ENDODONTOLOGY. Introduction. Original Research ABSTRACT. crucial for a wide variety of molecular biology applications.

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1 Original Research Quality control using Nano Drop 1000 in PCR molecular diagnostic method to measure Enterococcus faecalis from Secondary Endodontic Infections Moksha Nayak # Subbannayya Kotigadde ## Nithya Pushpanathan # Harish Shetty K. # ABSTRACT Aim: To evaluate the DNA quality using Nano Drop 1000 spectrophotometer in samples from root canal treated teeth with apical periodontitis prior to species- or genus-specific PCR procedure. Materials and Methods: Samples from 40 root canal treated teeth with apical periodontitis were collected and subjected to two groups, Group I(n=40): samples subjected to PCR amplification, Group II(n=40): samples subjected to Nano Drop followed by PCR amplification. Statistical analysis was done using Chi square test. Results: E. faecalis was detected in 70% (28/40)teeth in group 2 and 50% (20/40) in group 1. There was significant difference in the quality of DNA obtained in group 1 when compared to group 2(p<0.01). More number of clear bands were visible in group 2,and higher number of blurred bands (12/40)in group 1. Conclusion: The Nano Drop 1000 technique proved to be beneficial in the measurement of both quantity and purity of DNA in the samples. This technique not only designs better experiments but also ensures better reporting and significant savings in time and money spent for re-running samples. Abbreviations: ng - Nanogram, rpm - Rotations per minute, ml - Milliliter, µl - Microliter, mm - Millimeter, QC - Quality control, nm - Nanometer, mm - Millimolar. Introduction Mullis and colleagues in 1987 were the first to use the PCR in molecular biology;since then it has become one of the most powerful and widely used techniques in this field. Recently, molecular methods have been used to investigate the microbiota of endodontic infections 1. Molecular biology studies have confirmed the status of E. faecalis as the most frequently found species in previously filled root canals that have failed 2,3. Proper PCR amplification of the bacterial 16S or 23S rrna gene (rdna) is more sensitive and more efficient than culturing and biochemical identification of endodontic flora 4. Correct quantification of small amounts of DNA is very crucial for a wide variety of molecular biology applications. Although the use of DNA probes can be more sensitive and more efficient than culturing, it still requires the presence of >10 4 bacterial cells to ensure detection 5. The amount of genomic DNA required for PCR ranges, in general, from 50 to 500 ng, with each amplification requiring fresh DNA as template 6. By implementing the latest technology such as the NanoDrop 1000 spectrophotometer, measurement of DNA, RNA and protein concentration, can be done while also determining sample purity in accordance with A260/A280 and A260/A230 ratios. # Dept. of Conservative Dentistry & Endodontics, ## Dept. of Microbiology, KVG Dental College and Hospital, Sullia, Yenepoya Dental College, Mangalore 100

2 QUALITY CONTROL USING NANO DROP 1000 IN PCR MOLECULAR DIAGNOSTIC METHOD TO MEASURE ENTEROCOCCUS FAECALIS FROM SECONDARY ENDODONTIC INFECTIONS Therefore, the purpose of this study was to determine the presence of Enterococcus faecalis in samples from root canal treated teeth with apical periodontitis by using species- or genus-specific PCRprimers and to check the DNA quality using Nano Drop 1000 spectrophotometer. Materials and Methods The study population consisted of 40 patients presenting at the Endodontic Clinical Section of the Department of Conservative Dentistry & Endodontics, K.V.G Dental College, Karnataka, India, for endodontic retreatment. Medical histories revealed that all patients were in good general health. Patients who had received antibiotic therapy during the last two months before root canal therapy, third molars, lesions with periodontal pocket probing greater than 4 mm were excluded from the study. Termini of the root canal filling are at least 2mm short of the radiographic apex. The patient ages ranged