Effect of sample storage time on detection ofhybridization signals in Checkerboard DNA–DNAhybridization

Cássio do Nascimento, Katia Muller, Sandra Sato, andRubens Ferreira Albuquerque Junior

Abstract: Long-term sample storage can affect the intensity of the hybridization signals provided by molecular diagnosticmethods that use chemiluminescent detection. The aim of this study was to evaluate the effect of different storage times onthe hybridization signals of 13 bacterial species detected by the Checkerboard DNA–DNA hybridization method usingwhole-genomic DNA probes. Ninety-six subgingival biofilm samples were collected from 36 healthy subjects, and the inten-sity of hybridization signals was evaluated at 4 different time periods: (1) immediately after collecting (n = 24) and (2) afterstorage at –20 °C for 6 months (n = 24), (3) for 12 months (n = 24), and (4) for 24 months (n = 24). The intensity of hy-bridization signals obtained from groups 1 and 2 were significantly higher than in the other groups (p < 0.001). No differen-ces were found between groups 1 and 2 (p > 0.05). The Checkerboard DNA–DNA hybridization method was suitable todetect hybridization signals from all groups evaluated, and the intensity of signals decreased significantly after long periodsof sample storage.

Key words: Checkerboard DNA–DNA hybridization, genomic probes, sample storage, oral bacteria.

Résumé : L’entreposage à long terme peut affecter l’intensité des signaux d’hybridation générés par des méthodes molécu-laires de diagnostic qui comportent l’utilisation de la détection chimiluminescente. Le but de cette étude était d’évaluer l’ef-fet de différentes périodes d’entreposage sur les signaux d’hybridation de 13 espèces bactériennes détectées par hybridationADN–ADN en damier à l’aide de sondes d’ADN génomique total. Quatre-vingt-seize échantillons de biofilms sous-gingivaux ont été récoltés de 36 sujets sains et l’intensité des signaux d’hybridation a été évaluée lorsque les échantillonsétaient traités : (1) immédiatement après le prélèvement (n = 24), (2) après un entreposage à –20 °C pendant 6 mois (n =24), (3) 12 mois (n = 24) et (4) 24 mois (n = 24). L’intensité des signaux d’hybridation obtenus des groupes 1 et 2 était si-gnificativement plus élevée que celle des autres groupes (p < 0,001). Aucune différence n’a été trouvée entre les groupes 1et 2 (p > 0,05). La méthode d’hybridation ADN–ADN en damier était appropriée pour détecter les signaux d’hybridationde tous les groupes évalués, et l’intensité des signaux diminuait significativement après une longue période d’entreposage.

Mots‐clés : hybridation ADN–ADN en damier, sondes génomiques, entreposage d’échantillons, bactéries orales.

[Traduit par la Rédaction]

Introduction

Molecular diagnostic methods that employ whole-genomicDNA probes were developed during the last 2 decades andhave been extensively used in the detection and quantificationof pathogenic bacteria (Chen and Slots 1999; Socransky et al.2004; Sakamoto et al. 2005; do Nascimento et al. 2011).These methods are faster and more reliable than culture-dependent techniques, which fail to detect and identify sev-eral bacterial species often present in the oral cavity (Rolphet al. 2001; Moraes et al. 2002; Barbosa et al. 2009). Can-dida species have also been detected by this methodology(do Nascimento et al. 2009b). The Checkerboard DNA–DNA hybridization method utilizes whole-genomic DNA

probes to simultaneous identify and quantify several bacterialspecies in a large number of samples of subgingival plaque,applying a relatively simple and inexpensive technique(Socransky et al. 1994). Unlike conventional culture tech-niques, DNA hybridization methods detect both viable andnonviable microorganisms. Nevertheless, the preservation ofbacterial genome is an important factor in molecular diagnos-tic techniques, as degradation of DNA may occur during timein storage and this may interfere with detection of bacteria(Katsoulis et al. 2005a). Moreover, as with any molecular es-say, differences in specimen collection and storage, togetherwith several other factors, may influence the results, makingcomparisons between studies difficult, if at all possible. Stor-age criteria is particularly critical in subgingival plaque sam-

Received 8 November 2011. Revision received 13 January 2012. Accepted 13 January 2012. Published at www.nrcresearchpress.com/cjmon 27 March 2012.

C. do Nascimento, S. Sato, and R.F. Albuquerque Junior. Department of Dental Materials and Prosthodontics, Faculty of Dentistry ofRibeirão Preto, University of São Paulo, Ribeirão Preto, Brazil.K. Muller. Faculty of Dentistry, McGill University, Montréal, Quebec, Canada.

