Rapid Species-specific PCR Identification of Bactrocera tau (Diptera: Tephritidae)
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摘要:
目的 快速鉴定我国进境植物检疫性害虫南瓜实蝇Bactrocera tau（Walker），防止该虫传入扩散。 方法 基于物种特异性PCR（SS-PCR）技术，选取20种形态近似的常见实蝇，将南瓜实蝇作为阳性对照，提取基因组DNA模板，选用mt DNA COⅠ序列，检查同源系列，设计筛选1对可快速准确鉴定南瓜实蝇的种特异性引物NF404和NR610，并进行PCR检测和验证，建立一种利用种特异性引物的快速鉴定方法。 结果 南瓜实蝇在207 bp的位置扩增出一条清晰且单一的目标条带，其余果实蝇未见条带。将截获的南瓜实蝇与其同亚属的近缘种具条实蝇、瓜实蝇的不同虫态和成虫部分虫体组织的虫样，采用本实验室建立的SS-PCR鉴定方法进行验证，结果一致。 结论 建立的南瓜实蝇的SS-PCR鉴定方法种特异性和稳定性强，能快速鉴定目标种类，解决口岸进境果蔬中截获实蝇幼虫、蛹等不同虫态和残体难于快速鉴定问题。 -
关键词:
- 南瓜实蝇 /
- mt DNA COI /
- 种特异性引物 /
- 物种特异性PCR(SS-PCR) /
- 快速鉴定
Abstract:Objective A rapid method to detect Bactrocera tau (Walker) was established for inspection on produce to prevent potential introduction and spread of the invasive pest. Method Based on the species-specific PCR (SS-PCR) technology, B. tau as control and 20 common fruit flies of similar morphology were used to extract the genomic DNA templates, and the homology of the specimens checked with the COI mt DNA sequence. A rapid and accurate identification method applying the B. tau-specific primers NF404 and NR610 was designed and verified by PCR amplification and electrophoresis analysis. Result The newly developed SS-PCR detection method clearly amplified a single 207 bp band only on the target B. tau, not on any other fruit flies. Comparisons on the test results on the insects or body parts of all fruit flies with homogenous band sequence to B. tau validated the new methodology. Conclusion The newly established SS-PCR method exhibited high repeatability, specificity, and accuracy in detecting B. tau. It was considered applicable for the entry/exit and quarantine inspection on the invasive insect, larvae or dead body parts found on vegetables and fruits. -
图 1 利用LCO1490和HC02198 一对通用型引物对提取的实蝇DNA模板进行质量检测的结果
注：M：50 bp DNA Ladder，1：南瓜实蝇，2：洋桃实蝇，3：瓜实蝇，4：辣椒实蝇，5：腿端黑实蝇，6：番石榴果实蝇，7：二颜带实蝇，8：桔小实蝇，9：锈红果实蝇实蝇, 10：瘤胫实蝇，11：具条实蝇，12：何氏华实蝇，13：近黑颜实蝇，14：黑颜实蝇，15：黑膝实蝇，16：滇寡鬃实蝇，17：瑞丽果实蝇，18：五指山实蝇，19：瓜棍腹实蝇，20：枣实蝇，21：空白对照（双蒸水）。
Figure 1. Utilization of universal primers, LCO1490 and HC02198, for testing extracted DNA templates from fruit flies
Note: M: 50 bp DNA ladder; 1: B. tau; 2: B. carambolae; 3: B. cucurbitae; 4: B. latifrons; 5: B. atrifemur; 6: Bactrocera correcta; 7: B. cilifera; 8: B. dorsalis; 9: B. rubigina; 10: B. tuberculata; 11: B. scutellata; 12: B. hochii; 13: B. parater; 14: B. diaphora; 15: B. scutellaris; 16: B. modica; 17: B. ruiliensis; 18: B. wuzhishana; 19: Dacus longicornis; 20: Carpomya vesuviana; 21: CK (dd H2O).
图 2 8种果实蝇的ClustalW多重比对的部分结果（种特异性引物NF’404和NR’610序列位置用横线划出）
Figure 2. Partial result of ClustalW multiple comparisons on 8 fruit flies
图 3 引物NF404和NR610的种特异性验证
注：M：100 bp DNA Ladder，1：南瓜实蝇，2：洋桃实蝇，3：瓜实蝇，4：辣椒实蝇，5：腿端黑实蝇，6：番石榴果实蝇，7：二颜带实蝇，8：桔小实蝇，9：锈红果实蝇实蝇, 10：瘤胫实蝇，11：具条实蝇，12：何氏华实蝇，13：近黑颜实蝇, 14：黑颜实蝇，15：黑膝实蝇，16：滇寡鬃实蝇，17：瑞丽果实蝇，18：五指山实蝇，19：瓜棍腹实蝇，20：枣实蝇，21：空白对照（双蒸水）。
Figure 3. Verification on species-specificity of primers, NF404 and NR610
Note: M: 50 bp DNA Ladder，1: B. tau，2: B. carambolae，3: B. cucurbitae，4: B. latifrons，5: B. atrifemur，6: Bactrocera correcta，7: B. cilifera，8: B. dorsalis，9: B. rubigina，10: B. tuberculata，11: B. scutellata，12: B. hochii，13: B. parater，14: B. diaphora，15: B. scutellaris，16: B. modica，17: B. ruiliensis，18: B. wuzhishana，19: Dacus longicornis，20: Carpomya vesuviana，21: CK (dd H2O).
图 4 引物NF404和NR610的灵敏度验证
注：M：100 bp DNA Ladder，1−100，2−10−1，3−10−2，4−10−3，5–空白对照
Figure 4. Verification on sensitivity of primers, NF404 and NR610
Note: M: 100 bp DNA ladder; 1: 100; 2: 10−1; 3: 10−2; 4: 10−3; 5: dd H2O.
图 5 SS-PCR快速鉴定南瓜实蝇的应用与验证
注：M: 100 bp DNA Ladder，1～17: 南瓜实蝇，18：具条实蝇，19：瓜实蝇B. cucurbitae，20：空白对照（双蒸水）
Figure 5. Application and verification of SS-PCR for rapid identification of B. tau
Note: M:100 bp DNA ladder; 1-17: B. tau; 18: B. scutellata; 19: B. cucurbitae; 20: CK (dd H2O).
表 1 设计南瓜实蝇特异性引物所用实蝇种类及登录号
Table 1. Species and accession numbers of fruit flies used for designing B. tau-specific primers
物种
Species登录号
Accession No.南瓜实蝇 B. tau JN542420.1 瓜实蝇 B. cucurbitae HM561566.1 普通果实蝇 B. caudata JN542417.1 具条实蝇 B. scutellata AY530891.1 瘤胫实蝇 B. tuberculata AF423105.1 地中海实蝇 Ceratitis capitata JX855917.1 桔小实蝇 B. dorsalis KF998674.1 洋桃实蝇 B. carambolae KF318598.1 
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