Nested-PCR w czasie rzeczywistym, jako alternatywne molekularne narzędzie w detekcji Borrelia burgdorferi w porównaniu do klasycznej diagnostyki serologicznej krwi
[Nested-PCR real time as alternative molecular tool for detection of Borrelia burgdorferi compared to the classical serological diagnosis of the blood]

STRESZCZENIE

Trudności w diagnostyce boreliozy spowodowane są głównie heterogennością genogatunków, strategiami życiowymi Borrelia spp, oraz deficytami diagnostyki serologicznej. W niniejszej pracy podjęto próbę zwiększenia czułości detekcji B. burgdorferi poprzez opracowanie metody nested-PCR w czasie rzeczywistym oraz porównania uzyskanych wyników z klasycznym PCR w czasie rzeczywistym i rutynowo stosowaną diagnostyką serologiczną.

ABSTRACT

Introduction: Lyme disease, caused by Borrelia burgdorferi, is a multisystem disease that often makes difficulties to recognize caused by their genetic heterogenity. Currently, the gold standard for the detection of Lyme disease (LD) is serologic diagnostics based mainly on tests: ELISA and Western blot (WB). These methods, however, are subject to considerable defect, especially in the initial phase of infection due to the occurrence of so-called serological window period and low specificity. For this reason, they might be replaced by molecular methods, for example polymerase chain reaction (PCR), which should be more sensitivity and specificity. In the present study we attempt to optimize the PCR reaction conditions and enhance existing test sensitivity by applying the equivalent of real time PCR - nested PCR for detection B. burgdorferi DNA in the patient’s blood.

Methods: The study involved 94 blood samples of patients with suspected LD. From each sample, 1.5 ml of blood was used for the isolation of bacterial DNA and PCR real time amplification and its equivalent, in nested version. The remaining part earmarked for serological testing. Optimization of the reaction conditions made experimentally, using gradient of the temperature and gradient of the magnesium ions concentration for reaction real time in nested-PCR and PCR version.

Results: The results show that the nested-PCR real time, has a much higher sensitivity 45 (47.8%) of positive results for the detection of B. burgdorferi compared to the singlevariety, without a preceding pre-amplification 2 (2.1%). Serological methods allowed the detection of infection in 41 (43.6%) samples.

Conclusions: These results support of the nested PCR method as a better molecular tool for the detection of B. burgdorferi infection than classical PCR real time reaction. The nested-PCR real time method may be considered as a complement to ELISA and WB mainly in the early stages of infection, when in the blood circulating B. burgdorferi cells . By contrast, the results of serological and molecular tests should always be carried out taking into account the patient’s clinical status.