PDF(Pubmed)
Abstract：
The oriental fruit fly, Bactrocera dorsalis (Hendel), is a significant economic and quarantine pest due to its polyphagous nature. The accurate identification of B. dorsalis is challenging at the egg, maggot, and pupal stages, due to lack of distinct morphological characters and its similarity to other fruit flies. Adult identification requires specialized taxonomist. Existing identification methods are laborious, time consuming, and expensive. Rapid and precise identification is crucial for timely management. By analyzing the variations in the mitochondrial cytochrome oxidase-1 gene sequence (Insect barcoding gene), we developed a species-specific primer (SSP), DorFP1/DorRP1, for accurate identification of B. dorsalis. The optimal annealing temperature for the SSP was determined to be 66°C, with no cross-amplification or primer-dimer formation observed. The SSP was validated with B. dorsalis specimens from various locations in northern and eastern India and tested for cross-specificity with six other economically significant fruit fly species in India. The primer specificity was further confirmed by the analysis of critical threshold (Ct) value from a qPCR assay. Sensitivity analysis showed the primer could detect template DNA concentrations as low as 1 pg/µl, though sensitivity decreased at lower concentrations. Sequencing of the SSP-amplified product revealed over >99% similarity with existing B. dorsalis sequences in the NCBI GenBank. The developed SSP reliably identifies B. dorsalis across all developmental stages and sexes. This assay is expected to significantly impact pest identification, phytosanitary measures, and eradication programs for B. dorsalis.
摘要：
东方果蝇，dorsalis（Hendel），由于其多食性，是一种重要的经济和检疫性害虫。对蛋的准确识别是一个挑战，法师,和蛹阶段，由于缺乏独特的形态特征及其与其他果蝇的相似性。成人身份识别需要专门的分类学家。现有的识别方法费力，耗时,而且昂贵。快速准确的识别对于及时管理至关重要。通过分析线粒体细胞色素氧化酶-1基因序列（昆虫条形码基因）的变异，我们开发了一种物种特异性引物(SSP)，DorFP1/DorRP1，用于精确鉴别背芽孢杆菌。SSP的最佳退火温度确定为66°C，没有观察到交叉扩增或引物二聚体形成。SSP已通过来自印度北部和东部不同地点的B.dorsalis标本进行了验证，并与印度其他六种具有经济意义的果蝇物种进行了交叉特异性测试。通过分析来自qPCR测定的临界阈值(Ct)值进一步证实引物特异性。灵敏度分析显示引物可以检测模板DNA浓度低至1pg/µl，虽然灵敏度在较低浓度时下降。SSP扩增产物的测序显示与NCBIGenBank中现有的背芽孢杆菌序列的相似性超过99%。开发的SSP可靠地识别所有发育阶段和性别的背芽孢杆菌。预计该测定将对害虫鉴定产生重大影响，植物检疫措施,和背芽孢杆菌的根除计划。
