Comparison Of Real-time Fluorescent Quantitative PCR With Urine Flow Cytometry UF-1000i Bacteria Count

Objective:?1?Real-time fluorescence quantitative PCR?FQ-PCR?and urine flow cytometry UF-1000i two methods to compare the basic performance of the urine bacterial count to provide a reliable basis for rapid clinical diagnosis and treatment of urinary tract infection.Methods:?1?BacterialcountswereperformedonstandardstrainsATCC27853?Pseudomonas aeruginosa?and ATCC29212?Enterococcus faecalis?by UV-vis spectrophotometry and micropropagation.The correlation between the two methods and the relationship between absorbance and bacterial concentration were compared.?2?The bacterial DNA was extracted from the standard strains ATCC27853?Pseudomonas aeruginosa?and ATCC29212?Enterococcus faecalis?by boiling method,lye boiling pyrolysis method and chloroform extraction method,and amplified by FQ-PCR method.The amplified products were subjected to Agarose gel electrophoresis comparative analysis.?3?Bacterial suspensions?live and dead?of different concentrations were prepared with standard strains ATCC27853?Pseudomonas aeruginosa?and ATCC29212?Enterococcus faecalis?.FQ-PCR and bacterial culture were used to measure the effect of urinary flow cytometry UF-1000i urine bacterial count performance evaluation.Results:?1?There was a good linear relationship between the absorbance of Pseudomonas aeruginosa and Enterococcus faecalis?at the wavelength of 625nm?and Maxwell's turbidity in the range of 0.4-0.8 of Maxwell's turbidity.Pseudomonas aeruginosa,Enterococcus faecalis concentration of 10⁸CFU,the absorbance values at the wavelength of 625nm were 0.163 and 0.155 respectively,and that of the wheat was 0.5.?2?Three methods for the same effect on the extraction of Pseudomonas aeruginosa DNA;For Enterococcus faecalis,boiling amplification by PCR amplification no amplification products,chloroform extraction and lye boiling pyrolysis DNA extraction effect the same.?3?The linear ranges of UF-1000i for the two strains were 10³-10⁷CFU/ml,the Pseudomonas aeruginosa reproducibility CV was between 12.72%-19.46%,bacterial culture method was 25.1%,and the Enterococcus faecalis repeatability CV was between14.49%-15.57%,and bacterial culture method was 23.0%.UF-1000i and bacterial culture method for the determination of bacterial count no significant difference?p>0.05?;The linear range of FQ-PCR assay for both strains was between 10⁵-10¹¹CFU/ml.The reproducibility CV of Pseudomonas aeruginosa and Enterococcus faecalis were between11.38%-19.76%,12.86%-23.28%.There was no significant difference in bacterial counts between FQ-PCR andbacterial culture method?p>0.05?.To detection of dead bacteria,the accuracy of UF-1000i assay for detecting Pseudomonas aeruginosa and Enterococcus faecalis were 78.9%and 94.7%respectively.The accuracy of FQ-PCR for detecting Pseudomonas aeruginosa and Enterococcus faecalis were 81.5%and 98.4%.Bacteria culture method for the detection of two kinds of bacteria accuracy of 0%?can not cultivate dead bacteria?.Conclusion:?1?UV-visible spectrophotometry as Pseudomonas aeruginosa and Enterococcus faecalis fast counting method.?2?Boiling method,lye boiling pyrolysis method,chloroform extraction method can be extracted three Pseudomonas aeruginosa DNA;lye boiling pyrolysis method,chloroform extraction method can extract Enterococcus faecalis DNA,and lye boiling pyrolysis method Extraction of lower concentrations of bacteria DNA,boiling method can not extract Enterococcus faecalis DNA.?3?The performance of UF-1000i against Pseudomonas aeruginosa and Enterococcus faecalis was stable and its precision was higher than that of bacterial culture.The accuracy and FQ-PCR method were not significantly different between UF-1000i and Bacillus subtilis,and the dead bacteria could be counted accurately.