Patent ID: 6068974
Filing Date: 2000-05-30
Classification: C12Q

Abstract:
A method for detecting the presence or absence of pseudorabies virus in a sample suspected of being infected with said virus, said method comprising the steps of:a) isolating a sample nucleic acid mixture from said sample suspected of being infected with said virus;b) preparing a purified sample nucleic acid mixture from said sample nucleic acid mixture;c) performing a nested polymerase chain reaction to selectively amplify a targeted nucleotide sequence within said purified sample nucleic acid mixture, said polymerase chain reaction comprising the steps of:i) amplifying in a first-stage polymerase chain reaction mixture a first targeted nucleotide sequence, said first targeted nucleotide sequence comprising a 674 base-pair region of the pseudorabies virus gII glycoprotein gene, said 674 base-pair region having the nucleotide sequence set forth in SEQ ID NO: 1, wherein said first targeted nucleotide sequence is flanked by an outer oligonucleotide primer pair, said outer primer pair consisting of a 5' upper primer and a 3' lower primer, wherein said upper primer of the outer primer pair is the nucleotide sequence set forth in SEQ ID NO: 2 and said lower primer of the outer primer pair is the nucleotide sequence set forth in SEQ ID NO: 3 or SEQ ID NO:4, andii) removing an aliquot of the first-stage reaction mixture and amplifying in a second-stage polymerase chain reaction mixture a second targeted nucleotide sequence internal to the first targeted nucleotide sequence, said second targeted nucleotide sequence flanked by an inner oligonucleotide primer pair, said inner primer pair consisting of a 5' upper primer and a 3' lower primer, wherein said upper primer of the inner primer pair is a nucleotide sequence selected from the group consisting of the sequences set forth in SEQ ID NOS: 5 to 7 and said lower primer of the inner primer pair is a nucleotide sequence selected from the group consisting of the sequences set forth in SEQ ID NOS: 8 to 11; andd) analyzing said first and second-stage polymerase chain reaction mixtures following amplification to detect the presence or absence of said first and second targeted nucleotide sequences wherein the presence of the first and second targeted nucleotide sequences indicates the presence of said virus in said sample suspected of being infected with said virus.