Jiang F, Li Z H, Deng Y L, Wu J J, Liu R S, Buahom N
Department of Entomology, College of Agronomy and Biotechnology, China Agricultural University, Yuanmingyuan West Road 2, Haidian District, Beijing 100193, P.R. China.
Bull Entomol Res. 2013 Jun;103(3):363-71. doi: 10.1017/S0007485312000806. Epub 2013 Mar 5.
The guava fruit fly, Bactrocera correcta (Bezzi) (Diptera: Tephritidae), is an invasive pest of fruit and vegetable crops that primarily inhabits Southeast Asia and which has the potential to become a major threat within both the Oriental and Australian oceanic regions as well as California and Florida. In light of the threat posed, it is important to develop a rapid, accurate and reliable method to identify B. correcta in quarantine work in order to provide an early warning to prevent its widespread invasion. In the present study, we describe a species-specific polymerase chain reaction assay for the diagnosis of B. correcta using mitochondrial DNA cytochrome oxidase I (mtDNA COI) barcoding genes. A B. correcta-specific primer pair was designed according to variations in the mtDNA COI barcode sequences among 14 fruit fly species. The specificity and sensitivity of the B. correcta-specific primer pair was tested based on the presence or absence of a band in the gel profile. A pair of species-specific B. correcta primers was successfully designed and named BCOR-F/BCOR-R. An ∼280 bp fragment was amplified from specimens belonging to 17 geographical populations and four life stages of B. correcta, while no such diagnostic bands were present in any of the 14 other related fruit fly species examined. Sensitivity test results demonstrated that successful amplification can be obtained with as little as 1 ng μl⁻¹ of template DNA. The species-specific PCR analysis was able to successfully diagnose B. correcta, even in immature life stages, and from adult body parts. This method proved to be a robust single-step molecular technique for the diagnosis of B. correcta with respect to potential plant quarantine.
番石榴实蝇，即瓜实蝇（Bactrocera correcta (Bezzi)，双翅目：实蝇科），是一种侵袭果蔬作物的入侵性害虫，主要分布于东南亚地区，有可能成为东洋区和澳大利亚大洋区以及加利福尼亚州和佛罗里达州的重大威胁。鉴于其所构成的威胁，开发一种快速、准确且可靠的方法以在检疫工作中鉴定瓜实蝇，从而提供早期预警以防止其广泛入侵至关重要。在本研究中，我们描述了一种利用线粒体DNA细胞色素氧化酶I（mtDNA COI）条形码基因诊断瓜实蝇的物种特异性聚合酶链反应检测方法。根据14种实蝇物种的mtDNA COI条形码序列的差异设计了一对瓜实蝇特异性引物。基于凝胶图谱中条带的有无来测试瓜实蝇特异性引物对的特异性和灵敏度。成功设计了一对瓜实蝇物种特异性引物，并命名为BCOR-F/BCOR-R。从瓜实蝇17个地理种群和四个生活阶段的标本中扩增出了一条约280 bp的片段，而在所检测的其他14种相关实蝇物种中均未出现此类诊断条带。灵敏度测试结果表明，仅需1 ng μl⁻¹的模板DNA就能成功扩增。这种物种特异性PCR分析即使对于未成熟生活阶段以及成虫身体部位的瓜实蝇也能够成功诊断。就潜在的植物检疫而言，该方法被证明是一种用于诊断瓜实蝇的强大的单步分子技术。
