Urinary tract infection has been shown to be quite complicated and often difficult to diagnose and treat. For appropriate diagnosis, it is very important to find the correct Gram
stain classification as soon as possible, especially in severe cases where there is a possibility of
severe sepsis developing. In order to solve this problem, we developed a new method to detect a Gram
stain of bacteria obtained from 1 ml of urine from
urinary tract infection patients using a consensus real-time PCR protocol with a TaqMan probe that allows detection of spiked bacterial 16S
DNA from urine. We extracted
DNA of 55 urine samples obtained from patients with complicated
urinary tract infection and at the same time performed urine culture testing. After
DNA extraction, they were subjected to real-time PCR using a TaqMan discrimination system. Sixteen kinds of bacteria were cultured from the urine culture testing. Of these bacteria, eight were classified as Gram-positive bacteria and the other eight were classified as Gram-negative bacteria. Of the 55 samples, the TaqMan technique result showed 27 samples that were classified as Gram-negative bacteria; 11 samples that were Gram-positive, 10 that included both Gram-negative and -positive bacteria, and 7 that showed no amplification. The classifications of all samples corresponded exactly to those determined by urine culture testing. The present genotyping method of real-time PCR using a TaqMan discrimination system could be applied to the rapid detection of Gram-positive or -negative bacteria in urine of
urinary tract infection patients. This assay can differentiate those species tested, but whether the presence of other (untested) bacteria could lead to misinterpretation is unknown. For further investigation, it is important to test other (untested) bacteria in the near future.