MOLECULAR GENETIC ANALYSIS OF A MIXED ALPHAVIRUS RNA SAMPLE

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Authors
  1. Netolitzky, D.J.
  2. Bader, D.E.
Corporate Authors
Defence Research Establishment Suffield, Ralston ALTA (CAN)
Abstract
Reverse transcription polymerase chain reaction amplification (RT-PCR) and DNA sequence analysis were employed to detect, distinguish and identify nucleic acid from two closely related alphaviruses within a mixed sample, namely Highlands J (HJ), a non-human pathogen, and Western equine encephalitis (WEE), a human pathogen. HJ-specific and WEE-specific primers were designed from published sequence data for the E1 envelope glycoprotein structural gene to distinguish the two viruses. Each primer set amplified a cDNA fragment of the expected theoretical size of 264 bp as determined by agarose gel electrophoresis suggesting the presence of HJ and WEE nucleic acid in the mixed sample. Sequence analysis of the 264 bp RT-PCR products from each reaction confirmed the presence of HJ and WEE nucleic acid in the mixed sample. RT-PCR analysis of all vials in the DRES collection labelled "HJ RNA" revealed extensive contamination with WEE sequence. Evidence is provided which suggests that contamination of the putative HJ RNA sample with WEE nucleic acid occurred at DRES. The ability to detect, discriminate and identify nucleic acid from two closely related organisms within the same sample could only have been achieved using the molecular genetic technologies described in this study. TRUNCATED
Keywords
WEEV (Western Equine Encephalitis Virus);Biological strains;Sequencing;Genetic engineering;Alphaviruses;Polymerase chain reaction (PCR);Nucleic acid sequencing;Nucleocapsid Protein Gene;Reverse Transcription Polymerase Chain Reaction;Genetic analysis;Highlands J virus
Report Number
DRES-696 —
Date of publication
01 Jul 1998
Number of Pages
35
DSTKIM No
98-02277
CANDIS No
509140
Format(s):
Hardcopy;Document Image stored on Optical Disk

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