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      A PCR protocol to establish standards for routine mycoplasma testing that by design detects over ninety percent of all known mycoplasma species

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          Summary

          Mycoplasma infection leads to false and non-reproducible scientific data and poses a risk to human health. Despite strict guidelines calling for regular mycoplasma screening, there is no universal and widely established standard procedure. Here, we describe a reliable and cost-effective PCR method that establishes a universal protocol for mycoplasma testing. The applied strategy utilizes ultra-conserved eukaryotic and mycoplasma sequence primers covering by design 92% of all species in the six orders of the class Mollicutes within the phylum Mycoplasmatota and is applicable to mammalian and many non-mammalian cell types. This method can stratify mycoplasma screening and is suitable as a common standard for routine mycoplasma testing.

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          Highlights

          • •

            Efficient PCR/qPCR procedure to detect mycoplasma infections in cell culture

          • •

            Ultra-conserved primers covering by design >90% of all Mycoplasmatota species

          • •

            Simple, generally applicable and reliable protocol for routine mycoplasma testing

          Abstract

          Microbiology; Mycology; Sequence analysis

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          Most cited references57

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          MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform.

          K Katoh (2002)
          A multiple sequence alignment program, MAFFT, has been developed. The CPU time is drastically reduced as compared with existing methods. MAFFT includes two novel techniques. (i) Homo logous regions are rapidly identified by the fast Fourier transform (FFT), in which an amino acid sequence is converted to a sequence composed of volume and polarity values of each amino acid residue. (ii) We propose a simplified scoring system that performs well for reducing CPU time and increasing the accuracy of alignments even for sequences having large insertions or extensions as well as distantly related sequences of similar length. Two different heuristics, the progressive method (FFT-NS-2) and the iterative refinement method (FFT-NS-i), are implemented in MAFFT. The performances of FFT-NS-2 and FFT-NS-i were compared with other methods by computer simulations and benchmark tests; the CPU time of FFT-NS-2 is drastically reduced as compared with CLUSTALW with comparable accuracy. FFT-NS-i is over 100 times faster than T-COFFEE, when the number of input sequences exceeds 60, without sacrificing the accuracy.
          Article 0 comments Cited 2855 times – based on 0 reviews     Review now
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            The Human Genome Browser at UCSC

            a. Haussler, W. J. Kent, C. Sugnet … (2002)
            As vertebrate genome sequences near completion and research refocuses to their analysis, the issue of effective genome annotation display becomes critical. A mature web tool for rapid and reliable display of any requested portion of the genome at any scale, together with several dozen aligned annotation tracks, is provided at http://genome.ucsc.edu. This browser displays assembly contigs and gaps, mRNA and expressed sequence tag alignments, multiple gene predictions, cross-species homologies, single nucleotide polymorphisms, sequence-tagged sites, radiation hybrid data, transposon repeats, and more as a stack of coregistered tracks. Text and sequence-based searches provide quick and precise access to any region of specific interest. Secondary links from individual features lead to sequence details and supplementary off-site databases. One-half of the annotation tracks are computed at the University of California, Santa Cruz from publicly available sequence data; collaborators worldwide provide the rest. Users can stably add their own custom tracks to the browser for educational or research purposes. The conceptual and technical framework of the browser, its underlying MYSQL database, and overall use are described. The web site currently serves over 50,000 pages per day to over 3000 different users.
            Article 0 comments Cited 1451 times – based on 0 reviews     Review now
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              Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction

              Jian Ye, George Coulouris, Irena Zaretskaya … (2012)
              Background Choosing appropriate primers is probably the single most important factor affecting the polymerase chain reaction (PCR). Specific amplification of the intended target requires that primers do not have matches to other targets in certain orientations and within certain distances that allow undesired amplification. The process of designing specific primers typically involves two stages. First, the primers flanking regions of interest are generated either manually or using software tools; then they are searched against an appropriate nucleotide sequence database using tools such as BLAST to examine the potential targets. However, the latter is not an easy process as one needs to examine many details between primers and targets, such as the number and the positions of matched bases, the primer orientations and distance between forward and reverse primers. The complexity of such analysis usually makes this a time-consuming and very difficult task for users, especially when the primers have a large number of hits. Furthermore, although the BLAST program has been widely used for primer target detection, it is in fact not an ideal tool for this purpose as BLAST is a local alignment algorithm and does not necessarily return complete match information over the entire primer range. Results We present a new software tool called Primer-BLAST to alleviate the difficulty in designing target-specific primers. This tool combines BLAST with a global alignment algorithm to ensure a full primer-target alignment and is sensitive enough to detect targets that have a significant number of mismatches to primers. Primer-BLAST allows users to design new target-specific primers in one step as well as to check the specificity of pre-existing primers. Primer-BLAST also supports placing primers based on exon/intron locations and excluding single nucleotide polymorphism (SNP) sites in primers. Conclusions We describe a robust and fully implemented general purpose primer design tool that designs target-specific PCR primers. Primer-BLAST offers flexible options to adjust the specificity threshold and other primer properties. This tool is publicly available at http://www.ncbi.nlm.nih.gov/tools/primer-blast.
              Article 0 comments Cited 984 times – based on 0 reviews     Review now
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                Author and article information

                Contributors
                Dominik Siegl
                Journal
                Journal ID (nlm-ta): iScience
                Journal ID (iso-abbrev): iScience
                Title: iScience
                Publisher: Elsevier
                ISSN (Electronic): 2589-0042
                Publication date PMC-release: 26 April 2023
                Publication date Collection: 19 May 2023
                Publication date (Electronic): 26 April 2023
                Volume: 26
                Issue: 5
                Electronic Location Identifier: 106724
                Affiliations
                [1 ]Institute of Translational Immunology (TIM), University Medical Center of the Johannes Gutenberg-University, Mainz 55131, Germany
                [2 ]Department of Medicine III Hematology & Medical Oncology, University Medical Center of the Johannes Gutenberg-University, Mainz 55131, Germany
                [3 ]ImmuneNTech GmbH, Wendelsheim 55234, Germany
                [4 ]Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA
                [5 ]Research Center for Immunotherapy, University Medical Center of the Johannes Gutenberg-University, Mainz 55131, Germany
                Author notes
                [∗ ]Corresponding author domsiegl@ 123456uni-mainz.de
                [6]

                Lead contact

                Article
                Publisher Item ID: S2589-0042(23)00801-5 Publisher ID: 106724
                DOI: 10.1016/j.isci.2023.106724
                PMC ID: 10192841
                PubMed ID: 37216121
                SO-VID: 778aa0c9-43a0-4260-92b7-c8dda3369fc8
                Copyright © © 2023 The Author(s)
                License:

                This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

                History
                Date received : 26 October 2022
                Date revision received : 7 February 2023
                Date accepted : 19 April 2023
                Categories
                Subject: Article

                Keywords: microbiology,mycology,sequence analysis
                Data availability:
                Keywords: microbiology, mycology, sequence analysis

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