Rapid identification of Bactrocera latifrons (Dipt., Tephritidae) by real-time PCR using SYBR Green chemistry

Abstract

The solanum fruit fly, Bactrocera latifrons (Hendel), is a major agricultural pest in Asia and Hawaii, and it is important to prevent its widespread invasion in plant quarantine. In this study we introduced a real-time polymerase chain reaction (PCR) essay, using SYBR Green I dye, to rapidly identify B. latifrons on an ABI PRISM 7700 sequence detection system. A latifron-specific PCR primer set was obtained based on mtDNA COI gene of B. latifrons. Nine Bactrocera fruit flies, B. latifrons, Bactrocera dorsalis, Bactrocera papayae, Bactrocera carambolae, Bactrocera philippinensis, Bactrocera occipitalis, Bactrocera correcta, Bactrocera cucurbitae and Bactrocera tau, were used to determine the specificity of primers lati1 and lati2. A series of genomic DNA dilutions of B. latifrons (0.01, 0.1, 1, 10, 20, 40 and 100 ng) were used to assess the sensitivity of the SYBR Green PCR. Template DNA concentration was one of the sources of variability in cycle threshold values (CT) and the optimum DNA concentration was between 1 and 20 ng. Genomic DNA isolated from larvae, pupae and adult specimens of B. latifrons were used to assess the specificity of the SYBR Green PCR. Melting curve analysis and agarose gel electrophoresis was employed to check the specificity of PCR products. Similar amplification plots were obtained using DNA from the three different stages of B. latifrons with primer set lati1/lati2. The melting temperature (Tm) of PCR products was 77.5 ± 0.1°C, and the length of the amplified fragment 366 bp. Given the specificity and sensitivity of the assay, combined with high speed, low cost and the possibility of automating, SYBR Green PCR can be used as a rapid and specific technique for pest species identification in plant quarantine.