Detection of Mycoplasma in cell cultures and cell culture derived biologicals

• US 10,472,683 B2
• Filed: 05/31/2013
• Issued: 11/12/2019
• Est. Priority Date: 06/01/2012
• Status: Active Grant

• Current Assignee: Akron Biotechnology, LLC
• Original Assignee: Akron Biotechnology, LLC
• Inventors: Zylberberg, Claudia, Rodriguez, Juan Manuel, Hernando Insua, Andres Clemente, Lopez, Ricardo Ariel
• Primary Examiner(s): Salmon, Katherine D
• Application Number:
  US14/404,902
• Publication Number:
  US 20150111207A1
• Time in Patent Office: 2,356 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays

Abstract

Sequences having specificity for Mycoplasma and related Mollicutes genus strains and uses thereof. Methods of use include detection of samples contaminated with Mycoplasma. Kits are provided and comprise one or more oligonucleotides for the detection of Mycoplasma and related Mollicutes genus strains.

Claims

1. A Mycoplasma detection kit comprising at least one synthetic oligonucleotide selected from the group consisting of SEQ ID NOS:

10, 12, 13, 14, 15, and sequences complementary thereto;

a buffer;

a discriminatory positive control synthetic oligonucleotide (DPC),

wherein the DPC comprises at least one sequence selected from the group consisting of SEQ ID NOS;

4 and 6; and

an instruction for operation.

2. The kit of claim 1 comprising two or more synthetic oligonucleotides of claim 1 comprising a nucleic acid having at least 95% sequence identity or complementarity with either strand in the sense or anti-sense orientation of a sequence selected from the group consisting of SEQ ID NOS:

1 to 10 and 12 to 15.

4. The kit of claim 3 comprising two or more synthetic oligonucleotides of claim 1.

3. A Mycoplasma detection kit comprising at least one synthetic oligonucleotide selected from the group consisting of SEQ ID NOS:

10, 12, 13, 14, 15, and sequences complementary thereto;

a buffer;

a discriminatory positive control synthetic oligonucleotide (DPC),

wherein a detectable label is attached at a 5′

-end or a 3′

-end of the synthetic oligonucleotide and the detectable label is selected from the group consisting of a fluorophore, a chromophore, a luminescent compound, a radioactive isotope, an enzyme substrate, a co-factor, a ligand, a hapten, an enzyme, a magnetic particle and a barcode,

wherein the DPC comprises at least one sequence selected from the group consisting of SEQ ID NOS;

4 and 6; and

an instruction for operation.

5. A Mycoplasma detection kit comprising:

at least one synthetic oligonucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOS;

10, 12, 13, 14, 15, and sequences complementary thereto;

a lysis buffer;

a solution including a buffer, a dye, dNTPs, and a polymerase;

a discriminatory positive control synthetic oligonucleotide (DPC),

wherein the DPC comprises at least one sequence selected from the group consisting of SEQ ID NOS;

4 and 6; and

an instruction for operation.

6. The kit of claim 5, wherein the two or more synthetic oligonucleotides include at least one forward primer and at least one reverse primer.

7. The kit of claim 5, wherein the dye comprises at least one selected from the group consisting of SYBR green and ROX.