Detection of bacterial (mollicutes) contamination

Abstract

The present disclosure provides a method and system for the PCR amplification of a target sequence which suppresses non-specific amplification products. The disclosure concerns the use of a primer pair optimized to amplify a nucleic acid of a contaminant in the background of genomic DNA of a first organism. When DNA from a second organism suspected for comprising the contaminant is subjected to the same PCR-based amplification reaction, detection sensitivity and specificity of the contaminant is enhanced when an amount of genomic DNA of the first organism is present in the amplification reaction.

Description

RELATED APPLICATIONS[0001]This application is a continuation of U.S. application Ser. No. 13 / 364,050 filed Feb. 1, 2012, which is a continuation of International Application No. PCT / EP2010 / 004655, filed Jul. 29, 2010, which claims the benefit of European Patent Application No. 09009965.6, filed Aug. 1, 2009, the disclosures of which are hereby incorporated by reference in their entirety.SEQUENCE LISTING[0002]The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jan. 31, 2012, is named Sequence_Listing—26237_US.txt, and is 1,128 bytes in size.BACKGROUND[0003]1. Field of the Disclosure[0004]The present disclosure relates to contamination within biological samples. More specifically, the instant disclosure relates to a system and method for detecting bacterial contamination within biological samples.[0005]2. Description of the Related Art[0006]Mycoplas...

AI Technical Summary

Benefits of technology

[0013]The present disclosure provides an improved PCR-based amplification of a target sequence by suppressing non-specific amplification products. This improvement concerns the use of a primer pair optimized to amplify a nucleic acid of a contaminant in the background of genomic DNA of a first organism. When DNA from a second organism, suspected as comprising the contaminant, is subjected to the same polymerase chain reaction (PCR) based amplification reaction, detection sensitivity and specificity of the contaminant is enhanced when an amount of genomic DNA of the first organism is present in the amplification reaction.

[0015]According to some embodiments, relative to the volume of the sample, a predetermined amount of DNA from CHO cells is added to at least one of the sample, the processed sample, the purified nucleic acids, or the reaction mixture. In such embodiments, the added DNA from CHO cells reduces unspecific amplification in the step of detecting.

Problems solved by technology

Mycoplasma cell culture contamination can occur due to contamination from individuals or contaminated cell culture medium ingredients, for example.

Mycoplasma cells are physically small—less than 1 μm—and are difficult to detect with a conventional microscope.

Severe Mycoplasma infections have the potential to destroy a cell line.

However, such linkage is considered controversial (see, Alexeeva, I., et al., J Clin Microbiol 44 (2006) 91-97, were a blinded study of rRNA species failed to detect any footprint of Spiroplasma in scrapie-infected hamster brain).

Moreover, the possibility of Mycoplasma contamination during biopharmaceutical production, cell therapy, and tissue engineering is also a concern and potential major problem.

These culture-based techniques require a long time to achieve results.

Furthermore, cultivation of certain Mycoplasma species may not be possible or reliable.

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