Corresponding author: Rubens Ferreira de Albuquerque Junior (e-mail: ralbuque@forp.usp.br and rubens.albuquerque@mcgill.ca).

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Can. J. Microbiol. 58: 502–506 (2012) doi:10.1139/W2012-021 Published by NRC Research Press

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ples due to the presence of a complex mixture of bacteriathat generate DNA-degrading products causing bacterialDNA fragmentation and greater degradation of exogenousDNA (Kruszewska et al. 2004; Socransky et al. 2004).Few studies are found in the literature regarding the effect

of sample storage time on the consistency of results of mo-lecular analysis for bacterial identification. In a study of theuse of synthetic DNA probes for identification of bacteria,up to 15% of the samples were lost probably due to long-time storage (Moncla et al. 1990). The effect of storage timein subgingival bacteria detection was evaluated in a recentstudy, which suggested that a sample storage protocol of amaximum of 6 weeks at 4 °C should be followed to achieveresults qualitatively and quantitatively reliable. Although thestudy provides relevant information regarding the standard-ization of storage time and the effect it has on the results, aquestion remains whether samples kept at –20 °C since col-lection and stored for long periods of time would be suitablefor achieving accurate detection levels. Thus, the aim of thisstudy was to evaluate the effect of sample storage time on thehybridization signals of 13 bacterial species detected by theCheckerboard DNA–DNA hybridization method, keeping thetemperature constant throughout the experiment.

Materials and methods

Subject populationThirty-six healthy subjects (mean age 37 years) were se-

lected based on the following inclusion criteria: presence ofthe 2 first molar teeth in the posterior region, no clinicalsigns of disease in the oral mucosa, and a healthy gingivalsulcus <3 mm deep. Exclusion criteria were pregnancy, lacta-tion, periodontal treatment, or intake of antibiotics in the pre-vious 3 months; also excluded were current smokers orindividuals with any systemic disease, which could influencethe periodontal status or which would require premedication.The study was approved by the ethics committee of the Fac-ulty of Dentistry of Ribeirão Preto, and all the participantsgave written informed consent.

Sample collectionSubgingival biofilm samples were collected from each sub-

ject (n = 36) with sterile paper points left for 30 s in the gin-gival sulcus of all the first molars. Two paper points wereinserted in the vestibular and 2 in the palatal or lingual sidesof the teeth, in both the mesial and distal positions, totaling apool of 4 paper collections for each tooth. After collection,the 4 paper points from each tooth were transferred to an in-dividual microtube containing 150.0 µL of TE (10.0 mmol/LTris–HCl, 1.0 mmol/L EDTA, pH 7.6), followed by the addi-tion of 150.0 µL of 0.5 mol/L NaOH. At the end of collec-tion, samples from each microtube (a total of 144 tubes: 4teeth per subject × 36 subjects) were pooled in a single con-tainer and homogenized. Then, the pooled biofilm sample(300 µL) was divided into 4 groups (n = 24) according tothe time until laboratory processing: immediately after collec-tion (group 1), after storage at –20 °C for 6 months (group2), for 12 months (group 3), and for 24 months (group 4).

Probe preparationThe following 13 bacterial strains and respective ATCC

number were assessed by the Checkerboard DNA–DNA hy-bridization method: Capnocytophaga gingivalis (ATCC33624), Campylobacter rectus (ATCC 33238), Capnocyto-phaga sputigena (ATCC 33612), Fusobacterium nucleatum(ATCC 31647), Pseudomonas aeruginosa (ATCC 47053),Prevotella intermedia (ATCC 25611), Parvimonas micra(ATCC 33270), Prevotella nigrescens (ATCC 33563), Strep-tococcus mutans (ATCC 25175), Streptococcus oralis(ATCC 35037), Streptococcus sanguinis (ATCC 10556),Streptococcus sobrinus (ATCC 33478), and Veillonella par-vula (ATCC 10790).All species were cultivated in trypticase soy broth (Difco,

