Impact of temperature and time storage on the microbial detection of oral samples by Checkerboard DNA-DNA hybridization method.
OBJECTIVE
Molecular diagnosis methods have been largely used in epidemiological or clinical studies to detect and quantify microbial species that may colonize the oral cavity in healthy or disease. The preservation of genetic material from samples remains the major challenge to ensure the feasibility of these methodologies. Long-term storage may compromise the final result. The aim of this study was to evaluate the effect of temperature and time storage on the microbial detection of oral samples by Checkerboard DNA-DNA hybridization.


METHODS
Saliva and supragingival biofilm were taken from 10 healthy subjects, aliquoted (n=364) and processed according to proposed protocols: immediate processing and processed after 2 or 4 weeks, and 6 or 12 months of storage at 4°C, -20°C and -80°C.


RESULTS
Either total or individual microbial counts were recorded in lower values for samples processed after 12 months of storage, irrespective of temperatures tested. Samples stored up to 6 months at cold temperatures showed similar counts to those immediately processed. The microbial incidence was also significantly reduced in samples stored during 12 months in all temperatures.


CONCLUSIONS
Temperature and time of oral samples storage have relevant impact in the detection and quantification of bacterial and fungal species by Checkerboard DNA-DNA hybridization method. Samples should be processed immediately after collection or up to 6 months if conserved at cold temperatures to avoid false-negative results.
Faculty of Dentistry of Ribeirão Preto, Department of Dental Materials and Prosthodontics, Molecular Diagnosis Laboratory, University of São Paulo, Av. Café s/n°, Monte Alegre, Ribeirão Preto, SP 14040-904, Brazil.
Cássio
C
do Nascimento
Janine Navarro
JN
dos Santos
Vinícius
V
Pedrazzi
Murillo Sucena
MS
Pita
Nadia
N
Monesi
Ricardo Faria
RF
Ribeiro
Rubens Ferreira
RF
de Albuquerque
D000704:Analysis of Variance; D015169:Colony Count, Microbial; D015342:DNA Probes; D004269:DNA, Bacterial / Q000032:analysis*; D005260:Female; D005881:Gingiva / Q000382:microbiology*; D006801:Humans; D008297:Male; D009693:Nucleic Acid Hybridization / Q000379:methods*; D011309:Preservation, Biological / Q000379:methods*; D012463:Saliva / Q000382:microbiology*; D013696:Temperature; D013997:Time Factors; D055815:Young Adult
D015342:DNA Probes; D004269:DNA, Bacterial
Bacteria; Candida; Checkerboard DNA–DNA hybridization; Clinical assessment; DNA storage; Diagnosis