Helicobacter pylori and Chlamydia pneumoniae in root canals means of a #15 K-type file with the handle cut off. and negative controls consisting of sterile ultrapure The file was introduced to a level approximately water instead of sample were included with each 1mm short of the root apex, based on diagnostic batch of samples analyzed. DNA was amplified us- radiographs, and a gentle filing motion was ap- ing a DNA thermocycler (Mastercycler personal, plied. Afterwards, two sequential paper points were Eppendorff, Hamburg, Germany). placed to the same level and used to soak up the PCR products were subjected to electrophoresis fluid in the canal. Each paper point was retained in in a 1.5% agarose gel–Tris-borate-EDTA buffer. The position for 1 min. The cut file and the two paper gel was stained with GelRed (Biotium, Hayward, CA, points were transferred to cryotubes containing USA) and visualized under UV illumination. A 100- 1 ml of Tris-EDTA buffer (10 mM Tris-HCl, 1 mM bp DNA ladder (New England Biolabs, Beverly, MA) EDTA, pH 7.6) and immediately frozen at –20o C. was used as a molecular size standard. Abscesses were sampled by aspiration of puru- lent exudate from the swollen mucosa using a ster- rEsuL ts ile syringe. The overlying mucosa was disinfected All genomic extracts from clinical samples ex- with 2% chlorhexidine, and a sterile disposable sy- hibited the expected amplicon after PCR amplifica- ringe was used to aspirate pus, which was immedi- tion with broad-range 16S rRNA gene primers. This ately injected into cryotubes containing Tris-EDTA indicates that the DNA extraction and amplification buffer. Pus samples were then immediately frozen. procedures were adequate, bacteria were present DNA was extracted using the QIAamp DNA Mini in all examined samples, and significant PCR inhib- Kit (Qiagen, Valencia, CA, USA) according to the itors were not present. No PCR products were ob- manufacturer’s instructions. DNA extracted from H. served in negative controls using sterile ultrapure pylori ATCC 43629 and C. pneumoniae ATCC VR1310 water instead of sample. Positive controls yielded served as positive controls. the amplicons of predicted sizes for each tested DNA extracts from clinical samples and positive species. No clinical sample was positive for either controls were subjected to multiple displacement H. pylori or C. pneumoniae. amplification (MDA) by using the Illustra Genomi- Phi V2 DNA Amplification kit (GE Healthcare, Pis- dIscuIon cataway, NJ, USA) following the manufacturer´s To increase the sensitivity of the method, the instructions. A negative control with sterile ultra- present study made use of nonspecific whole ge- pure water instead of sample was included in ev- nomic amplification by means of MDA so as to gen- ery batch of MDA. This MDA step was carried out erate large quantity of assay-ready DNA. MDA is of to achieve a better performance of the subsequent special value in experiments involving samples with PCR assays, particularly for samples with low num- low amount of DNA and has been used as a pre- ber of bacteria. PCR strategy for the detection of low copy number In order to check if bacterial DNA was available sequences.27-28 This can be the case for endodontic for analysis after extraction, aliquots of the ampli- infections, because of the recognized limitations of fied DNA extracts from clinical samples were eval- root canal sampling techniques.29-30 uated by polymerase chain reaction (PCR) using a In the present study, endodontic samples were broad-range 16S rRNA gene primer set. taken from healthy patients with no informed gas- Primers and PCR cycling conditions used for tric infection and all samples were negative for H. broad-range analysis or specific detection of H. pylori. Although the oral cavity has been suggest- pylori and C. pneumoniae were as described previ- ed to be a reservoir for H. pylori, there appears to ously.11,22,26 PCR amplifications were performed in a be no consensus in this regard. Prevalence of H. 50 µl of reaction mixture containing 5 µl of DNA ex- pylori in the oral cavity has been shown to range tract, 0.8 µM of each primer, 5 µl of 10× PCR buffer from 0% 31 to 97%.10 Specifically, detection of H. py- (Fermentas, Burlington, Canada), 2 mM MgCl2, 1.3U lori in subgingival biofilms has also been demon- of Taq DNA polymerase (Fermentas) and 0.2 mM of strated to vary, with studies showing absence32 and each deoxyribonucleoside triphosphate (dNTPs) others finding this species in about 25-40% of the (Biotools, Madrid, Spain). Positive control compris- samples.7-9,11 A study concluded that the oral cavity ing DNA extracted from H. pylori and C. pneumoniae may not be a reservoir for H. pylori in patients with 160 European Journal of Dentistry