基金Supported by Department Education of Heilongjiang Province of China (10531146)
文摘Three pairs of primers were designed and synthesized from nucleotide sequences of garlic latent virus （GLV）, onion yellow dwarf virus （OYDV）, and leek yellow stripe virus （LYSV） by using PCR primer design software. The expected fragments about 170 bp, 287 bp, and 191 bp were amplified by RT-PCR for GLV, OYDV, and LYSV, respectively in disease-infected plants of potato onion （Allium cepa L., Aggregatum group）, but such fragments were not obtained from healthy-looking plants and virus-free seedlings of shoot-tips. The amplified products ofGLV, OYDV and LYSV were cloned into pGEM-T vectors, and transformed into Escherichia coli. JM109. The recombinant plasmids were obtained and sequenced. The nucleotide sequences were compared with corresponding viral nucleotide sequences reported in GenBank by performing a NCBI BLAST. The analysis showed that their homology attained 75% to 90%,89.5% to 96.1%,and 91.6% to 96.3% in GLV, OYDV, and LYSV, respectively. The total RNA of 6.34 ug·uL^-1 from infected plants was diluted to a series of 10^-1 to 10^5 and the detection sensitivity of RT-PCR was 10^4 （about 4 ng）. Thus, a method of identification and detection by RT-PCR of GLV, OYDV, and SLYV was established.
