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Amplification of a Full‐Length Borna Disease Virus (BDV) cDNA from Total RNA of Cells Persistently Infected with BDV
Author(s) -
Shoya Yuko,
Kobayashi Takeshi,
Koda Toshiaki,
Lai Patrick K.,
Tanaka Hidetoshi,
Koyama Tsukasa,
Ikuta Kazuyoshi,
Kakinuma Mitsuaki,
Kishi Masahiko
Publication year - 1997
Publication title -
microbiology and immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.664
H-Index - 70
eISSN - 1348-0421
pISSN - 0385-5600
DOI - 10.1111/j.1348-0421.1997.tb01881.x
Subject(s) - complementary dna , primer (cosmetics) , biology , virology , reverse transcriptase , rna , microbiology and biotechnology , oligonucleotide , rapid amplification of cdna ends , viral quasispecies , virus , rna virus , dna , gene , genetics , molecular cloning , chemistry , hepatitis c virus , organic chemistry
We have developed a novel reverse transcriptase‐polymerase chain reaction (RT‐PCR) to amplify the full‐length 8.9 kilobase (kbp) cDNA of the Borna disease virus (BDV) RNA genome from the total cellular RNA of MDCK cells persistently infected with BDV (MDCK/BDV). Antigenomic BDV cDNA was reverse transcribed using a 53‐mer oligonucleotide primer, corresponding to the 5′‐terminus of a putative 3′‐leader sequence of the BDV RNA genome, for 2 hr at 42 C followed by 30 min at 55 C. PCR was performed in the presence of this 53‐mer antigenomic primer and a 25‐mer primer, corresponding to the 3′‐terminus of the BDV antigenomic cDNA, by use of an rTth DNA polymerase with proof‐reading activity. The amplified full‐length BDV cDNA was detected in as little as 20 ng of total cellular RNA of MDCK/BDV. This RT‐PCR method should be a useful technique to study the molecular quasispecies of BDV.