Avaliação de primers universais bacteriano em PCR in silico

Matheus Silva Alves, Luiz Alfredo Torres Sales, João Victor Nogueira Nojosa, Roberval Nascimento Moraes Neto, Alexsander Rodrigues Carvalho Junior, Ruth Flávia Barros Setúbal, Lucas Weba Soares, Luís Claudio Nascimento da Silva

Resumo


As técnicas de biologia molecular são indispensáveis em diversas áreas de pesquisa, dentre elas está a identificação de espécies bacterianas como Staphylococcus aureus, Streptococcus pneumoniae, Pseudomona auruginosa, Klebsiella pneumoniae, Corynebacterium diphtheriae, Mycobacterium tuberculosis e Escherichia coli e para isso utilizam primers universais. Este trabalho busca avaliar a qualidade deste primers universais em amplificar e diferenciar as bactérias descritas. Para isso foi utilizado uma PCR in silico e observados os resultados das amplificações. As amplificações demonstraram uma redundância ou mesmo ineficiência de vários dos primers testados.

 

Palavras-chave: Primers; Bacterias; PCR in silico.


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Referências


Challagundla L, Luo X, Tickler IA, Didelot X, Coleman DC, Shore AC, et al. Range Expansion and the Origin of USA300 North American Epidemic Methicillin-Resistant Staphylococcus aureus. mBio. 2018;9(1).

Hau SJ, Kellner S, Eberle KC, Waack U, Brockmeier SL, Haan JS, et al. Methicillin-Resistant Staphylococcus aureus Sequence Type (ST) 5 Isolates from Health Care and Agricultural Sources Adhere Equivalently to Human Keratinocytes. Applied and environmental microbiology. 2018;84(2).

Gillis HD, Demczuk WHB, Griffith A, Martin I, Warhuus M, Lang ALS, et al. PCR-based discrimination of emerging Streptococcus pneumoniae serotypes 22F and 33F. Journal of microbiological methods. 2018;144:99-106.

Hare KM, Leach AJ, Smith-Vaughan HC, Chang AB, Grimwood K. Streptococcus pneumoniae and chronic endobronchial infections in childhood. Pediatric pulmonology. 2017;52(12):1532-45.

Santos-Juanes J, Fernandez-Vega I, Vivanco-Allende B, Fresno Forcelledo M. Perivascular basophilic rim as a histopathological clue of cutaneous Pseudomona aeruginosa sepsis. The American Journal of dermatopathology. 2015;37(5):427-8.

Grundmann H, Glasner C, Albiger B, Aanensen DM, Tomlinson CT, Andrasevic AT, et al. Occurrence of carbapenemase-producing Klebsiella pneumoniae and Escherichia coli in the European survey of carbapenemase-producing Enterobacteriaceae (EuSCAPE): a prospective, multinational study. The Lancet Infectious diseases. 2017;17(2):153-63.

Hong KW, Asmah Hani AW, Nurul Aina Murni CA, Pusparani RR, Chong CK, Verasahib K, et al. Comparative genomic and phylogenetic analysis of a toxigenic clinical isolate of Corynebacterium diphtheriae strain B-D-16-78 from Malaysia. Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases. 2017;54:263-70.

Stutz MD, Clark MP, Doerflinger M, Pellegrini M. Mycobacterium tuberculosis: Rewiring host cell signaling to promote infection. Journal of leukocyte biology. 2018;103(2):259-68.

Saputra S, Jordan D, Mitchell T, Wong HS, Abraham RJ, Kidsley A, et al. Antimicrobial resistance in clinical Escherichia coli isolated from companion animals in Australia. Veterinary microbiology. 2017;211:43-50.

Santos ALd, Santos DO, Freitas CCd, Ferreira BLA, Afonso IF, Rodrigues CR, et al. Staphylococcus aureus: visitando uma cepa de importância hospitalar. Jornal Brasileiro de Patologia e Medicina Laboratorial. 2007;43:413-23.

Vieira AC, Gomes MC, Rolo Filho M, Eudes Filho J, Bello EJ, de Figueiredo RB. Streptococcus pneumoniae: a study of strains isolated from cerebrospinal fluid. Jornal de pediatria. 2007;83(1):71-8.

Sangal V, Tucker NP, Burkovski A, Hoskisson PA. The draft genome sequence of Corynebacterium diphtheriae bv. mitis NCTC 3529 reveals significant diversity between the primary disease-causing biovars. Journal of bacteriology. 2012;194(12):3269.