from 25 to 75 years. Ethical clearance was obtained from the institutional ethical committee and written informed consent was obtained from each patient before inclusion in the study. Samples from 40 root canal treated teeth with apical periodontitis were collected and evaluation of E.faecalis was done by PCR techniques (Group I,n=40) and Nano Drop followed by PCR amplification (Group II, n=40). Clinical Sampling After rubber dam isolation, the operative field was sterilized with 30% hydrogen peroxide and 2.5% sodium hypochlorite solution. These medicaments were inactivated by using 5% sodium thiosulphate solution. Access cavity preparations were made using sterile burs with sterile water spray. Gutta-percha was preliminary removed without chemical solvents with the use of # 4, 3 and 2 Gates Glidden burs (Dentsply-Maillefer, Ballaigues, CH) and # 10-15K-files. To obtain microbial samples, two or more paper points (ADA products-mynol, Milwaukee, WI, USA) were placed into the root canal and retained inside for 40 seconds. The paper points were then immediately transferred to sterile 5ml tubes (Eppendorf AG, Hamburg, Germany) containing RTF transport medium. Samples were frozen immediately at - 20 C and stored up to one-two months until assayed by PCR.. The tube was vortexed for 60s.The Eppendorf tube were transferred on dry ice to the Neurobiology Laboratory, National Centre for Biological Sciences (Tata Institute of Fundamental Research, Bangalore) Nano Drop and PCR analysis. Pre Operative Radiograph Removal of old filling and Working length Samples in transport media Isolation & swabbing with H 2 O 2 and NaOCL Root canal sampling 101

3 MOKSHA NAYAK, SUBBANNAYYA KOTIGADDE, NITHYA PUSHPANATHAN, HARISH SHETTY K. DNA extraction DNA extraction was done for all the 40 samples in both group 1 and group 2.The vials with paper point specimens were vortexed for 2 min to disperse microbial cellular material into suspension. The suspension was removed from the original vial and transferred to 2-ml sterile vials, which were then centrifuged at 3000 rpm (all centrifuge procedures were carried out with Eppendorf [Westbury, N.Y.] scientific microcentrifuge model 5417C) for 10 min, and the supernatant was again removed. DNAs were extracted from the cellular pellet by one of two methods. The pellets were resuspended in 100µl 1x PCR buffer and heated for 10mins for 114ºC in a dry heater and chilled at 4ºC for 5 minutes. The mixture was then centrifuged at 13,000 rpm for 4 to 5 minutes. The solutions were collected and frozen at -20 C until use. Reference DNA was extracted and used as positive control. After DNA extraction, the samples in group 1 were subjected to PCR amplification, whereas samples in group 2 were then quantified on the NanoDrop 1000 to ensure adequate template prior to amplification. The Nano Drop Spectrophotometer By using fiber optic technology and the inherent surface tension properties of liquid samples to hold a droplet of sample in place between two optical surfaces during the measurement cycle, 1ul of sample is pipetted directly onto the lower optical (measurement) surface. The upper optical surface is automatically engaged to the sample to form a liquid column of mechanically-controlled path length. Once the measurement is complete, both the optical surfaces are cleaned with a standard laboratory wipe to prepare for the next sample. Cleaning the Sample Retention System 2µl water aliquots is used to clean the measurement surfaces after particularly high concentration samples to ensure no residual sample is retained on either pedestal. A final cleaning of all surfaces with de-ionized water is done after the last measurement. The optical density (OD) is measured at different wave lengths: 230 nm (absorption of contaminants & background absorption), 260 nm (absorption maxima of nucleic acids), 280 nm (absorption maxima of proteins), and 320 nm (absorption of contaminants & background absorption). The OD260/280 ratio is used as indicator for DNA purity. A ratio higher than 1.8 is assumed suitable for gene expression measurements. The OD260/230 and the OD260/230 should be maximized as these represent the degree of background absorption and contaminants.the DNA concentrations were obtained graphically. Extracted DNA The Nano Drop Spectrophotometer PCR Amplification Procedures Amplification procedures were performed in group 1 and group 2 Aliquots of each sample (1.0 ml) were centrifuged at 13,000 g for 10 minutes. The 102

