MODYFIKACJA I OPTYMALIZACJA METOD PCR DO WYKRYWANIA REGIONU MIE LUDZKIEGO HERPESWIRUSA TYPU 5
[MODIFICATION AND OPTIMIZATION OF PCR METHODS FOR DETECTION OF MIE REGION FROM HUMAN HERPESVIRUS 5]

Streszczenie.

Celem pracy była modyfikacja i optymalizacja wariantów real-time PCR do wykrywania DNA ludzkiego herpeswirusa typu 5 oraz porównanie ich czułości analitycznej z konwencjonalną metodą PCR i komercyjnym zestawem diagnostycznym jako testem odniesienia.

Abstract.

Human herpesvirus 5 (HHV-5, formerly known as CMV) is a β-herpesvirus widely spread within a population. Thus, HHV-5 infections are a serious matter of concern in a group of immunocompromised patients. The goal of the study was modification and optimization of conventional PCR method developed for the detection of HHV-5 DNA to the real-time variant (RTmPCR) and determination of analytical resolution of the modified methods.
Thirty plasma samples were tested for the presence of HHV-5 DNA using the LightCycler® system with two different methods – one with SYBR Green I fluorochrome method and second one using TaqMan fluorescent probes and a qualitative in-house gel-stained PCR assay using primers that amplify part of HHV-5 MIE gene. The analytical sensitivity of real-time PCR assay was tested using serial dilutions of HHV-5 DNA in range between 100 and 10-6. For comparison typical end-point
detected PCR for cytomegalovirus detection with the same DNA dilutions was made. The sensitivity of novel method was about 100-fold higher than older one.
Both LightCycler® assays detected HHV-5 DNA in 27 samples, also which were negative by the gel-stained PCR. Analysis of the available clinical and serological data associated with these samples suggested that the real-time results in all of these cases were true positive.
The conclusion is that real-time PCR methods are more sensitive than the conventional PCR used in this study. The additional sensitivity was valuable for detection of patients with low-copy viremia. The high level of sensitivity, specificity, accuracy, and rapidity provided by the LightCycler instrument are favorable for the use of this system in the detection of HHV-5 DNA in clinical specimens.