Abstract
The genera Anastrepha, Bactrocera, Ceratitis, Dacus and Rhagoletis in the family Tephritidae order Diptera are economically important, worldwide distributed and cause damage to a large number of commercially produced fruits and vegetables. China had regulated these five genera as quarantine pests, including the species Carpomya vesuviana. An accurate molecular method not depending on morphology able to detect all the quarantine fruit flies simultaneously is required for quarantine monitoring. This study contributes a comparative analysis of 146 mitochondrial genomes of Diptera species and found variable sites at the mt DNA cox2 gene only conserved in economically important fruit flies species. Degenerate primers (TephFdeg/TephR) were designed specific for the economically important fruit flies. A 603 bp fragment was amplified after testing each of the 40 selected representative species belonging to each economically important Tephritid genera, no diagnostic fragments were detected/amplified in any of the other Tephritidae and Diptera species examined. PCR sensitivity assays demonstrated the limit of detection of targeted DNA was 0.1 ng/μl. This work contributes an innovative approach for detecting all reported economically important fruit flies in a single-step PCR specific for reported fruit fly species of quarantine concern in China.
Highlights
Rapid detection of economically important fruit flies is a pivotal measure to prevent the introduction of potentially invasive species
Jiang et al developed a standardized reaction system for simultaneous detection of 27 fruit fly species intercepted at ports in China based on a microfluidic dynamic array of mt DNA cox[1] gene that uses species-specific primers and probes for quarantine concern[16]
The complete mitochondrial genomes of 146 Diptera species were sourced from GenBank and the pool included 16 economically important Tephritidae species: Bactrocera (Bactrocera) arceae (Hardy & Adachi)[24], B. (B.) carambolae Drew & Hancock, B. (B.) correcta (Bezzi), B. (B.) dorsalis[25], B. (B.) latifrons (Hendel)[26], B. (B.) melastomatos Drew & Hancock[26], B. (B.) tryoni (Froggatt)[27], B. (B.) umbrosa (Fabricius)[26], B. (B.) zonata (Saunders)[28], B. (Daculus) oleae (Gmelin)[27,29], B. (Tetradacus) minax (Enderlein)[30], B. (Z.) cucurbitae[31], B. (Z.) diaphora (Hendel)[32], B. (Z.) scutellata (Hendel), B. (Z.) tau (Walker)[33] and Ceratitis (Ceratitis) capitata (Wiedemann)[34]
Summary
Rapid detection of economically important fruit flies is a pivotal measure to prevent the introduction of potentially invasive species. Jiang et al developed a standardized reaction system for simultaneous detection of 27 fruit fly species intercepted at ports in China based on a microfluidic dynamic array of mt DNA cox[1] gene that uses species-specific primers and probes for quarantine concern[16]. These molecular methods are yet insufficient to detect all economically important fruit flies at the border during quarantine inspections. Mitochondrial genomes of Diptera sourced in GenBank (https://www.ncbi.nlm.nih.gov/genbank/) were aligned aiming for variable landmarks in Diptera but conserved in economically important fruit flies to develop a ‘specific broad detection’ pair of primers for simultaneous broad detection of all the economically important fruit flies in a single-step end point PCR
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