4 QUALITY CONTROL USING NANO DROP 1000 IN PCR MOLECULAR DIAGNOSTIC METHOD TO MEASURE ENTEROCOCCUS FAECALIS FROM SECONDARY ENDODONTIC INFECTIONS Graphical representation of Nano drop analysis Primers resulting pellets were washed with 500 µl of phosphate-buffered saline, and placed in 200 µl of TE buffer (10mM Tris-Cl ph 7.5, 1 mm EDTA). The DNA concentrations in clinical samples and the concentrations of the reference DNA were determined by the Nano Drop 1000 spectropho2tometric measurement of the absorbance at 260 nm, which is an optimal solution for application of very small volumes. Serial 10-fold dilutions of known concentration of reference DNA of the target species were processed to determine PCR assay sensitivity. The lowest DNA concentration that resulted in a positive PCR product was regarded as indicative of the sensitivity of the assay. Primer specificity was further tested against reference. Insufficient amplification can result if the initial amount of template is too low, resulting in no bands, blurred or thin bands. PCR primers, with expected amplicon size and thermocycling parameters used in the present study are shown in Table 1. The PCR reaction used to assess the occurrence of all target taxa, was performed in 50µl of reaction mixture containing 10 µl DNA, 5µl 10x PCR buffer, 2 mm MgCl2, 1.25 µltaq DNA polymerase, 0.2 mmdntp, 1µM specific primer. Negative controls consisting of ultrapure water instead of sample were included with each batch of samples analyzed. DNA amplification was performed in a thermal cycler Gene Amp PCR system (Applied Biosystems). Amplicons were stored at 20 o C. The amplification products were analyzed through the use of electrophoresis in a 1.5% agarose gel conducted at 4V/cm in Tris-borate EDTA buffer. The gels were stained with 0.5µg/ml ethidium bromide and the PCR products were visualized under 300 nm ultraviolet light. GeneRuler DNALadder Mix (Fermentas GmbH, Germany) served as the molecular weight marker. The identity of each band was determined by visual comparison with a molecular weight ladder. Reactions were deemed positive in the presence of bands of the appropriate size. A successful amplification for E.faecalis reaction Primer pair Sequence (5 to 3)a Size (bp) Annealing temp ( C) or organism Enterococcus TAC TGA CAA ACC ATT CAT GAT G (forward primer) spp. AAC TTC GTC ACC AAC GCG AAC (reverse primer) 103

5 MOKSHA NAYAK, SUBBANNAYYA KOTIGADDE, NITHYA PUSHPANATHAN, HARISH SHETTY K. should yield a readily visible DNA fragment corresponding to sizes of 320bp to 420bp. If all is well, lanes of the gel containing samples of the positive control and the template DNA under test should contain a prominent band of DNA of the appropriate molecular weight. This band should be absent from the lanes containing samples of the negative control.the results were categorized as amplification products obtained with and without photometric analysis and compared using Chi square test. GEL PICTURE OBTAINED IN GROUP 1 RESULTS OBTAINED IN GROUP1 Results Obtained Without Nano Drop Analysis (n=40) Clear Bands Blurred Imaged No bands Presence of E.faecalis % RESULTS OBTAINED IN GROUP2 Results Obtained after Nano Drop Analysis(n=40) Clear Bands Blurred Imaged No bands Presence of E.faecalis % Chi square test showed significance of p<0.01 The DNA Concentrations obtained using Nano Drop 1000 Spectrophotometer ranged from67.4 to Gel picture(presence of blurred bands) obtained without the use of Nano Drop spectrophotometer GEL PICTURE OBTAINED IN GROUP 2 Figure 3: Gel picture obtained after using the Nano Drop 1000 spectrophotometer Results The presence of E. faecalis was evaluated by 16S r DNA gene-based PCR in these 40 samples. Out of which E. faecalis was detected in 28teeth (70%). As DNA quality consists of DNA integrity and DNA purity, also the OD260/280 