探討利用PCR技術鑑別遺傳工程庫斯蘇力菌EG7841菌株

Use of Novel Primers and PCR Technology to Identify Genetically Engineered Bacillus thuringiensis subsp. kurstki EG7841

謝玉貞(Yu-Chen Hsieh) ； 林芳妘(Fang-Yun Lin) ； 蔡韙任(Wei-Ren Tsai)

《臺灣農藥科學》 5期 (2018/12) Pp. 75-89

https://doi.org/10.6671/TPS.201812_(5).05

摘要

蘇力菌（Bacillus thuringiensis，Bt）為全世界最常使用之生物農藥，內含的殺蟲結晶蛋白分別對某些鱗翅目、雙翅目和鞘翅目昆蟲具有毒效，是一種已發展成熟且被廣泛使用的微生物殺蟲劑。目前在臺灣登記上市的多種蘇力菌生物農藥產品，又可分成庫斯蘇力菌（Bt subsp. kurstaki，Btk）和鮎澤蘇力菌（Bt subsp. aizawai，Bta），但其中有一Btk EG7841菌株產品是屬於遺傳工程蘇力菌。本研究針對此菌株設計BTSS-F4與BTSS-R2專一性引子對，利用聚合酶連鎖反應（polymerase chain reaction，PCR）進行檢測分析，並針對9種不同品系蘇力菌，包括Btk EG2371、Btk ABTS351、Btk SA12、Btk E911、Bta GC91、Bta ABTS1857、Bta NB200、Btk SA11和Btk EG7841菌株，進行PCR分析。結果此專一性引子對，僅對Btk EG7841增幅出特異性的858bp核酸片段，而其他品系蘇力菌菌株則無，顯示此專一性引子對可用來快速檢測該特定菌株遺傳工程基因片段。利用此引子對進行田間十字花科甘藍葉及土壤樣本中Btk EG7841菌PCR檢測，結果顯示噴施Btk EG7841菌株後1個月仍可於甘藍葉及土壤測得Btk EG7841遺傳工程基因片段（858bp），於第63天則無檢出。另在臺灣中部地區採集田間土壤，進行該菌株之Btk EG7841遺傳工程基因片段環境流佈調查，分析100處土壤樣本，結果有30個樣本檢出蘇力菌基因片段，但均尚未檢出Btk EG7841遺傳工程基因片段。未來可利用此檢測方法，持續監控此菌株於環境中流佈之情形。

關鍵字

蘇力菌 ； 聚合酶連鎖反應 ； 遺傳工程 ； 殺蟲結晶蛋白質 ； cry1C基因

並列摘要

Bacillus thuringiensis is a biological insecticide that is among the most commonly used in the world. The insecticidal crystal protein it contains is toxic to certain lepidopteran, dipteran and coleopteran insects. At present, a variety of Bacillus thuringiensis bio-pesticide products are registered in Taiwan, and these products can be subdivided into Bt subsp. kurstaki (Btk) and Bt subsp. aizawai (Bta). Of these, Btk EG7841 is a genetically recombinant strain. In this study, we designed novel BTSS-F4 and BTSS-R2 primer pairs that can be employed to detect Btk EG7841 using polymerase chain reaction (PCR). To test the specificity of these primer pairs, nine strains of Bacillus thuringiensis, including Btk EG2371, Btk ABTS351, Btk SA12, Btk E911, Bta GC91, Bta ABTS1857, Bta NB200, Btk SA11, and Btk EG7841 were analyzed by PCR. Results of this analysis showed that the specific primers only successfully increased the specific nucleic acid fragment by 858 bp to the Btk EG7841 strain. This confirms that our proposed primer pairs can be used to quickly identify genetically engineered Btk EG7841 gene fragments. Hence it is applied to the establishment of the field gene environment distribution detection method. We then sought to determine whether the field gene distribution detection method could be used as a residual regression test to detect Btk EG7841 gene fragments in leaf and soil samples from cabbage fields. Results showed that, after spraying the cabbage field with Btk EG7841, Btk EG7841 gene fragments could still be detected in the leaf and soil samples within 1 month, but could not be detected on the 63rd day. In addition, 100 soil samples were collected from central Taiwan in order to perform an environmental flow investigation of Btk EG7841 gene fragments. Results of this investigation revealed that, although 30 samples contained Bt, genetically modified Btk EG7841 gene fragments were not found in any of the 100 samples. In the future, the methods for Btk EG7841 detection proposed in this study may be used to monitor the emergence of this strain in target areas.

並列關鍵字

Bacillus thuringiensis ； Polymerase chain reaction ； Genetic engineering ； Insecticidal crystalline protein ； cry1C gene