Abstract
BackgroundPCR-based detection and identification of viruses assumes a known, relatively stable genome. Unfortunately, high mutation rates may lead to extensive changes in viral nucleic acid sequences making dedicated PCR primer use problematic. Furthermore, in bioterrorism, viral consensus sequences can be genetically modified as a countermeasure to RT-PCR and DNA chip detection. Accordingly, there is a great need for the development of rapid and universal virus detection and identification technologies.ResultsWe report herein that viral genomic DNA or RNA can be separated from host nucleic acids in plasma by filtration and nuclease digestion, and randomly amplified in a single PCR using a mixture of primers designed to be resistant to primer-dimer amplification (5'-VVVVVVVVAA-3', V = A, G or C; 38 or 6561 primers). We have termed this novel PCR method Random Multiplex (RT)-PCR since hundreds of overlapping PCR amplifications occur simultaneously. Using this method, we have successfullydetected and partially sequenced 3 separate viruses in human plasma without using virus-specific reagents (i.e., Adenovirus Type 17, Coxsackievirus A7, and Respiratory Syncytial Virus B). The method is sensitive to ~1000 genome equivalents/ml and may represent the fastest means of detection of unknown viruses.ConclusionThese studies suggest that the further development of random multiplex (RT)-PCR may lead to a diagnostic assay that can universally detect viruses in donated blood products as well as in patients suffering with idiopathic disease states of possible viral etiology.
Highlights
PCR-based detection and identification of viruses assumes a known, relatively stable genome
A recent example involved a case of fatal yellow fever (YF) in a traveler returning from Amazonas, Brazil in March, 2002 [3]
The disease did not respond to empirical antibiotic treatment, and no known bacterial or viral pathogens were identified using serological and (RT)-PCR
Summary
PCR-based detection and identification of viruses assumes a known, relatively stable genome. DNA chips that allow the simultaneous measurement of literally thousands of genes through hybridization are being developed as the next-generation rapid diagnostic test for all known human pathogens [4] Both of these technologies rely on a relatively stable genome, and several human pathogens display a high mutation rate (e.g., HIV 2 × 10-5/base, 9 kB genome [5]). The ability to recombine "non-pathogenic" viruses in vitro introduces the potential for de novo pathogenicity and for enhanced stealth These considerations suggest that it may be impossible to design DNA micro-arrays which detect nucleic acids from all known and unknown viruses, including less obvious vehicles of bioterrorism such as adenovirus or rhinovirus recombined with a single gene for enhanced virulence. There is a great need for the development of techniques that enable the universal amplification of viral nucleic acids
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