PCR MP Method for Differentiation of Clavibacter michiganensis Subsp. sepedonicus Isolates

The published data indicate that the bacterium Clavibacter michiganensis subsp. sepedonicus (Cms) shows a notably low degree of intraspecific variation, and most of the molecular diagnostic methods could differentiate among subspecies groups but lacked the resolution to detect genetic diversity within subspecies. The present study describes effective differentiation of 50 isolates of C. michiganensis subsp. sepedonicus by the PCR MP method using five restriction enzymes (ApaI, PstI, BamHI, XmaI, HindIII). By analysing the electrophoretic patterns obtained by the PCR MP method performed with the applied restriction enzymes, each of the 50 tested isolates could be identified and distinguished from the remaining isolates. The PCR MP method of bacterial strain identification can be used in epidemiological studies, particularly for excluding the isolates of C. michiganensis subsp. sepedonicus as a source of primary infection.

Application of Microsatellite-Primed PCR (MSP-PCR) and PCR Melting Profile (PCR-MP) Method for Intraspecies Differentiation of Dermatophytes

In this study, two PCR-based methods (MSP-PCR and PCR-MP) were compared for their abilities to identify intraspecies variations of 23 isolates of Trichophyton rubrum, 78 isolates of Trichophyton interdigitale and 22 isolates of Microsporum canis, obtained mainly from patients in Lódź city. The results allowed to distinguish four types (containing two subtypes) characteristic for T. interdigitale and three types characteristic for T. rubrum using PCR-MP method. Analysis conducted using MSP-PCR with (GACA)4 primer revealed four types for T. rubrum and three types (containing one subtype) for T. interdigitale and with (GTG), primer showed two types (containing one subtype) for T. rubrum and six types (containing one subtype) for T. interdigitale. No differentiation was observed for the M. canis isolates with either method.

Evaluation of a PCR melting profile method for intraspecies differentiation of Trichophyton rubrum and Trichophyton interdigitale

In order to identify the source of infections caused by dermatophytes, as well as the pathogen transmission pathway, there is a need to determine methods that allow detailed genetic differentiation of the strains within the dermatophyte genera. In this work, a PCR melting profile (PCR-MP) technique based on the ligation of adaptors and the difference in melting temperatures of DNA restriction fragments was used for the first time for intraspecies genotyping of dermatophytes. Clinical isolates and reference strains of dermatophytes isolated from skin, scalp, toenails and fingernails were used for this study. PCR-MP and random amplification of polymorphic DNA (RAPD) were used to type 11 isolates of Trichophyton rubrum, 40 isolates of Trichophyton interdigitale and 14 isolates of Microsporum canis. The results distinguished five types (containing one subtype) characteristic for T. rubrum and seven types characteristic for T. interdigitale using the PCR-MP technique. Analysis conducted using RAPD revealed five types for T. rubrum and four types for T. interdigitale isolates. No differentiation was observed for the M. canis isolates with either method. These results demonstrate that PCR-MP is a reliable method for the differentiation of T. rubrum and T. interdigitale strains and yields a discriminatory power that is at least equal to that of RAPD.

Molecular analysis of predation on parasitized hosts

Predation on parasitized hosts can significantly affect natural enemy communities, and such intraguild predation may indirectly affect control of herbivore populations. However, the methodological challenges for studying these often complex trophic interactions are formidable. Here, we evaluate a DNA-based approach to track parasitism and predation on parasitized hosts in model herbivore-parasitoid-predator systems. Using singleplex polymerase chain reaction (SP-PCR) to target mtDNA of the parasitoid only, and multiplex PCR (MP-PCR) to additionally target host DNA as an internal amplification control, we found that detection of DNA from the parasitoid, Lysiphlebus testaceipes, in its aphid host, Aphis fabae, was possible as early as 5 min. post parasitism. Up to 24 h post parasitism SP-PCR proved to be more sensitive than MP-PCR in amplifying parasitoid DNA. In the carabid beetles Demetrias atricapillus and Erigone sp. spiders, fed with aphids containing five-day-old parasitoids, parasitoid and aphid DNA were equally detectable in both predator groups. However, when hosts containing two-day-old parasitoids were fed to the predators, detection of parasitoid prey was possible only at 0 h (immediately after consumption) and up to 8 h post consumption in carabids and spiders, respectively. Over longer periods of time, post-feeding prey detection success was significantly higher in spiders than in carabid beetles. MP-PCR, in which parasitoid and aphid DNA were simultaneously amplified, proved to be less sensitive at amplifying prey DNA than SP-PCR. In conclusion, our study demonstrates that PCR-based parasitoid and prey detection offers an exciting approach to further our understanding of host-parasitoid-predator interactions.