COMPOSITIONS AND METHODS FOR DETECTING A BIOLOGICAL CONTAMINANT

Abstract

Provided are compositions and methods useful to the determination of whether a microbial contaminant is present in a biological therapeutic production process. Specifically, an artificial positive amplification control plasmid and unique quantitative PCR detection probe are provided, which enables the rapid and real-time detection of a false positive result.

Description

Claims

1. A positive amplification control (PAC) plasmid comprising: a unique artificial plasmid-specific sequence (UAPS) comprising SEQ ID NO: 10, anda target amplification polynucleotide (TAP) sequence, wherein said TAP comprises all or part of a parvovirus NS-1 sequence.

2. The PAC plasmid of claim 1, wherein said UAPS does not recognize or anneal to any natural parvovirus.

3. The PAC plasmid of claim 1, wherein the parvovirus NS-1 sequence is at least 88% identical to any one of SEQ ID NOs: 12-37 or 97% identical to SEQ ID NO: 9.

4. The PAC plasmid of claim 1, wherein the parvovirus NS-1 sequence comprises SEQ ID NO: 9 or any one of SEQ ID NOs: 12-37.

5. The PAC plasmid of claim 1, wherein the parvovirus NS-1 sequence comprises SEQ ID NO: 37.

6. The PAC plasmid of claim 1, comprising a nucleic acid extraction control (NEC) nucleotide sequence, wherein said NEC comprises an M13 bacteriophage nucleotide sequence.

7. The PAC plasmid of claim 6, wherein said M13 bacteriophage nucleotide sequence is a M13K07 nucleotide sequence.

8. The PAC plasmid of claim 7, wherein said M13K07 nucleotide sequence comprises SEQ ID NO. 8.

9. The PAC plasmid of claim 1, further comprising sequences of one or more oligonucleotide primers.

10. The PAC plasmid of claim 9, wherein said one or more oligonucleotide primers is selected from the group consisting of a forward oligonucleotide primer, a reverse oligonucleotide primer, and a combination thereof.

11. The PAC plasmid of claim 9, wherein said one or more oligonucleotide primers are rodent parvovirus oligonucleotide primers.

12. The PAC plasmid of claim 11, wherein said rodent parvovirus oligonucleotide primers is selected from the group consisting of a forward oligonucleotide primer, a reverse oligonucleotide primer, and a combination thereof.

13. The PAC plasmid of claim 1, further comprising M13 oligonucleotide primers.

14. The PAC plasmid of claim 13, wherein said M13 oligonucleotide primers is selected from the group consisting of a forward oligonucleotide primer, a reverse oligonucleotide primer, and a combination thereof.

15. The PAC plasmid of claim 1, wherein said NS-1 sequence is selected from the group consisting of minute virus of mice prototype strain (MVMp), minute virus of mouse immunosuppressive strain (MVMi), minute virus of mouse Cutter strain (MVMc); mouse parvovirus 1b (MPV-1b), mouse parvovirus 1a (MPV-1a), mouse parvovirus 1c (MPV-1c), hamster parvovirus (HaPV), Toolan’s parvovirus (H-1), Kilham rat virus (KRV), rat parvovirus 1a, rat minute virus, and Umass strain of Rat virus L (RV-Umass).