Paul,30 used two pairs of specific primers for Van A and Van B to screen clinical

isolates, and found it was more sensitive than culture methods. In our study, the sensitivity of PCR was the same as that of routine test. It seems that the quality and quantity of DNA, which is related to DNA extraction method, is critical to the higher sensitivity. Kariyama,31 used Multiplex-PCR with seven pairs of primers to screen many clinical isolates and standard strains, Inhibitors,research,lifescience,medical and found that it was simpler and more efficient than the routine method. It is also compatible with our study. False-positive cases is mainly the result of amplifying DNA of dead bacteria in the sample and amplifying resistance genes like Van A and Van B that are present in some other bacteria. This is critical for fecal samples that contain different bacteria, but not Inhibitors,research,lifescience,medical for blood ones.25 Regulating the concentrations of several primers in PCR mix is a technical problem for multiplex-PCR. Kariyama found that inhibition of Van A primers can be neutralized by increasing their concentrations to two-folds.31 Angeletti,29 and Stake,18 performed the

multiplex-PCR in two steps, and in one step they only used Van A primers. We used one step and the same concentrations of primers, which seems to be the cause of weak view of bands (figure 3). The other cause of false-positivity is the specificity of primers. Primers designed by Ke,28 Inhibitors,research,lifescience,medical from Inhibitors,research,lifescience,medical tuf gene could amplify two species of Abiotrophia and four species of Lysteria. However, they are not the usual causes of bacteremia and this problem is important for fecal screening. It has been recommended to use molecular typing methods such as RFLP on PCR product,26 or very specific primers for E. faecalis,19,27 for characterization. The present study used the latter method (figure 2). It has been recommended to use genus specific or universal primers as the internal LY2835219 solubility dmso control for detecting false-negative cases.27 However, we used species specific primers Inhibitors,research,lifescience,medical to diagnose E. Faecalis and as an internal control (figure 2). One of the main technical problems in the diagnosis of bacteremia by PCR is the obtainment of high quality and quantity

bacterial DNA from the whole blood. This may not be easily possible because of high contents of PCR inhibitors. Therefore, DNA extraction method is critical.25,32 Zang,33 used Quiagen kit, and detected five cfu/ml bacteria in the blood.33 Newcomb,34 used Boom method and Klausegger,35 used DNA ZOl buffer for lysis bacteria in Montelukast Sodium blood. However, Anthony,36 used double distilled H20 for lysis blood cells and boiling for extraction bacterial DNA. Rothman,37 recommended initial enrichment process of blood sample in TSB before DNA extraction. We used sterile double distilled H2O and red cell lysis buffer for lysis blood cell, proteinase K for eliminating PCR inhibitors, phenol-cholroform and alcohol precipitation to extract DNA of bacteria in blood, however, the quality of extracted DNAs was not very good.