Application of the PCR-RFLP method for the rapid differentiation of Spodoptera exigua nucleopolyhedrovirus genotypes.
Quality control during mass production of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV), and studies on environmental fate following the use of this virus as a biological pesticide, would be facilitated by a rapid method for the detection and identification of isolates. A molecular biology tool was developed that combined the polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) to differentiate SeMNPV isolates. Oligonucleotide primers were designed to amplify five variable SeMNPV genomic regions (V01, V02, V03, V04, V05). Four wild-type SeMNPV strains isolated from the United States (US2) and Spain (SP1, SP2, and SP3), and a laboratory cloned genotype (US1A), were analyzed with 36 different primer-endonuclease combinations. BglII digestion of the variable region 1 (V01) amplicon was the only combination that differentiated each of the five virus isolates tested, although genetic heterogeneity limited the discriminatory power of the technique. Six novel SeMNPV isolates originating from greenhouse soils in southern Spain were successfully identified using this method. As judged by sequence analysis, the V01 region, which comprises the homologous region 1 (hr1), is the most variable genomic region among the genotypes present in the Spanish isolates. This method constitutes a useful tool for processing large number of environmental samples and could be used to address environmental biosafety concerns.
Murillo
Rosa
R
Departamento de Producción Agraria, Universidad Pública de Navarra, Pamplona 31006, Spain.
Muñoz
Delia
D
Williams
Trevor
T
Mugeta
Nora
N
Caballero
Primitivo
P
D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D016172:DNA Fingerprinting / Q000379:methods; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D018740:Genetic Heterogeneity; D016679:Genome, Viral; D008969:Molecular Sequence Data; D017924:Nucleopolyhedroviruses / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods; D011110:Polymorphism, Genetic; D012150:Polymorphism, Restriction Fragment Length; D017422:Sequence Analysis, DNA; D012988:Soil Microbiology; D018411:Spodoptera / Q000821:virology*
D001426:Bacterial Proteins; D017931:DNA Primers; D004279:DNA, Viral; C024404:BglII endonuclease; D015252:Deoxyribonucleases, Type II Site-Specific