Abstract:
Objective To establish a digital PCR system for accurate and sensitive quantitative detection of Helicobacter pylori (Hp).
Methods Specific primer and probe were designed based on the Hp 16S rRNA sequence, and reference bacteria was used to conduct specificity testing. The sensitivity was detected using gradient diluted DNA as templates. The accuracy was detected using different concentration templates with multiple repeated tests. The gradient method was used to set the primer concentration and annealing temperature for optimizing reaction conditions.
Results The designed primers and probes could detect Hp 16S rRNA genes specifically without the interference from Escherichia coli and other bacteria. The optimal reaction temperature for the detection was 57.1 ℃, and the optimal primer concentration was 550 nmol/L. The detection limit was 0.35 copies/μL, the coefficient of variation (CV) of the detection was < 10%, and the coefficient of determination for the linear regression between dilution ratio and DNA concentration was 0.9968.
Conclusion The established digital PCR system is specific, sensitive, and highly accurate for Hp detection.
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