Quantitative and bioluminescent assay to measure efficacy of conventional and DNA vaccinations against Helicobacter pylori

Vaccination against Helicobacter pylori using DNA sequences encoding Urease A and B subunits was compared to immunization with urease antigen and MTP- PE in a liposome formulation. To determine the effectiveness of a vaccine a gainst H. pylori in a mouse model it is essential to quantify the number of H. pylori remaining in the stomachs following challenge with an inoculum o f live bacteria. Culture assays and enzymatic assays produce inconsistent r esults often unsuitable to conclude if vaccine candidates are protective. T o overcome this problem, we developed two assays: 1) a competitive quantita tive PCR using a colorimetric readout and 2) a non-competitive direct quant itative PCR using a highly sensitive bioluminescent readout. The competitiv e PCR requires coamplification of a segment of the urease C sequence and an internal control standard in a competitive manner using a single set of pr imers. PCR products were quantified colorimetrically by an enzyme-linked im munosorbent assay and compared with known quantities of the internal contro l standard added to the PCR reaction. The highly sensitive, bioluminescent assay measures the amplified DNA directly using a flash-type luminescent ta g and a specific probe. The Sydney strain of H. pylori was used for the mou se infection model. Quantification of H. pylori by either the bioluminescen t assay or the competitive PCR was reliable, specific and sensitive compare d to quantitative growth assays which often gave false results. The biolumi nescent assay was much more sensitive and less labor/time intensive than th e competitive PCR. The bioluminescent assay was able to quantitate as few a s 100 bacteria, while the competitive assay could not detect less than 10(3 ) bacteria per mouse stomach. Quantification of H. pylori by bioluminescent assay was superior to the competitive assay and may be used for research a pplications, such as the development of vaccines, pathogenesis of gastric d isease and monitoring of antibiotic treatment.