Cerdeno-Tarraga AM, Efstratiou A, Dover LG, Holden MT, Pallen M, Bentley SD, et al. The complete genome sequence and analysis of Corynebacterium diphtheriae NCTC13129. Nucleic acids research. 2003;31(22):6516-23.

Camus JC, Pryor MJ, Medigue C, Cole ST. Re-annotation of the genome sequence of Mycobacterium tuberculosis H37Rv. Microbiology. 2002;148(Pt 10):2967-73.

Glickman MS, Jacobs WR, Jr. Microbial pathogenesis of Mycobacterium tuberculosis: dawn of a discipline. Cell. 2001;104(4):477-85.

Reid G, Howard J, Gan BS. Can bacterial interference prevent infection? Trends in microbiology. 2001;9(9):424-8.

Eckburg PB, Bik EM, Bernstein CN, Purdom E, Dethlefsen L, Sargent M, et al. Diversity of the human intestinal microbial flora. Science. 2005;308(5728):1635-8.

Ebili HO, Hassall JC, Fadhil W, Ham-Karim H, Asiri A, Raposo TP, et al. "Squirrel" Primer-Based PCR Assay for Direct and Targeted Sanger Sequencing of Short Genomic Segments. Journal of biomolecular techniques : JBT. 2017;28(3):97-110.

Nathalang O, Intharanut K, Sasikarn W, Nathalang S, Kupatawintu P. A new polymerase chain reaction: sequence-specific primer method for the Augustine blood type. Blood transfusion = Trasfusione del sangue. 2016;14(6):577-9.

Spencer M, Barnes S, Parada J, Brown S, Perri L, Uettwiller-Geiger D, et al. A primer on on-demand polymerase chain reaction technology. American journal of infection control. 2015;43(10):1102-8.

Ritchie LE, Burke KF, Garcia-Mazcorro JF, Steiner JM, Suchodolski JS. Characterization of fecal microbiota in cats using universal 16S rRNA gene and group-specific primers for Lactobacillus and Bifidobacterium spp. Veterinary microbiology. 2010;144(1-2):140-6.

Saggu SK, Mishra PC. Characterization of thermostable alkaline proteases from Bacillus infantis SKS1 isolated from garden soil. PloS one. 2017;12(11):e0188724.

Mao DP, Zhou Q, Chen CY, Quan ZX. Coverage evaluation of universal bacterial primers using the metagenomic datasets. BMC microbiology. 2012;12:66.

Ito T, Suzaki K. Universal detection of phytoplasmas and Xylella spp. by TaqMan singleplex and multiplex real-time PCR with dual priming oligonucleotides. PloS one. 2017;12(9):e0185427.

Nadkarni MA, Martin FE, Jacques NA, Hunter N. Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set. Microbiology. 2002;148(Pt 1):257-66.

Hidalgo M, Prieto I, Abriouel H, Villarejo AB, Ramirez-Sanchez M, Cobo A, et al. Changes in Gut Microbiota Linked to a Reduction in Systolic Blood Pressure in Spontaneously Hypertensive Rats Fed an Extra Virgin Olive Oil-Enriched Diet. Plant foods for human nutrition. 2017.

Takahashi S, Tomita J, Nishioka K, Hisada T, Nishijima M. Development of a prokaryotic universal primer for simultaneous analysis of Bacteria and Archaea using next-generation sequencing. PloS one. 2014;9(8):e105592.

Bahl MI, Bergstrom A, Licht TR. Freezing fecal samples prior to DNA extraction affects the Firmicutes to Bacteroidetes ratio determined by downstream quantitative PCR analysis. FEMS microbiology letters. 2012;329(2):193-7.

De Laveleye M, Huang TD, Bogaerts P, Berhin C, Bauraing C, Sacre P, et al. Increasing incidence of carbapenemase-producing Escherichia coli and Klebsiella pneumoniae in Belgian hospitals. European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology. 2017;36(1):139-46.

Larsen N, Vogensen FK, van den Berg FW, Nielsen DS, Andreasen AS, Pedersen BK, et al. Gut microbiota in human adults with type 2 diabetes differs from non-diabetic adults. PloS one. 2010;5(2):e9085.

Bikandi, J., San Millán, R., Rementeria, A., and Garaizar, J. 2004. In silico analysis of complete bacterial genomes: PCR, AFLP-PCR, and endonuclease restriction. Bioinformatics 20:798-9. DOI: 10.1093/bioinformatics/btg491




DOI: https://doi.org/10.24863/rib.v9i2.124

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