Polymerase chain reaction (PCR) has for many years been regarded as the new standard procedure in detecting the genes of microorganisms, plants and animals. Detecting the reacting products is reliant upon agarose gel electrophoresis, ethidium bromide staining, ultraviolet irradiation and comparing the size to a DNA size marker. This has slowed progress in adopting the PCR method in diagnostic laboratories. Developing new methods of PCR product detection without opening a reaction tube has opened new possibilities in applying this technique during routine diagnosis. Monitoring PCR product accumulation as amplification progresses is possible through using labeled primers and/or oligonucleotide probes or DNA binding fluorophores. Real-Time PCR methods can be subdivided into two groups: sequence dependent or sequence independent DNA detection. All of these involve using fluorophores linked to oligonucleotides or their presence in the reaction mixture. Their common feature is measuring fluorescence in each PCR cycle and calculating the threshold cycle that corresponds to the DNA copy number in that sample.
Powszechnie stosowane metody identyfikacji drożdży przemysłowych opierają się głównie na ich właściwościach fizjologicznych i biochemicznych. Zasadniczą wadą tych metod jest czasochłonność i niska czułość. W ostatnich kilku latach obserwuje się bardzo szybki i dynamiczny rozwój metod identyfikacji mikroorganizmów z wykorzystaniem technik biologii molekularnej np. łańcuchowej reakcji polimerazy (PCR). Różnorodność tych technik sprawia, że mikroorganizm może być charakteryzowany bardzo dokładnie i na różne sposoby nawet w obrębie jednego gatunku.
The aim of the study was to develop multiplex PCR (mPCR) assays which allow identification of shigatoxigenic Escherichia coli (STEC) strains and a characterization of their virulence properties. As an internal control, a fragment of 16S rRNA gene - two amplicons of 230 bp (stx gene) and 401 bp (16S rRNA) were obtained in the test, designated as mPCR-1 for the amplification of the stx gene, characteristic for all known Shiga toxin variants. The mPCR-2 assay was developed to characterize the stx-positive bacterial colonies, which allowed the detection of Shiga toxin 1 and/or 2, the affiliation of STEC O157:H7 serotype (rfbO157 and fliCн₇ genes), and internal control, resulting in amplicons of 348 bp (stx1), 584 (stx2), 420 bp (rfbO157), 247 bp (fliCн₇) and 798 bp (16S rRNA). The detection of markers O26wzx, rfbO111 and rfbO113, typical for E. coli O26, O111 and O113, respectively, was performed with mPCR-3. The products of molecular masses 153, 406 and 593 bp were observed. All STEC’s were also tested using mPCR-4, for the presence of enterohemolysin (ehlyA) and intimin (eaeA) markers generating specific bands of 837 bp (eaeA gene), 534 bp (ehlyA) and 401 (16S rRNA). The mPCR assays developed allow STEC to be identified and characterized and may be used in routine diagnosis of these bacteria.
The aim of the study was to detect Mycobacterium paratuberculosis DNA in raw milk samples. DNA from 103 udder milk samples was isolated using the eQIAamp DNA Mini Kit (Qiagen). IS-900 - a part of genome characteristic for MAP - was detected in 21 samples.
Reverse transcription coupled with the polymerase chain reaction (RT-PCR) was used for the detection of HCV and BVDV in virus cell cultures. Reverse transcription reaction was performed with RNA isolated by means of the guanidine method or with cell culture supernatant precipitates by eliminating the RNA isolation step. Both reactions, RT and PCR, were carried out by using rTth DNA polymerase in one tube. The RT-PCR products were analysed by a gel electrophoresis and hybridization with the digoxygenine labelled DNA probe. It was demonstrated that it is possible to omit the RNA isolation step and that applied primers are suitable for diagnosing HCV and BVDV.