A drawback is that amplicons have to be identified on agarose gels. We have simplified and quickened the Carattoli PCR by the incorporation of fluorescent dye SYBR-green in a real time PCR. This dye intercalates in the amplicons during the PCR, and is thereby quenched.
It is released from the amplicons at specific melting temperature points. Upon release, quenching is Tariquidar order abolished and fluorescence can be measured. The use of this dye eliminates the need to detect the amplicons by agarose gel electrophoresis, which means that a time-consuming step is eliminated. Furthermore, since it is not necessary to open the PCR vials for analysis, the risk of contamination by other PCR replicons is decreased. Another advantage of the method we present here is that it is possible to use crude cell lysates in the PCR, with no need to purify plasmid DNA, which is also time and cost saving. The use of crude cell lysates has been described
in previous studies and has been shown to provide solid data [15, 16]. A third benefit of real time PCR with SYBR-green is its high analytical sensitivity. This is desirable because plasmids can be low-copy-number plasmids and because plasmid numbers vary per bacterial cell and SC79 growth phase . In 2011 for instance, Waltner-Toews et al. described a wild-type TEM-1-carrying strain, where the plasmid occurred at an average of 3.5 learn more to 4.1 copies per cell . We have shown that we can detect replicons in samples containing as little as 50 fg of DNA (50•10-15 g), hence even low-copy-number plasmids can be detected. The dry weight of the average E. coli genome of 5 mBp is approximately 5 fg, which means that in theory 10 bacterial cells are needed to
be able to detect the replicon 17-DMAG (Alvespimycin) HCl . The PCR can be performed with single primer sets or in a multiplex setting. This allows the user to choose between the advantage of high sensitivity or the advantage of multiplexing. Moreover, 96-wells plates can be used to test 10 strains for up to 8 different plasmid types. Of course, the multiplex setting has its limitations due to an overlap in melting temperatures of some of the replicons. Combinations of replicons should therefore be carefully chosen to allow to discriminate between melting peaks. Recently, a commercial kit for plasmid typing was introduced (PBRT kit, Diatheva, Fano, Italy). This kit provides the primers and controls needed to run the multiplex PCR, but still requires agarose gels as read out. This makes the kit a less attractive alternative for labs that have access to RT-PCR equipment. The prevalence of the different plasmid types is variable. For high prevalent plasmids several reference strains are available which can be used as positive controls. For the less prevalent plasmids it is difficult to obtain wild type reference strains.