Lawrence, Kansas, USA) at 35 ºC in anaerobic conditions.Briefly, cells from 50.0 mL of liquid cultures were harvestedby centrifugation at 1000g. Pellets of Gram-negative strainswere resuspended in 3.0 mL of lysis buffer (10.0 mmol/LTris–HCl, 0.1 mmol/L EDTA, pH 8.0), and pellets of Gram-positive strains were lysed in buffer solution containing lyso-zyme (100.0 mg/mL). After incubation for 1 h at 37 ºC, 5 vol-umes of GES buffer (5.0 mol/L guanidine thiocyanate,0.1 mol/L EDTA, 0.5% Sarkosyl) were added, followed bythe addition of 2.5 volumes of ammonium acetate (5.0 mol/L). After extracting with 1 volume of chloroform – 2-penta-nol (24:1), genomic DNA was precipitated with ethanol. Thepellets were resuspended in TE (10.0 mmol/L Tris–Cl,1.0 mmol/L EDTA, pH 8.0) and re-extracted once with 1 vol-ume of phenol–chloroform solution (1:1) and once with 1volume of chloroform. DNA was reprecipitated with sodiumacetate, pH 5.2, (final concentration 0.3 mol/L) and 2 vol-umes of ethanol. After 3 washes with 70% ethanol the DNApellets were air-dried and the DNA was resuspended in TE(10.0 mmol/L Tris–HCl, 1.0 mmol/L EDTA, pH 8.0). TotalDNA concentrations were estimated by absorbance at260.0 nm in RNase-treated samples, assuming that 50.0 µg/mL DNA has an absorbance of 1 (GeneQuant Pro, Amer-sham Pharmacia Biotech, Buckinghamshire, UK). Thewhole-genomic DNA probes from the 13 bacterial specieswere direct labeled with thermostable alkaline phosphataseenzyme using the AlkPhos Direct Labeling and DetectionSystem (GE Healthcare, UK). Briefly, 100 ng of denaturedDNA was mixed with the labeling buffer and alkaline phos-phatase enzyme. Formaldehyde was then added to covalentlycross-link the enzyme to the probe. Resulting alkalinephosphatase-labeled probes were adjusted to a final concen-tration of 1 ng/µL. Tests were performed for each probe tooptimize the amount of probe needed to detect both 105 and106 bacterial cells of each species with the lowest possiblebackground (Socransky et al. 1994). The same set of labeledgenomic probes was used to evaluate all the biofilm samplesin the 4 proposed protocols.

Microbiological analysisThe samples were processed according to protocol estab-

lished by do Nascimento et al. (2010). All the probe setswere tested for sensitivity and specificity before sample hy-bridization in each protocol. Microtubes containing subgingi-val biofilm samples were vortexed for 2 min at roomtemperature, and the paper points were removed using sterilepliers. Tubes were boiled for 5 min, followed by the additionof 800.0 µL of 5.0 mol/L ammonium acetate. After process-ing, the content of each tube was individually applied into the

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extended slots of the MiniSlot apparatus (Immunetics, Cam-bridge, Massachusetts, USA), and then concentrated onto a15 cm × 15 cm nylon membrane (Hybond N+, GE Health-care, Buckinghamshire, UK), followed by baking for 2 h, at80 ºC. For standard samples, mixtures of genomic DNA cor-responding to either 105 or 106 bacterial cells of each ana-lyzed species were assembled, denatured, precipitated, andapplied into 2 standard slots. The membranes were prehybri-dized and then hybridized in the checkerboard format, andchemiluminescent signals were detected by exposing themembrane to ECL Hyperfilm-MP (GE Healthcare) for20 min.

Data analysisThe image of hybridization reaction obtained on the Hy-

perfilm was digitized and analyzed with the ImageQuant TLsoftware (GE Healthcare). The hybridization samples signalswere recorded as count cells by comparing the intensity withthe standard lanes. The counts obtained for the 4 proposedprotocols were analyzed the first time for each of the bacte-rial species independently, and thereafter, as a pool of the 13species. The data were analyzed using the Generalized LinearModel, with time as the fixed factor and each of the 13 bac-terial species and the pool of bacteria as dependent variables.When significant differences were found, pair-wise compari-sons among the estimated marginal means were carried outwith the Sidak adjustment for multiple comparisons. All theanalyses were done with the software PASW Statistics ver-sion 17 (SPSS, Chicago, Illinois, USA), and differenceswere considered significant when p was <0.05.

ResultsFigure 1 represents the results of the Checkerboard analy-

sis for each of the species evaluated independently according

to the proposed protocols. Significant differences in totalcounts were observed for all species evaluated in the 4 pro-posed protocols (C. gingivalis; C. rectus, C. sputigena, F. nu-cleatum, P. aeruginosa, P. intermedia, P. micros,P. nigrescens, S. mutans, S. oralis, S. sanguinis, S. sobrinus,and V. parvula). Samples processed immediately or after6 months of storage had higher bacterial counts than the sam-ples stored for 12 and 24 months. Capnocytophaga sputi-gena, F. nucleatum, P. aeruginosa, P. intermedia, andS. mutans had higher counts after immediate processing, andC. rectus, P. nigrescens, and S. sanguinis had higher countsafter 6 months. The samples processed after 24 months hadthe lowest counts for F. nucleatum, P. aeruginosa, P. inter-media, S. mutans, and V. parvula.When microorganisms were analyzed as a pool of bacteria,

significant differences in bacterial count were found amongdifferent storage times (p < 0.001; Fig. 2). Higher countswere observed in samples processed immediately and inthose processed after 6 months, with nonsignificant differ-ence between them. Samples analyzed after 12 and 24months had the lowest counts, also with nonsignificant differ-ences between them.

