आण्विक जीवविज्ञान: खुली पहुंच

Topoisomerase I Improve JC virus DNA detection

Abstract

Ana Matos, Vitor Duque and Cristina Luxo

Introduction: Progressive Multifocal Leukoencephalopathy (PML) is a severe and often fatal CNS disease caused by JC human polyomavirus infection. Diagnosis of PML is based upon suggestive clinical symptoms and brain images, supported by the presence of JC virus genome in CSF samples.

Objective: The main objective of our study was the search for an alternative method for JC virus DNA detection in CSF samples, with sensitivity and specificity characteristics close to that of standard techniques, but feasible at any clinical laboratory.

Methods: In order to evaluate the effect of topoisomerase I treatment in the detection of JC virus genome, and its feasibility in laboratory diagnosis of PML, 129 CSF samples were examined for the presence of JC virus DNA by a nested-PCR protocol, with and without previous treatment with topoisomerase I. All CSF samples were also evaluated through a real-time PCR protocol.

Results: Eleven CSF samples presented detectable JC virus DNA with all used protocols. On 9 CSF samples, JC virus DNA was only detectable with topoisomerase I modified nested-PCR and real-time PCR protocols. Real-time PCR was the only protocol able to detect JC virus genome in 4 CSF samples. One CSF sample revealed the presence of the expected amplified fragment only when tested with topoisomerase I modified nested-PCR protocol.

Conclusion: The results of the present study point towards the benefit of using topoisomerase I DNA treatment before amplification reactions in JC virus DNA detection on CSF samples, and confirm that topoisomerase I modified nested-PCR protocol represents a good alternative method to detect JC virus DNA in CSF samples from patients with clinical signs and brain images suggestive of PML.