ZASTOSOWANIE TECHNIKI REAL-TIME PCR DO WYKRYWANIA DNA LUDZKIEGO WIRUSA OPRYSZCZKI TYPU 1
[APPLICATION OF REAL-TIME PCR ASSAY FOR INVESTIGATING THE PRESENCE OF HERPES SIMPLEX VIRUS TYPE 1 DNA]

Streszczenie

Celem pracy było zaprojektowanie i zbadanie użyteczności metody real-time PCR do diagnostyki zakażeń ludzkim wirusem opryszczki typu 1, zwłaszcza u pacjentów poddanych immunosupresji oraz z zakażeniami ośrodkowego układu nerwowego.

Abstract

Herpes simplex virus type 1 is a member of the Alphaherpesviridae subfamily, as it can infect both skin and nerves and develop latent infection within the dorsal root and trigeminal ganglia. Infection with this virus is common and causes a wide range of clinical syndromes. Although HSV-1 infect healthy children and adults, disease is more severe and extensive in the immunocompromised individuals. The goal of the study was development of real-time PCR assay for detection of herpes simplex virus type 1 DNA in clinical samples, using primers targeting a conserved region of the viral DNA glycoproteine G gene and a specific TaqMan hydrolyzing probe. The analytical sensitivity of assay was tested using serial dilutions of HSV-1 DNA in range between 100 and 10-6 (4,35x105 - 4,00x101 copies/ml). Fifteen cell line isolates and twenty plasma samples taken from a group of adult recipients of allogeneic HSCT were tested for the presence of HSV-1 DNA in the LightCycler® system. For comparison commercial quantitative HSV-1/2 LC PCR Kit (Artus/Qiagen) was used, according to the manufacturer’s instructions. Both LightCycler® assays, including in-house real-time PCR, detected HSV-1 DNA in 23 specimens. The conclusion is that developed TaqMan-based probe real-time PCR test is very reliable and valuable for detection of HSV-1 viremia in different kind of samples. The high level of sensitivity and accuracy provided by the LightCycler® instrument is favorable for the use of this method in the detection of herpes simplex virus 1 DNA also in clinical specimens.