Discussion

Diagnostic methods involving the analysis of genetic mate-rial have been widely employed in many areas of health re-search, such as epidemiological studies (Gizani et al. 2009),contamination of dental implants (do Nascimento et al.2009a), pathogens detection (Yang et al. 2002,) and studiescomparing the effectiveness of treatments on pathogens re-duction (Máximo et al. 2009). However, in most experimen-tal protocols, immediate sample processing is not possible,and storage conditions of samples until laboratory processingmay cause DNA degradation mostly due to temperature var-iation and (or) contamination (Smith and Burgoyne 2004).The loss of DNA integrity and quality may affect the chemi-luminescent signals provided by the hybridization reaction,producing incorrect test outcomes. In this study, we evaluated

Fig. 1. Comparison of sample storage time on the mean counts(×105) of individual species in subgingival sulcus (*, p < 0.01;**, p < 0.001; different letters mean significant differences amonggroups, p < 0.01).

Fig. 2. Comparison of sample storage time on the mean counts(×105) for the pool of 13 bacterial species (different letters meansignificant differences between groups, p < 0.01).

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the effect of sample storage time on the intensity of hybrid-ization signals from 13 bacterial species detected by whole-genomic probes in the Checkerboard DNA–DNA hybridiza-tion method. Our findings suggest that the period of time thesample remains in storage has an important impact on the fi-nal results of hybridization signals, when storage temperatureis kept constant at –20 °C. Overall, chemiluminescent hybrid-ization signals from bacterial DNA detection was signifi-cantly lower in samples that were processed after 12 or24 months of storage at –20 °C than in samples processedimmediately after collection or stored at –20 °C for 6 months.This reduction was observed when species were evaluated in-dividually as well as when considered as a pool of bacteria.Some possibilities have been reported as the potential cause(s)of DNA loss, such as nuclease activity, oxidation, UV dam-age, and microbial or fungal degradation (Smith andBurgoyne 2004). If DNA is stored at room temperature foran extended period of time, proteolytic activities caused bybacterial or fungal actions could result in DNA alterations.Oral microbiota represent one of the largest collections ofpathogen and nonpathogen species. Kruszewska et al.(2004) have shown that several bacterial species can de-grade exogenous DNA to nucleic bases or their derivates.Moreover, bacterial diversity in the samples can changeover time, as was reported by Roesch et al. (2009) whofound a 10% change in bacterial community between sam-ples stored at room temperature and those frozen at–80 °C. Storage of DNA samples in frozen solution mayprevent material degradation caused by nucleases or en-zymes released after microbial lyses, although the preserva-tion by freezing may also be detrimental for sample quality.The repeated process of freezing–thawing may cause exten-sive fragmentation of DNA. In addition, this process can in-directly cause DNA degradation, probably due to a celllysis mechanism that exposes the genetic material to the en-vironment (Moncla et al. 1990; Vu et al. 1999; Rajendramet al. 2006). Our data are in agreement with the results re-ported by Katsoulis et al. (2005a, 2005b) who found moretotal bacterial DNA in samples processed immediately orafter 6 weeks of storage, although temperature variationduring storage time in the different groups in that studymay have affected the results.The impact of storage on the genetic material was also re-

ported by Nedel et al. (2009) who evaluated qualitatively theeffect of sample storage time before DNA extraction frombuccal cells. The authors observed more degraded DNA ingroups where samples were stored at room temperature or at4 °C for 72 hours than in those with samples evaluated im-mediately after collection. Ng et al. (2004) examined the im-pact of different storage conditions on the quality of genomicDNA from saliva samples and demonstrated that DNA canbe successfully extracted using PCR for at least 1 monthwhen stored at –70 °C. However, these methods use DNAfor identification as a result of amplification reactions. Con-versely, Checkerboard DNA–DNA hybridization is a quanti-tative method, and the preservation of total genetic materialis primordial for a correct analysis.Within the limitations of this study, it can be concluded

that the Checkerboard DNA–DNA hybridization method issuitable to detect hybridization signals from all groups eval-uated and that the intensity of hybridization signals decreases

significantly after long periods of sample storage at –20 °C.According to the protocols established in this study, samplesshould be processed immediately after collection to avoidfalse-negative results. Further studies are necessary to evalu-ate the impact of different temperatures and shorter storagetime protocols on Checkerboard DNA hybridization methods.

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