Method of detecting adeno-associated virus (aav)

A method of detecting adeno-associated virus (AAV) in a sample comprising recombinant adeno-associated virus (rAAV) particles which may be pseudo-wild type rAAV particles, said method comprising:

• a. obtaining the sample comprising rAAV particles;
• b. partitioning the sample into a plurality of compartments;
• c. performing assays for indicating the presence of a nucleotide sequence corresponding to at least a portion of a rep gene and the presence of a nucleotide sequence corresponding to at least a portion of a cap gene within each of the plurality of compartments;
• and
• d. detecting any compartments, within the plurality of compartments, that comprise, as indicated by the assays of step (c):
• i) a nucleotide sequence corresponding to the at least a portion of the rep gene but do not comprise a nucleotide sequence corresponding to the at least a portion of the cap gene;
• ii) a nucleotide sequence corresponding to the at least a portion of the cap gene but do not comprise the nucleotide sequence corresponding to the at least a portion of the rep gene;
• and
• iii) a nucleotide sequence corresponding to the at least a portion of the rep gene and a nucleotide sequence corresponding to the at least a portion of the cap gene;
• wherein the detection of compartments that comprise both a nucleotide sequence corresponding to the at least a portion of the rep gene and a nucleotide sequence corresponding to the at least a portion of the cap gene indicates that the sample comprises rAAV particles which may be pseudo-wild type rAAV particles, and wherein step (d) comprises determining the number of compartments that comprise a nucleotide sequence corresponding to the at least a portion of the rep gene and a nucleotide sequence corresponding to the at least a portion of the cap gene, wherein the number of compartments that comprise both a nucleotide sequence corresponding to the at least a portion of the rep gene and a nucleotide sequence corresponding to the at least a portion of the cap gene indicates the quantity of rAAV in the sample which may be pseudo-wild type rAAV particles.

A method of quantifying adeno-associated virus (AAV) in a sample comprising recombinant adeno-associated virus (rAAV) particles which may be pseudo-wild type rAAV particles, said method comprising:

• a. obtaining the sample comprising rAAV particles;
• b. partitioning the sample into a plurality of compartments;
• c. performing assays for indicating the presence of a nucleotide sequence corresponding to at least a portion of a rep gene and the presence of a nucleotide sequence corresponding to at least a portion of a cap gene within each of the plurality of compartments;
• and
• d. determining the number of compartments, within the plurality of compartments, that comprise, as indicated by the assays of step (c):
• i) a nucleotide sequence corresponding to the at least a portion of the rep gene but not a nucleotide sequence corresponding to the at least a portion of the cap gene;
• ii) a nucleotide sequence corresponding to the at least a portion of the cap gene but not a nucleotide sequence corresponding to the at least a portion of the rep gene;
• and
• iii) a nucleotide sequence corresponding to the at least a portion of the rep gene and a nucleotide sequence corresponding to the at least a portion of the cap gene;
• wherein the number of compartments that comprise both a nucleotide sequence corresponding to the at least a portion of the rep gene and a nucleotide sequence corresponding to the at least a portion of the cap gene indicates the quantity of rAAV in the sample which may be pseudo-wild type rAAV particles.
• wherein step (d) comprises detecting any compartments or determining the number of compartments, within the plurality of compartments, that do not comprise, as indicated by the assays of step (c), either a nucleotide sequence corresponding to a portion of the rep gene or a nucleotide sequence corresponding to a portion of the cap gene.

A kit comprising:

• (a) forward and reverse PCR primers capable of hybridising to an AAV rep gene;
• and
• (b) forward and reverse PCR primers capable of hybridising to an AAV cap gene;
• optionally wherein the forward and/or reverse PCR primers capable of hybridising to the AAV rep gene are forward and/or reverse PCR primers, and/or wherein the forward and/or reverse PCR primers capable of hybridising to the AAV cap gene are forward and/or reverse PCR primers.