Wykorzystanie nowej techniki multiplex real-time PCR do wykrywania i różnicowania DNA wirusów opryszczki typu 1 i 2
[Novel multiplex real-time PCR assay for detection and differentiation of herpes simplex virus type 1 and 2 DNA]

Streszczenie.

Celem pracy było optymalizacja nowego wariantu techniki multiplex real-time PCR, który może być wykorzystany zarówno do wykrywania, jak i różnicowania zakażeń HSV-1/2, zwłaszcza w próbkach pobranych od osób z zakażeniami narządów płciowych. Specyficzność opracowanej metody, w połączeniu z szybkością oznaczenia, daje możliwość rutynowego jej stosowania, zwłaszcza u pacjentów z zaburzeniami odporności lub objawami niespecyficznymi, w przypadku których szybka i trafna diagnoza jest szczególnie istotna.

Abstract.

Introduction: Herpes simplex viruses type 1 and 2 are the cause of world spread multiple infections with different course and severity. The aim of this work was to design and to optimize multiplex real-time PCR assay for simultaneous detection of HSV-1 and HSV-2. The second aim of the project was to check if the designed method is laboratory useful analyzing different clinical specimens.Materials and methods: In this experiment primers and probes were designed to specific viral sequences: for HSV-1 to the gene of viral DNA polymerase; for HSV-2 to the UL5 sequence. For performing qPCR assay TaqMan chemistry was used. Reference strains HSV-1 McIntyre and HSV-2 MS were used as a positive control. To test laboratory utility of the designed method 58 different clinical specimens were analyzed. Results: Developed multiplex real-time PCR gave positive result only in the samples containing genetic material of HSV-1/2. Of the 58 clinical samples tested, 27 proved to be positive for HSV-1 and 17 for HSV-2. The 7 samples showed the presence of both types of DNA herpes simplex virus, and 7 others were found for both HSV-1 and HSV-2 negative. Conclusions: Obtained results show that the designed method is highly specific and can possibly be used to simultaneously detect and differentiate HHV-1/2. Both high specificity and very short time of analysis have great importance in diagnosing immunocompromised patients, which ought to be diagnosed quickly and effectively in order to provide appropriate treatment.