This study use QPCR on gastric mucosa to quickly detect Helicobacter pylori, and its antibiotic resistance, offering a fast, accurate diagnostic tool with great clinical significance for treating this infection. It is critical to ensure quality contrast standards for QPCR. Consequently, the QPCR IC for detecting Helicobacter pylori as held to specific criteria, and each condition must be satisfied concurrently.
Early deviation invalidate test requiring rerun. We need a powerful and comprehensive diagnostic tool to detect Helicobacter pylori and drug resistance. We use gastric mucosa QPCR.
We use specific primers for this purpose. We believe future studies with larger patient groups will validated and realize the full potential of gastric mucosa QPCR, enhancing Helicobacter pylori diagnosis, monitoring and treatment. To begin, open the biopsy clamp valve and move it slowly to the dominant hand.
When the head of the forceps is in the field of vision, open the clamp flap and manipulate the endoscope at the gastric mucosa of the sampling site. Apply slight pressure and close the biopsy forceps to clamp the gastric mucosa tissue. Using pliers, extract the tissue samples and place them in the sampling tube containing preservation fluid.
Once all sites have been sampled, tighten the sampling tube cover and seal it to prevent drying. Label the tubes with a unique identification number on the outside. Submit the collected samples for testing as soon as possible, avoiding storage.
Adjust the temperature of a metal bath to 100 degrees Celsius in advance. If the sample is frozen, remove it, and allow it to reach room temperature. Mix the lysate thoroughly to suspend the aminodiacetic acid resin.
Take 100 microliters of lysate into a centrifuge tube with a filter element, and add gastric mucosa. Mix the contents using vortex oscillation. Place the centrifuge tube in the metal bath at 100 degrees Celsius for 10 minutes.
After heating, remove the tube and let it cool to room temperature. Centrifuge the sample at 9, 500G for five to 10 minutes, and carefully transfer the supernatant into another sterilized labeled centrifuge tube. If not proceeding to the next step immediately, temporarily store the sample at four degrees Celsius.
Remove the primer probe, mixed enzyme solution and detection buffer from the kit. Melt all components on ice, or at two to eight degrees Celsius. To mix the samples, gently shake them and perform a brief centrifugation at low speed.
Mix 12.5 microliters of detection buffer, seven microliters of primer probe, and 0.5 microliters of enzyme solution. Mix the contents in the tube and briefly centrifuge it. Dispense 20 microliters of the PCR reaction solution and add five microliters of nucleic acid into each PCR reaction well.
Insert the reaction tube into the fluorescent PCR thermocycler. Set the cycle parameters and run the program. Collect fluorescence signals at 58 degrees Celsius for the desired markers.
After the reaction, save the data and analyze it using specific QPCR software. The QPCR assays confirmed the presence of Helicobacter pylori in samples A, B, C, and E, while sample F tested negative. Sample A was susceptible to both antibiotics.
Sample B exhibited resistance to both clarithromycin and levofloxacin. Sample C showed resistance to clarithromycin, but remains susceptible to levofloxacin. Sample E was resistant to levofloxacin, but susceptible to clarithromycin.
