Detection and typing of viruses using broadly sensitive cocktail-PCR and mass spectrometric cataloging

Demonstration with dengue virus

Charul Gijavanekar, Rafal Drabek, Mithil Soni, George W. Jackson, Ulrich Strych, George E. Fox, Yuriy Fofanov, Richard C. Willson

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Virus detection and taxonomic identification of serotypes, strains, or genotypes provide important information relevant for diagnosis, and for the epidemiological characterization and tracking of new strains in an endemic region. In the specific case of dengue virus, rapid serotype identification can also be useful in the treatment of secondary infections that may cause the more severe dengue hemorrhagic fever and dengue shock syndrome. In this work, dengue virus was used as a model to test a new approach of combining broadly sensitive RT-PCR amplification of nearly any virus strain with subsequent serotype- and finer-level identification by mass spectrometry. PCR primers were appended with promoter sequences, such that the resulting PCR products could be transcribed into RNA. RNA fragments generated by guanosine-specific RNase T 1 digestion were analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Viral serotypes were identified by comparing the pattern of observed fragment masses to a mass database. The database was created by computationally fragmenting 2517 dengue strains after each guanosine residue using the same primers. Computationally, all 2517 strains in the mass database were correctly identified at the serotype level from the predicted PCR product. The methodology was successfully demonstrated experimentally by identifying the serotypes of eight test strains using mosquito cell cultures infected with strains of all four serotypes and with full-length cDNA clones.

Original languageEnglish (US)
Pages (from-to)402-407
Number of pages6
JournalJournal of Molecular Diagnostics
Volume14
Issue number4
DOIs
StatePublished - Jul 2012
Externally publishedYes

Fingerprint

Cataloging
Dengue Virus
Severe Dengue
Viruses
Polymerase Chain Reaction
Guanosine
Databases
Mass Spectrometry
RNA
Ribonuclease T1
Dengue
Serogroup
Culicidae
Coinfection
Digestion
Lasers
Complementary DNA
Clone Cells
Cell Culture Techniques
Genotype

ASJC Scopus subject areas

  • Molecular Medicine
  • Pathology and Forensic Medicine

Cite this

Detection and typing of viruses using broadly sensitive cocktail-PCR and mass spectrometric cataloging : Demonstration with dengue virus. / Gijavanekar, Charul; Drabek, Rafal; Soni, Mithil; Jackson, George W.; Strych, Ulrich; Fox, George E.; Fofanov, Yuriy; Willson, Richard C.

In: Journal of Molecular Diagnostics, Vol. 14, No. 4, 07.2012, p. 402-407.

Research output: Contribution to journalArticle

Gijavanekar, Charul ; Drabek, Rafal ; Soni, Mithil ; Jackson, George W. ; Strych, Ulrich ; Fox, George E. ; Fofanov, Yuriy ; Willson, Richard C. / Detection and typing of viruses using broadly sensitive cocktail-PCR and mass spectrometric cataloging : Demonstration with dengue virus. In: Journal of Molecular Diagnostics. 2012 ; Vol. 14, No. 4. pp. 402-407.
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