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Journal of Clinical Microbiology, February 2005, p. 716-720, Vol. 43, No. 2
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.2.716-720.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Characterization of Virus Isolates by Particle-Associated Nucleic Acid PCR

Alexander Stang,1 Klaus Korn,2 Oliver Wildner,1 and Klaus Überla1*

Department of Molecular and Medical Virology, Ruhr-University Bochum,1 Institute of Clinical and Molecular Virology, University Erlangen-Nürnberg, Erlangen, Germany2

Received 2 June 2004/ Returned for modification 27 August 2004/ Accepted 1 October 2004

Diagnostic virus isolation is still frequently used, particularly from respiratory tract secretions. Testing positive virus cultures for all possible viruses is time-consuming, and unexpected or unknown viruses may escape detection. Therefore, a novel random PCR approach was developed that allows sequence-independent amplification of viral nucleic acids from virus isolation-positive cultures. Selectivity for viral sequences is obtained by preferential isolation of nucleic acids that are particle associated and resistant to nucleases. Using primers with a degenerated 3' end, the isolated nucleic acids are amplified and the randomly amplified PCR products are cloned and sequenced. As proof of the concept, the PAN-PCR approach was applied to supernatants of coxsackievirus B3 and murine adenovirus type 1-infected cells. Enterovirus and adenovirus sequences were obtained, demonstrating that the random PCR approach allows detection of RNA and DNA viruses. As a first application of this PAN-PCR approach, we characterized a virus isolate from mouth-washing material of a patient with chronic fatigue syndrome and high antibody titers to coxsackievirus B2. The virus isolate had tested negative for enteroviruses and respiratory viruses (influenza viruses A and B, parainfluenza virus types 1 to 3, respiratory syncytial virus, and adenovirus) by immunofluorescence and PCR. Particle-associated, nuclease-resistant RNA and DNA were prepared from the supernatant of infected cells. The DNA and the reverse-transcribed RNA were randomly amplified, and PCR products were cloned and sequenced. Of 25 sequences obtained from the DNA preparation, 24 contained herpes simplex virus type 1 (HSV-1) sequences from 14 different loci spread over the HSV-1 genome. This result was confirmed by using a standard diagnostic HSV-PCR, demonstrating that the PAN-PCR correctly identified the virus isolate. Although the identification of HSV-1 in mouth-washing material is not surprising in retrospect, it clearly demonstrates the applicability of the PAN-PCR approach. This method should be particularly useful for characterizing virus isolates that have tested negative for all expected viruses and for identifying unknown viruses.

* Corresponding author. Mailing address: Department of Molecular and Medical Virology, Ruhr University Bochum, D-44780 Bochum, Germany. Phone: 49-234-3223189. Fax: 49-234-3214352. E-mail: klaus.ueberla{at}ruhr-uni-bochum.de.

Journal of Clinical Microbiology, February 2005, p. 716-720, Vol. 43, No. 2
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.2.716-720.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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