Development and use of a PCR assay for detection of the reproductive virus in wild populations of helicoverpa zea (Lepidoptera: noctuidae).
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abstract
Helicoverpa zea reproductive virus (HzRV) is a nonoccluded bacilliform virus that affects both female and male moths of the corn earworm H. zea. In order to study the biology and host range of HzRV, a bioassay was previously developed to detect the presence of this virus in infected insects. A drawback of this bioassay is that it is time consuming and requires more than a month to complete. Here we describe the development of a polymerase chain reaction (PCR) assay for the rapid detection of HzRV in infected corn earworms. The genome of HzRV was digested with PstI and cloned into a plasmid vector. Sequences from two different clones, P4 and P13, were selected for designing two sets of primers. These primers were used for PCR and their sensitivity and specificity in detecting HzRV DNA were examined. Both sets of primers produced the expected amplification product in samples containing HzRV DNA but not in uninfected corn earworm samples, Spodoptera frugiperda (Sf-9) cells, or samples from the Autographa californica nuclear polyhedrosis virus. In addition, the primer pair of the clone P13 was sensitive enough to detect approximately 175 copies of viral DNA. We then used this assay to examine feral populations of H. zea from seven geographical locations in the United States. HzRV was detected primarily in the Mississippi populations and to a lesser extent in Iowa and Georgia, but none in Maryland, Missouri, and Texas populations. This PCR assay provides a highly specific, sensitive, and rapid way of detecting the presence of HzRV and will be useful in further studying the host range, tissue specificity, and incidence of this virus in wild populations of the corn earworm.