ratio and the OD260/230 ratio have been checked photo metrically to ensure good DNA purity. The results obtained in group 1 and group 2 was statistically significant (P<.01). Discussion In the present study the prevalence of Enterococcus faecalis was 70% using PCR with Nano spectrophotometer and 50% in PCR without Nano drop. The high prevalence of E.faecalis in root filled teeth is in accordance with previous studies. Siqueira and Rocas detected E.faecalis in 77% of the root filled teeth from Brazilian patients and 64% from South Korean population, Pecuiliene et al demonstrated that E.faecalis is the dominant species in retreatment cases of apical periodontitis in Lithuanian patients that is 71% 5. This minor discrepancy can be related to differences in methodologies but may have occurred as a result of geographical influence in the composition of the root canal microbiota 6. The presence of E.faecalis is notable because it possesses certain virulence factors including lytic enzymes, cytolysin, aggregation substance, pheromones and lipoteichoic acid. E.faecalis is able to suppress the action of lymphocytes, potentially contributing to endodontic failure. It overcomes the challenges of survival within the root canal system in several ways. It is postulated that a virulence 104

6 QUALITY CONTROL USING NANO DROP 1000 IN PCR MOLECULAR DIAGNOSTIC METHOD TO MEASURE ENTEROCOCCUS FAECALIS FROM SECONDARY ENDODONTIC INFECTIONS factor of Enterococcus faecalis in failed endodontically treated teeth may be related to the ability of Enterococcus faecalis cells to maintain the capability to invade dentinal tubules and adhere to collagen in the presence of human serum (Love et al 2001). It has been shown to adhere to host cells, express proteins that allow it to compete with other bacterial cells, and alter host responses 7. The 30% blurred bands in group 1 signify the presence of contaminants in DNA. By using this method, pure DNA samples were obtained showing clear bands in 70% of samples in group 2, as the NanoDrop 1000 spectrophotometer proved to be ideal to assess the quantity and purity of the DNA prior to amplification, whereas group 1 showed 50% clear bands and 30% blurred bands, due to presence of contaminants. False positive results have the potential to occur because of PCR amplification of contaminant DNA. The most important means of contamination is through carryover of amplification product and special precautions should be taken to avoid this. False negatives may occur because of enzyme inhibitors or nucleases present in clinical samples, which may abort the amplification reaction and degrade the DNA template, respectively. Analysis of small sample volumes may also lead to false negative results, particularly if the target species is present in low numbers 1. The spectrophotometers are able to determine nucleic acid concentration and generate full absorbance spectral data. The spectral data provided can offer analysts additional information regarding the presence of potential chemical contaminants such as phenol, glycogen, guanidine, and Ethylenediaminetetraacetic (EDTA), that may be introduced by extraction procedures and have the potential to inhibit downstream applications. Advantages of Nano Drop 1000 spectrophotometer PCR methods have confirmed the presence of E.faecalis from failed root filled teeth. The most successful PCR results are achieved when the amplification reaction is performed using purified primers and templates that are essentially free of extraneous salts. When DNA of known concentration is available, amounts of ng of DNA template/100 µl reaction are typically used for amplifications of single-copy chromosomal targets 8. Therefore, it is essential that effective quality control (QC) measures for each molecular technique are used to minimize failure of downstream steps in the workflow. The study design allowed the analysis of DNA integrity and purity using Nano Drop 1000 with regard to detection of E. faecalis in root-filled teeth. The OD260/280 ratio and the OD260/230 ratio have been checked photometrically to ensure good DNA purity. DNA quantification is the key to good molecular biology results, and failure to produce high-quality evidence can sometimes be directly attributed to an incorrect estimate of the concentration of DNA template used. It is timeconsuming and unwarranted to apply complicated 105

