A highly sensitive internally-controlled real-time PCR assay for mycoplasma detection in cell cultures.

Mycoplasma contamination of cell lines is a common occurrence and may affect the cell line behaviour in a variety of ways. Contamination with mycoplasma is usually not obvious so cell lines should be frequently tested. Several commercially available kits for mycoplasma detection exist, however the Ph. Eur. culture method which can take several weeks to yield results is still considered to be the 'gold standard' method. There is therefore a need for rapid alternative methods with comparable sensitivity, specificity and species range. Here, we describe an internally-controlled Taqman-based real-time PCR assay for cell culture medium without the need for DNA extraction. The assay can detect less than 10 CFU of the most frequently encountered mycoplasma contaminants in mammalian cell cultures. The validated assay has the potential to be used as a routine test in the production of cell culture-based Biologicals.

Preparation and validation of the first WHO international genetic reference panel for Fragile X syndrome.

Fragile X syndrome is the most common inherited form of mental retardation. It is caused by expansion of a trinucleotide (CGG)n repeat sequence in the 5' untranslated region of the FMR1 gene, resulting in promoter hypermethylation and suppression of FMR1 transcription. Additionally, pre-mutation alleles in carrier males and females may result in Fragile X tremor ataxia syndrome and primary ovarian insufficiency, respectively. Fragile X is one of the most commonly requested molecular genetic tests worldwide. Quality assessment schemes have identified a wide disparity in allele sizing between laboratories. It is therefore important that clinical laboratories have access to characterized reference materials (RMs) to aid accurate allele sizing and diagnosis. With this in mind, a panel of genotyping RMs for Fragile X syndrome has been developed, which should be stable over many years and available to all diagnostic laboratories. Immortalized cell lines were produced by Epstein-Barr virus transformation of lymphocytes from consenting patients. Genomic DNA was extracted in bulk and RM aliquots were freeze-dried in glass ampoules. Twenty-one laboratories from seventeen countries participated in a collaborative study to assess their suitability. Participants evaluated the samples (blinded, in triplicate) in their routine methods alongside in-house and commercial controls. The panel of five genomic DNA samples was endorsed by the European Society of Human Genetics and approved as an International Standard by the Expert Committee on Biological Standardization at the World Health Organization.