Abstract
Mycoplasma contamination in cell culture affects the properties of cell lines. Gold standard detection by microbiological culture takes days and requires specialists. The polymerase chain reaction and loop-mediated isothermal amplification (LAMP) are fast molecular options, but LAMP only requires one heating block for DNA amplification. This study presents a comparative genomic analysis of Mycoplasma species to identify common target genes different from the rrsA gene, which encodes 16 S rRNA. The aim is to implement a LAMP assay to detect Mycoplasma species, reducing the time and specialized equipment required for detection. We performed a comparative genomic analysis through Mauve software and the GView server and selected infB and clpB genes as target candidates for designing LAMP primers. We evaluated both genes by multiple sequence alignment (MSA). The infB gene presented the best score MSA assessment with lower odd-log values (5,480,281) than other genes. We selected the infB gene to design LAMP primers specific to Mycoplasma spp. We used these primers to implement LAMP at 63 °C for 30 min, which showed 100% positive amplifications for detecting Mycoplasma spp. In conclusion, we present a methodology utilizing the infB gene-based LAMP assay to detect three of the six most prevalent Mycoplasma species in cell culture.
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The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.
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Acknowledgements
We are grateful for the support granted for the Innovation project “Desarrollo de un agente antimicoplasma basado en tecnología CRISPR-dCas9 para aplicación en la industria biofarmacéutica”, Benjamín Luna Callejas, PhD student in Biomedicine and Molecular Biotechnology from the Escuela Nacional de Ciencias Biológicas of the Instituto Politécnico Nacional. The project received approval under the call for “Desarrollo Tecnológico e Innovación para Alumnos 2021”. BL received a CONACyT and BEIFI scholarship; ER holds COFAA, EDI, and SNI grants.
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This project was funded by DGAPA-UNAM grant PAPIIT IT100922, by Secretaría de Investigación y Posgrado (SIP-IPN) through project 20221169, and by Consejo Nacional de Ciencia y Tecnología (CONACyT) through Fondo Sectorial de Investigación para la Educación through project No. A1-S-21548.
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All authors contributed to this research’s study conception and design. BL, ER, and LO performed research, material preparation, data collection, and analysis. BL wrote the first draft of the manuscript, and all authors commented on previous versions. All authors read and approved the final manuscript.
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Luna-Callejas, B., Oropeza-Ramos, L. & Ramón-Gallegos, E. Comparative genomic analysis of Mycoplasma related to cell culture for infB gene-based loop-mediated isothermal amplification. World J Microbiol Biotechnol 39, 355 (2023). https://doi.org/10.1007/s11274-023-03794-y
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DOI: https://doi.org/10.1007/s11274-023-03794-y