Gastric Fluid Sampling, DNA Extraction, and qPCR for H. pylori Detection and its Antibiotic Resistance

To begin, instruct the patient to fast overnight before the sample collection. The next day, open the string test kit. Take the capsule and hold the loop at the end of the string.

Tape the string onto the patient’s cheek. Then guide the patient to place the capsule on their tongue near the back of the throat and swallow it with a sip of water. After the capsule has been dissolved in the patient’s stomach for an hour instruct a trained assistant to carefully pull out the string from the patient.

Next place the end soaked in the gastric fluid in a TSE sample preservation solution and cut off the lower end of the string. Place the labeled specimen carrying collection tubes on a test tube rack and vortex for 10 seconds. Turn the 32 well plate upside down several times to resuspend the magnetic beads, vortex for 10 seconds.

Then carefully remove the aluminum foil ceiling from the plate. Transfer 200 microliters of gastric fluid from each sample and helicobacter pylori DNA as a positive control into the separate wells. Place the 32 well plate in the corresponding sample slot of the nucleic acid extractor machine for automatic DNA extraction, after DNA extraction and confirmation.

Place the excess gastric fluid and extracted DNA samples at minus 20 degrees Celsius until use, thaw the qPCR reaction mixture on ice and mix it by flicking and spinning to avoid reagent loss For each sample in a 32 well plate, mix 20 microliters of the PCR reaction mixture with urea forward and reverse primers, a urea probe and five microliters of the extracted DNA. Place the plate on the qPCR machine and set the thermal cycler program. Set the fluorescent signal acquisition to FAM the data acquisition, to the amplification extension period and start the run, upon completion of the reaction.

Save the data for analysis. Analyze the data with specialized software for qPCR as the instrument automatically selects baseline thresholds. If the test results for h.

pylori are positive replace the reagent kit with detection reagents for 23S rRNA gene and gyrA gene mutation in the h. pylori kit and start the drug resistance testing on the sample. This study shows the detection of h.

pylori and its antibiotic resistance in stomach fluid by qPCR. Among h. pylori positive samples.

S2 had CT values within the detection range indicating dual resistance to clarithromycin and Levofloxacin. The S3 CT values were within the detection for h. pylori infection and Levofloxacin resistance, but not for clarithromycin resistance, indicating levofloxacin resistance.

Similarly, S4 had detectable CT values for h. pylori infection and clarithromycin resistance but not for levofloxacin resistance suggesting clarithromycin resistance. The S5 had detectable CT values only for h.

pylori infection indicating sensitivity to both antibiotics and allowing treatment with either drug.