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      High-throughput detection and multiplex identification of cell contaminations

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      Author(s): * ,
      Publication date (Electronic):
      Journal: Nucleic Acids Research
      Publisher: Oxford University Press

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          Abstract

          Unnoticed cell culture contamination by viruses, Mycoplasma, or other cell lines is not uncommon and a threat to laboratory safety and the quality of scientific results. We developed and validated a novel high-throughput Multiplex cell Contamination Test (McCT), which is currently able to detect 37 contamination markers in a single reaction. The assay is based on multiplex PCR with target-specific primers and subsequent hybridization of amplimers to specific oligonucleotide probes. McCT proved to be highly specific, sensitive and robust, and allows to analyze more than 1000 cell lysates per week. In conclusion, the novel McCT assay is a powerful high-throughput tool in assessing cell line purity.

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          Bead-based multiplex genotyping of human papillomaviruses.

          Markus Schmitt, I. G. Bravo, Peter J. F. Snijders … (2006)
          Typing of human papillomaviruses (HPV) by DNA hybridization procedures, such as reverse line blot (RLB) assay, is sensitive and well validated. However, the application of these assays to high-throughput analyses is limited. Here, we describe the development of multiplex human papillomavirus genotyping (MPG), a quantitative and sensitive high-throughput procedure for the identification of multiple high- and low-risk genital HPV genotypes in a single reaction. MPG is based on the amplification of HPV DNA by a general primer PCR (GP5+/6+) and the subsequent detection of the products with type-specific oligonucleotide probes coupled to fluorescence-labeled polystyrene beads (Luminex suspension array technology). Up to 100 different HPV types can be detected simultaneously with MPG, and the method is fast and labor saving. We detected all 22 HPV types examined with high specificity and reproducibility (the median interplate coefficient of variation was below 10%). Detection limits for the different HPV types varied between 100 and 800 pg of PCR products. We compared the performance of MPG to an established RLB assay on GP5+/6+-PCR products derived from 94 clinical samples. The evaluation showed an excellent agreement (kappa = 0.922) but also indicated a higher sensitivity of MPG. In conclusion, MPG appears to be highly suitable for large-scale epidemiological studies and vaccination trials as well as for routine diagnostic purposes.
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            Short tandem repeat profiling provides an international reference standard for human cell lines.

            J Masters, J. A. Thomson, B Daly-Burns … (2001)
            Cross-contamination between cell lines is a longstanding and frequent cause of scientific misrepresentation. Estimates from national testing services indicate that up to 36% of cell lines are of a different origin or species to that claimed. To test a standard method of cell line authentication, 253 human cell lines from banks and research institutes worldwide were analyzed by short tandem repeat profiling. The short tandem repeat profile is a simple numerical code that is reproducible between laboratories, is inexpensive, and can provide an international reference standard for every cell line. If DNA profiling of cell lines is accepted and demanded internationally, scientific misrepresentation because of cross-contamination can be largely eliminated.
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              Homogeneous amplification of genital human alpha papillomaviruses by PCR using novel broad-spectrum GP5+ and GP6+ primers.

              Markus Schmitt, Bolormaa Dondog, Tim Waterboer … (2008)
              Human papillomavirus (HPV) DNA detection and typing are important for diagnosis and management of HPV-associated diseases. One of the most commonly used PCR methods, GP5+/6+, shows weaknesses in amplifying certain types. To circumvent this limitation, we developed and validated broad-spectrum primers targeting the GP5+/6+ region. The addition of eight upstream and two downstream BSGP5+/6+ (BS) primers improved amplification of plasmids of 14 genital HPV types 10- to 1,000-fold versus GP5+/6+ PCR without altering sensitivity for the 10 others. For these 24 types, an analytic sensitivity of < or = 1,000 plasmid copies in the presence of 100 ng cellular DNA was obtained. Additionally, we integrated an internal beta-globin PCR into both HPV PCR systems, allowing simultaneous DNA quality control without affecting the sensitivity of HPV detection. Furthermore, we describe five additional low-risk HPV probes used in multiplex HPV genotyping (MPG) for simultaneous identification of all 15 high-risk, 3 putative high-risk, and 9 low-risk HPV genotypes. The performance of BSGP5+/6+ multiplexed with beta-globin primers was compared to that of standard GP5+/6+ with DNA from 1,112 cervical scrapings. There was 79% overall agreement (kappa = 0.816). BSGP5+/6+ was significantly more sensitive than GP5+/6+ for detection of HPV 30, 39, 42, 44, 51, 52, 53, 68, 73, and 82, detecting 212 additional HPV infections and increasing the proportion of multiple infections from 17.2 to 26.9% in cancer patients. In conclusion, BSGP5+/6+ multiplexed with beta-globin PCR provides an improvement in type-specific amplification sensitivity and homogeneity compared to GP5+/6+ and offers simultaneous internal control of DNA quality. BSGP5+/6+-MPG, therefore, is suitable for epidemiologic and also diagnostic applications.
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                Author and article information

                Journal
                Journal ID (nlm-ta): Nucleic Acids Res
                Journal ID (iso-abbrev): Nucleic Acids Res
                Journal ID (publisher-id): nar
                Journal ID (hwp): nar
                Title: Nucleic Acids Research
                Publisher: Oxford University Press
                ISSN (Print): 0305-1048
                ISSN (Electronic): 1362-4962
                Publication date (Print): October 2009
                Publication date (Electronic): 9 July 2009
                Publication date PMC-release: 9 July 2009
                Volume: 37
                Issue: 18
                Page: e119
                Affiliations
                Research Program Infection and Cancer, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 242, 69120 Heidelberg, Germany
                Author notes
                *To whom correspondence should be addressed. Tel: +0049 (0)6221 424937; Fax: +0049 (0)6221 424932; Email: markus.schmitt@ 123456dkfz.de
                Article
                Publisher ID: gkp581
                DOI: 10.1093/nar/gkp581
                PMC ID: 2764421
                PubMed ID: 19589807
                SO-VID: 3b610631-db47-45d0-aa09-b4dedf777312
                Copyright © © 2009 The Author(s)
                License:

                This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                Date received : 30 April 2009
                Date revision received : 10 June 2009
                Date accepted : 24 June 2009
                Categories
                Subject: Methods Online

                ScienceOpen disciplines: Genetics
                Data availability:
                ScienceOpen disciplines: Genetics

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