7 MOKSHA NAYAK, SUBBANNAYYA KOTIGADDE, NITHYA PUSHPANATHAN, HARISH SHETTY K. molecular biology methods to DNA that has not been properly quantified. The latest microvolume quantification Nano Drop 1000 can be implemented as a routine QC step to minimise consumption of precious samples and provide fast assessment of nucleic acid concentration and purity. It enables the analysis of sample volumes as small as µl, with a dynamic range of 2 15,000 ng / µl for nucleic acids and without the need for a cuvette or dilutions 9. In conclusion, it could be shown that the Nano Drop 1000 technique proved to be beneficial in the measurement of both quantity and purity of DNA in the samples. DNA analysis using molecular diagnostic methods is influenced by the overall total DNA integrity. This technique not only designs better experiments but also ensures better reporting. Taking the bench to the clinic is a crucial aim in today s biomedical environment. However, the effective translation of molecular techniques from the laboratory to a clinical setting presents significant challenges, with assays requiring extensive optimization before results can be interpreted correctly in the clinic. By implementing these processes, patients will be provided with improved screening, and treatment of various disease conditions. References: 1.Siqueira J. F, Jr,, and Roˆc as I. N, Exploiting Molecular Methods to Explore Endodontic Infections: Part 1 Current Molecular Technologies for Microbiological Diagnosis., J Endod 2005;31: SiqueiraJr F J. and. Rôças I J, Polymerase chain reactionbased analysis of microorganisms associated with failed endodontic treatment, Oral Surg Oral Med Oral Pathol Oral RadiolEndod 2004; 97: Rolph HJ, Lennon A, Riggio MP et al. Molecular identification of microorganisms from endodontic infections. J ClinMicrobiol 2001; 39: Siqueira, J. F., Jr., Rocas I. N, Souto R, Uzeda M, and Colombo A.P. Checkerboard DNA-DNA hybridization analysis of endodontic infections. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod 2000; 89: Sheikh S.N and Lazarus P. Re-usable DNA template for the polymerase chain reaction (PCR). Nucleic Acids Research, 1997, Vol. 25, No Sundqvist G, Figdor D, Persson S, Sjögren U. Microbiologic analysis of teeth with failed endodontic treatment and the outcome of conservative retreatment. Oral Surg Oral Med Oral Pathol 1998; 85: Siqueira JF, Jr., Jung IY, Rôças IN, Lee CY. Differences in prevalence of selected bacterial species in primary endodontic infections from two distinct geographic locations. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2005; 99: Hubble T.S., Hatton J.F., Nallapareddy S.R., Murray B.E., Gillespie M.J. Influence of Enterococcus faecalis proteases and the collagen-binding protein, Ace, on adhesion to dentin. Oral Microbiol Immunol.2003; 18, Fouad F.A et al. PCR-Based Identification of Bacteria Associated with Endodontic Infections. J ClinMicrobiol 2002;40, Curet I.G. Molecular Diagnostics: Critical Issues for Successful Translation from the Bench to the Clinic. Pharma Bio World. 2011;8, Becker C, Fickinger A H, Riedmaier I, Pfaffl M W. mrna and microrna quality control for RT-qPCR analysis. Methods 2010; 50, Cheng S, Fockler C, Barnes W M, Higuchi R. Effective amplification of long targets from cloned inserts and human genomic DNA. Proc. Natl Acad. Sci. 1994;91: Portenier I, Waltimo T M T and Haapasalo M, Enterococcus faecalis- the root canal survivor and star in post treatment disease, Endod Topics 2003; 6: Fouad A F, Zerella J, Barry J, BA, MT(ASCP), and Spangberg L S, Molecular detection of Enterococcus species in root canals of therapy-resistant endodontic infections, Oral Surg Oral Med Oral Pathol Oral RadiolEndod 2005;99: Williams J M, Trope M, Caplen D J and Shugars D C, Detection and Quantitation of E.faecalis by Real-time PCR (qpcr), Reverse Transcription-PCR (RT-PCR), and Cultivation during Endodontic treatment, J Endod 2006;32: