Comparative genomic analysis of Mycoplasma related to cell culture for infB gene-based loop-mediated isothermal amplification
Por:
Luna-Callejas, Benjamin, Oropeza-Ramos, Laura, Ramon-Gallegos, Eva
Publicada:
1 dic 2023
Resumen:
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 degree
celsius 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.
Filiaciones:
Facultad de Ingenieria, Universidad Nacional Autonoma de Mexico, Mexico City, 04510, Mexico
Department of Morphology, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Mexico City, 07738, Mexico
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