Establishment of Digital PCR Detection System for Helicobacter pylori ureC and 23S rDNA
This study aimed to establish the digital PCR system for detection of Helicobacter pylori(Hp)genes ureC and 23S rDNA,providing an reliable reference detection method for Hp diagnosis.Specific primers and probes were designed based on the Hp ureC and 23S rDNA sequences,and quality control bacteria was used to conduct specificity testing.The gradient method was used to set the primer concentrations and annealing temperatures to optimize the reaction conditions.The sensitivity was detected using gradient diluted DNA as templates.The accuracy was detected using different concentration templates with multiple repeated tests.The designed primers and probes could specifically detect Hp ureC and 23S rDNA without the interference from E.coli and other bacteria.The optimal reaction temperature for ureC and 23S rDNA was both 55.0℃,the optimal primer concentration was 550 and 650 nmol·L-1,the linear fitting degree R2 was 0.999 1 and 0.999 7,and the CV values of samples with different concentrations was less than 10%.The Hp digital PCR detection system for ureC and 23S rDNA established in this study has the advantages of high specificity,sensitivity,and repeatability,which can provide accurate and reliable technical support for Hp pathological detection and scientific research.
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