RESUMO
A real-time PCR assay is reported for identification of Lobesia botrana (Denis and Schiffermüller) collected in California. This assay multiplexes two independent TaqMan probe systems in a single reaction tube to reduce handling time and sample exposure to environmental contaminants. One probe system targets a segment of DNA located in the internal transcribed spacer region 2 (ITS2) that is present in the L. botrana genome but absent in native North American Tortricidae. The second probe system serves as a control for DNA quality by targeting a segment of the 18S rDNA gene that is conserved in L. botrana and all of the tested nontarget species. The assay successfully diagnosed 70 Lobesia botrana specimens and 95 nontarget specimens. No false-positive or false-negative results were observed supporting its application for identification of this pest in California.
Assuntos
Controle de Insetos/métodos , Mariposas/classificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Animais , California , DNA Espaçador Ribossômico/análise , Larva/classificação , Larva/genética , Mariposas/genética , Pupa/classificação , Pupa/genética , RNA Ribossômico 18S/análiseRESUMO
European gypsy moth populations (Lymantria dispar L.) are well established and a proven destructive force in hardwood trees throughout the United States and Canada. Introduction of the exotic Asian gypsy moth into North America would be even more impactful, as Asian gypsy moth populations have wider host ranges, and are capable of naturally dispersing more rapidly due to female flight ability. To support early detection and exclusion of Asian gypsy moth, the U.S. Department of Agriculture (USDA) uses molecular techniques to screen moths trapped in North America for evidence of common Asian genotype. In order to strengthen U.S. domestic capacity to screen moths quickly and efficiently, we report a real-time PCR assay for this pest. A probe system using TaqMan 5' nuclease chemistry is reported for detection of an allele associated with common Asian gypsy moth genotypes. The targeted allele is located at the nuclear FS1 locus currently used by the USDA in conventional PCR tests to screen for evidence of Asian gypsy moth introductions or introgression. The diagnostic probe is successfully multiplexed with a conserved 18S probe system to detect reaction failure due to poor sample quality or quantity. The specificity, sensitivity, and repeatability of the FS1-18S multiplex real-time PCR assay were tested on laboratory-reared and field-collected moths to demonstrate diagnostic utility. Implications of the new assay as a screening tool for evidence of Asian gypsy moth introgression and introduction are discussed.
Assuntos
Genótipo , Mariposas/classificação , Mariposas/genética , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Animais , Ásia , Proteínas de Insetos/genética , RNA Ribossômico 18S/genética , Sensibilidade e Especificidade , Análise de Sequência de DNA , Estados UnidosRESUMO
A molecular assay for diagnosis of light brown apple moth, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae), in North America is reported. The assay multiplexes two TaqMan real-time polymerase chain reaction (RT-PCR) probe systems that are designed to target DNA segments of the internal transcribed spacer region 2 (ITS2) and 18S rRNA gene. The RT-PCR probe designed for the 18S target recognizes a DNA sequence conserved in all of the moths included in the study and functions as a control in the assay. The second probe recognizes a segment of the ITS2 specifically found in E. postvittana and not found in the other moths included in the study, i.e., this segment is not conserved. Inclusion of the two markers in a single multiplex reaction did not affect assay performance. The assay was tested against 637 moths representing > 90 taxa in 15 tribes in all three subfamilies in the Tortricidae. The assay generated no false negatives based on analysis of 355 E. postvittana collected from California, Hawaii, England, New Zealand, and Australia. Analysis of a data set including 282 moths representing 41 genera generated no false positives. Only three inconclusive results were generated from the 637 samples. Spike experiments demonstrated that DNA contamination in the assay can affect samples differently. Contaminated samples analyzed with the ITS2 RT-PCR assay and DNA barcode methodology by using the cytochrome oxidase I gene can generate contradictory diagnoses.
Assuntos
Código de Barras de DNA Taxonômico/métodos , Mariposas/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Animais , California , Complexo IV da Cadeia de Transporte de Elétrons/genética , Marcadores Genéticos , Dados de Sequência Molecular , Mariposas/classificação , América do Norte , RNA Ribossômico 18S/genética , Alinhamento de SequênciaRESUMO
A molecular protocol using a hemi-nested polymerase chain reaction (PCR) of the internal transcribed spacer region 2 (ITS2) is reported for the diagnosis of light brown apple moth, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae), in California. This protocol distinguishes the light brown apple moth from other moths in California based on size differences of PCR amplicons that are visualized on agarose gels. The molecular diagnostic tool generated no false negatives based on analysis of 337 light brown apple moths collected from California, Hawaii, England, New Zealand, and Australia. Analysis of a data set including 424 moths representing other tortricid species generated correct identification for >95% of the samples and only two false positives. Of the 761 moths tested only fourteen produced no PCR amplicons and five generated inconclusive data.
