Detection of Helicobacter pylori in dental plaque using a DNA biosensor for noninvasive diagnosis

Abstract

Noninvasive diagnosis of Helicobacter pylori (H. pylori) infection is very attractive. This study investigated the single strand DNA (ssDNA) acquisition method from H. pylori in dental plaque, and the integration of our previously developed 43-mer H. pylori DNA biosensor with the obtained target ssDNA (tDNA). Dental plaque samples were collected from 34 patients/volunteers, whose gastric H. pylori infection statuses were tested with the ¹³C urea breath test (UBT). The samples were treated with colony polymerase chain reaction (PCR) to obtain double strand DNA (dsDNA) of 104 basepairs (bp) long. A blocker ssDNA was designed and used in thermal treatment of the dsDNA to release the 104-mer tDNA, which contains the 43-mer DNA sequence in the middle. PCR primers were designed, and the tDNA releasing and detection conditions with the biosensor were optimized. The limit of detection with the biosensor was 12 fM dsDNA. The dental plaque detection results correlated quite well with the UBT results, with a sensitivity of 100%, and specificity of 97%. These results indicate that the residence of H. pylori in dental plaque is highly associated with gastric H. pylori infection, and detection of dental plaque samples with our DNA biosensor is promisingly applicable in noninvasive diagnosis of H. pylori infection.

Scheme 1. Schematic illustrations of (A) the amplification and acquirement of target dsDNA from H. pylori and releasing of the single strand tDNA from the dsDNA, and (B) the construction of the electrochemical sandwich DNA biosensor and the tDNA detection with the biosensor.

Fig. 1. The agarose (3.5%) gel electrophoresis results of the colony PCR products from (A) the H. pylori culture, and (B) the E. coli culture. M is denoted for the DNA marker. Lane 1, lane 2 and lane 3 were the repetitive loadings from the PCR products. The gels were stained with GoldView I for observation.

Fig. 2. DPV curves of the DNA biosensors in detection of (a, dash line) 1 pM target dsDNA treated with 100 nM blocker, (b, dash line) 1 pM target dsDNA treated without the presence of blocker, (c, dash-dot line) 100 nM blocker, and (d, dotted line) blank PBS. Inset: the statistic peak current (I_p) of the corresponding DPV curves (n = 3 repetitive biosensor fabrications and detections). *** represents that the p value in the t test is less than 0.001.

Fig. 3. The DPV I_p values of the biosensor in detection of 1 pM target dsDNA, and blank samples, treated and captured at various conditions. (A) The blocker concentration was varied in the immobilization buffer containing 0.6 M NaCl, and the renaturation was at 30 °C for 15 min. (B) The NaCl concentration was varied in the immobilization buffer containing 0.1 μM blocker, and the renaturation was at 30 °C for 15 min. (C) The renaturation time at 30 °C was varied, and the immobilization buffer contained 0.1 μM blocker and 0.6 M NaCl. Insets: the corresponding ΔI_p values. Error bars represent the standard deviation for 3 repetitive biosensor fabrications and detections.

Fig. 4. The DPV response curves of the biosensor to the target dsDNA with the concentrations of (a) 0, (b) 3.28 × 10⁻¹⁴, (c) 6.55 × 10⁻¹⁴, (d) 3.28 × 10⁻¹³, (e) 6.55 × 10⁻¹³, and (f) 6.55 × 10⁻¹² M. Inset: the plot of the DPV I_p values versus the logarithm of the dsDNA concentration (C_dsDNA). Error bars represent the standard deviation for 3 repetitive and independent biosensors.

Fig. 5. (A) Representative DPV curves of the developed biosensors in detecting H. pylori DNA in dental plaque samples collected from (a) gastric H. pylori negative, and (b and c) gastric H. pylori positive patients/volunteers (classified based on the UBT results). (B) Correlations between the detection results using the developed biosensing method in this work, and the UBT measurement in hospital. The lines represent the H. pylori infection thresholds based on the tests. The circled spot represents the inconsistent testing result between the two methods.