Compositions and methods for detecting a biological contaminant
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.
1. A positive amplification control (PAC) plasmid comprising:
a unique artificial plasmid-specific sequence (UAPS), wherein said UAPS comprises SEQ ID NO: 10;
a target amplification polynucleotide (TAP) sequence, wherein said TAP comprises all or part of a parvovirus NS-1 sequence, and
a nucleic acid extraction control (NEC) nucleotide sequence, wherein said NEC comprises an M13 bacteriophage nucleotide sequence.
2. 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.
3. The PAC plasmid of claim 1 , wherein the parvovirus NS-1 sequence comprises SEQ ID NO: 37.
4. The PAC plasmid of claim 1 , wherein said M13 bacteriophage nucleotide sequence is a M13K07 nucleotide sequence.
5. The PAC plasmid of claim 4 , wherein said M13K07 nucleotide sequence comprises SEQ ID NO. 8.
6. The PAC plasmid of claim 1 , further comprising binding sites that hybridize to one or more oligonucleotide primers.
7. The PAC plasmid of claim 6 , 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.
8. The PAC plasmid of claim 6 , wherein said one or more oligonucleotide primers are rodent parvovirus oligonucleotide primers.
9. The PAC plasmid of claim 8 , wherein said rodent parvovirus oligonucleotide primers are selected from the group consisting of a forward oligonucleotide primer, a reverse oligonucleotide primer, and a combination thereof.
10. The PAC plasmid of claim 1 , further comprising binding sites that hybridize to M13 oligonucleotide primers.
11. The PAC plasmid of claim 10 , wherein said M13 oligonucleotide primers are selected from the group consisting of a forward oligonucleotide primer, a reverse oligonucleotide primer, and a combination thereof.
12. 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).
13. The PAC plasmid of claim 1 , wherein
the unique artificial plasmid-specific sequence (UAPS) comprises SEQ ID NO: 10;
the parvovirus nucleic acid sequence comprises SEQ ID NO: 37; and
the M13 bacteriophage nucleotide sequence comprises SEQ ID NO: 8.
14. The PAC plasmid of claim 1 , wherein the plasmid sequence comprises SEQ ID NO: 11.