Rapid determination of the nucleotide sequences of potyviral coat protein genes using semi-nested RT-PCR with universal primers

Abstract

A rapid method (<20 h) for determining the nucleotide sequences of potyviral coat protein (CP) genes was developed. The procedure involves reverse transcription using total RNA from a potyvirus-infected plant, followed by polymerase chain reaction (PCR) with the universal primers Sprimer/M4. The expected fragment, purified from an agarose gel, is used in semi-nested PCR with three degenerate primer sets: Sprimer/D1000, Sprimer/RYAFDFY-C and U341/M4. The three purified fragments are directly sequenced using the respective primers D1000, RYAFDFY-C and U341, then the complete CP sequence is assembled. All 11 potyviruses tested were efficiently sequenced by this method.