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PCR w czasie rzeczywistym. Istota metody i strategie monitorowania przebiegu reakcji

100%

Studzinska A. , Tyburski J. , Daca P. , Tretyn A.

Biotechnologia

2008

nr 1

71-85

Wykrywanie oraz roznicowanie wirusow opryszczki typu 1 i 2 metoda real-time PCR z wykorzystaniem sond HybProbe

100%

Chudzik E. , Karabin K. , Dzieciatkowski T. , Majewska A. , Przybylski M. , Midak-Siewirska A. , Luczak M. , Mlynarczyk G.

nr 3

255-262

Celem pracy była optymalizacja metody real-time PCR do wykrywania oraz różnicowania zakażeń ludzkimi wirusami opryszczki typu 1 i 2, zwłaszcza w przypadkach infekcji przebiegających z niewielkim nasileniem replikacji patogenu.

Herpes simplex viruses types 1 and 2 are members of the Alphaherpesviridae subfamily, as they can infect both skin and nerves and develop latent infection within the dorsal root and trigeminal ganglia. Infections with these viruses are common worldwide and cause wide range of clinical syndromes. Although HSV-1/2 infect healthy children and adults, disease is more severe and extensive in the immunocompromised individuals and/or during neuroinfections. The aim of the study was development of real-time PCR assay for detection and differentiation of herpes simplex viruses type 1 and 2. DNA in clinical samples, using specific dual-channel HybProbe chemistry. The nalytical sensitivity of assay was tested using serial dilutions of HSV-1 and HSV-2 DNA in range between 100 and 10-5 (4,35x105 - 4,00x102 copies/ml and 4,18x105 - 3,82x102 copies/ml, respectively). Thirty four cell line isolates and sixteen clinical samples taken from a group of adult patients with neurological signs were tested for the presence of HSV-1/2 DNA in the LightCycler® instrument. Described in- house real-time PCR assay detected herpesviral DNA in all cell line isolates (31 of them were HSV-1 positive; 3 were HSV-2 positive) and in 10 clinical samples (positive only for HSV-1). The conclusion is that developed HybProbe-based real-time PCR test is very reliable and valuable tool for detection and differentiation of HSV-1/2 viremia in different kind of samples. The high level of sensitivity and accuracy provided by this assay is favorable for the quantification of herpes simplex virus 1 and 2 DNA in clinical specimens, especially during low-copy infections.