Detection of Helicobacter pylori Infection and Antibiotic Resistance via Stool Quantitative Polymerase Chain Reaction Analysis

This study demonstrates a qPCR-based fecal test to detect helicobacter pylori infection and antibiotic resistance, enabling personalized treatment and improving H. pylori eradication rate at the population level.

Fecal samples combined with quantity PCR enabled delayed detection of helicobacter pylori infections and antibiotic resistance without bacterial culture, reducing diagnosis done from three weeks to one day.

[Narrator] To begin, invert the specimen preservation tube and mix thoroughly. Then transfer one milliliter of the fecal specimen solution into a microcentrifuge tube. Mix the centrifuge tubes by inversion. Place the tubes into a metal bath set at 80 degrees Celsius for 10 minutes, and mix intermittently for 30 seconds at the fifth minute. After cooling the tubes to room temperature, centrifuge them at 10,000 G for five minutes. Collect the supernatant for further processing. Take a 96-well plate containing lysis buffer and invert it several times to resuspend the magnetic beads. Carefully remove the aluminum foil seal without shaking the plate to prevent spillage. Add 200 microliters of the prepared sample into each well of the 96-well plate, ensuring each well corresponds to a single sample. Place the 96-well plate into the designated sample compartment of the nucleic acid extraction instrument for automated extraction. Store any remaining specimens and the extracted nucleic acid samples at minus 20 degrees Celsius for long-term preservation and future use. Centrifuge the tube containing the lyophilized reagent to ensure the lyophilized powder settles at the bottom of the tubes. Open the lid of the reagent tube carefully to avoid spilling the powder. Add 25 microliters of the extracted nucleic acids from the samples to be tested. Positive control and negative control to each well. Close the tubes tightly. Vortex the qPCR reagents for eight to 10 seconds. Then centrifuge them briefly for three to five seconds to avoid bubble formation. Place the 96-well qPCR plate into the qPCR machine. Set the cycling program as shown on the screen. Then set the fluorescence detection parameters as shown. After saving the data, analyze it using qPCR-specific software as the instrument automatically selects the baseline threshold. Set all diagnostic criteria, including the presence of Helicobacter pylori infection or resistance to a CT value of less than or equal to 30. Confirm that they exhibit a characteristic S-shaped curve. The quality qPCR control results of negative and positive controls are shown in this figure. The results confirmed no amplification in negative controls and clear amplification curves in all detection channels in positive controls, validating assay reliability. This image depicts the amplification curves from five fecal samples analyzed by qPCR to detect Helicobacter pylori infection and its resistance to clarithromycin and quinolones using color-coded fluorescent probes. Sample S1 showed no significant amplification in any detection channel, confirming the absence of Helicobacter pylori infection. Sample S2 showed amplification only in the ROX channel, indicating the presence of Helicobacter pylori without antibiotic resistance. Sample S3 showed amplification in the ROX and HEX channels, but not in FAM, indicating quinolone resistance. Sample S4 showed amplification in the ROX and FAM channels, but not in HEX, indicating clarithromycin resistance. Sample S5 showed amplification in all three channels, indicating dual resistance to clarithromycin and quinolones.