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Utilization of the polymerase chain reaction in the diagnosis of nuclear polyhedrosis virus infections of gypsy moth ( Lymantria dispar , Lep., Lymantriidae) populations
Author(s) -
Charpentier G.,
Desmarteaux D.,
Bourassa J.P.,
Belloncik S.,
Arella M.
Publication year - 2003
Publication title -
journal of applied entomology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.795
H-Index - 60
eISSN - 1439-0418
pISSN - 0931-2048
DOI - 10.1046/j.1439-0418.2003.00759.x
Subject(s) - biology , polymerase chain reaction , lymantria dispar , nuclear polyhedrosis virus , virology , genome , microbiology and biotechnology , virus , pest analysis , genetics , gene , botany
Three specific DNA probes were used for the detection of the nuclear polyhedrosis (NPV) virus of Lymantria dispar ( Ld NPV) genome. Two of these probes, H2 and H3 were obtained by classical cloning method and one (TR6) by polymerase chain reaction (PCR). These probes, used individually or in a pool in the standard slot–blot hybridizations, were able to detect 10 9 genome copies. By performing 35 cycles of PCR amplification before hybridization with primers specific to Ld NPV genome on DNA extracted from infected larvae, the sensitivity of the hybridization technique was increased, so that as little as 10 copies of the Ld NPV genome could be detected. Using these methods, L. dispar naturally infected by Ld NPV were identified among field populations in Canada and in the United States near the eastern Canadian border. Using a combination of PCR and hybridization, Ld NPV contamination of egg masses were also detected. By disinfecting the eggs with sodium hypochlorite prior to PCR amplification and hybridization, it was also demonstrated that transmission of viral infection in the natural populations is mainly caused by external contamination of the egg and is unlikely to occur through the transovarial route.