Methods and reagents for assessing the presence or absence of replication competent virus

A method for detecting a presence, absence, or amount of a viral DNA, comprising:

• (a) amplifying two or more viral DNA sequences from DNA from at least one cell that contains a heterologous nucleic acid introduced by transduction of the at least one cell with a lentiviral vector comprising the heterologous nucleic acid, wherein:
• the two or more viral DNA sequences include at least a portion of a VSV-G env gene and a pol gene or at least a portion of a VSV-G env gene and a rev gene;
• and
• wherein the amplifying is carried out using:
• (i) forward and reverse oligonucleotide primers, each pair of forward and reverse oligonucleotide primer is specific for one of the two or more viral DNA sequences,
• (ii) an oligonucleotide probe specific for one of the two or more viral DNA sequences, and
• (iii) a DNA polymerase, under conditions sufficient to amplify the two or more viral DNA sequences by polymerase chain reaction (PCR), if present in the biological sample;
• and
• (b) detecting the presence, absence, amount or concentration of the amplified viral nucleic acid,
• wherein the forward and reverse oligonucleotide primers comprise:
• a forward oligonucleotide primer specific for the VSV-G env gene comprising a sequence with at least 90% identity and/or at least 15 contiguous nucleotides of a sequence set forth in SEQ ID NO:
• 35, 50, 51, or 54 and a reverse oligonucleotide primer specific for the env gene comprising a sequence with at least 90% identity and/or at least 15 contiguous nucleotides of a sequence set forth in SEQ ID NO:
• 36, 52, 53, or 55;
• and
• (i) a forward oligonucleotide primer specific for the pol gene comprising a sequence with at least 90% identity and/or at least 15 contiguous nucleotides of a sequence set forth in SEQ ID NO:
• 42, 45, or 46, and a reverse oligonucleotide primer specific for the pol gene comprising a sequence with at least 90% identity and/or at least 15 contiguous nucleotides of a sequence set forth in SEQ ID NO:
• 43 or 47;
• or
• (ii) a forward oligonucleotide primer specific for the rev gene comprising a sequence with at least 90% identity and/or at least 15 contiguous nucleotides of a sequence set forth in set forth in SEQ ID NO:
• 38 and a reverse oligonucleotide primer specific for the rev gene comprising a sequence with at least 90% identity and/or at least 15 contiguous nucleotides of a sequence set forth in set forth in SEQ ID NO:
• 39.