Nghiên cứu ứng dụng kỹ thuật LAMP phát hiện nhanh vi khuẩn E. coli gây bệnh tiêu chảy ở người

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Thông tin xuất bản: Tạp chí Khoa học và Công nghệ Việt Nam, 2020

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Hiện nay, có rất nhiều phương pháp khác nhau để phát hiện vi sinh vật gây bệnh, bao gồm cả những phương pháp truyền thống và phương pháp phân tử hiện đại. Tuy nhiên, trong chẩn đoán tiêu chảy do vi khuẩn Esherichia coli, các phương pháp phân lập trên môi trường chọn lọc, thử các tính chất sinh, hóa giúp phát hiện vi khuẩn E. coli nhưng không thể phân biệt giữa các vi khuẩn E. coli hội sinh cư trú bình thường ở đường ruột với các vi khuẩn E. coli mang gen độc lực gây bệnh. Bài báo này đề cập đến phương pháp LAMP (Loop-mediated isothermal amplification) khuếch đại DNA với độ đặc hiệu và độ nhạy cao được ứng dụng để phát hiện nhanh vi khuẩn E. coli ở giới hạn phát hiện là 15 pg, cao gấp hơn 100 lần so với phương pháp PCR. Một bộ 4 cặp mồi đặc hiệu được thiết kế để khuếch đại đoạn gen eae là một trong những gen độc lực của vi khuẩn E. coli có kích thước 288 bp trong DNA genome vi khuẩn và với sự có mặt chất chỉ thị kim loại calcein (C30H26N2O13). Phản ứng diễn ra ở điều kiện đẳng nhiệt 60oC trong khoảng 30 đến 60 phút, sản phẩm phản ứng là các băng DNA phân bố giống như dạng bậc thang với kích thước thấp nhất là băng cơ sở 288 bp khi điện di trên gel agarose. Kết quả phản ứng có thể được quan sát bằng mắt thường do sự thay đổi màu của dung dịch phản ứng từ màu cam (âm tính) sang màu xanh huỳnh quang (dương tính). Do đó đây là một kỹ thuật nhạy và đặc hiệu, có khả năng phát hiện nhanh, chính xác vi khuẩn E. coli gây bệnh có mang gen độc tố eae. Phương pháp phát hiện này mở ra tiềm năng ứng dụng trong nghiên cứu cơ bản về y học và dược phẩm, vệ sinh môi trường, xét nghiệm điểm chăm sóc..., Tóm tắt tiếng anh, Nowadays, many techniques are used for detecting pathogenic agents, including both traditional methods and modern molecular techniques. However, pathogenic Escherichia coli are not distinguishable from other common strains due to their appearance on culture plates or by the results of the usual biochemical tests. In this study, a new method called LAMP (Loop-mediated isothermal amplification) was applied to amplify DNA with high specificity and sensitivity at the detection limit of 15 pg, which was over 100 times higher than that of the PCR method. Four specific primers were designed to amplify the eae gene frogment - one of the toxic genes of E. coli with the length of 288 bp. Calcein (C30H26N2O13) is a fluorescent metal indicator for Magnesium detection. In this case, Calcein was initially quenched by manganese, then it emitted a yellowish-green fluorescence. In eae LAMP reaction, a large amount of DNA was synthesized, yielding a large pyrophosphate ion by-product. LAMP-generated pyrophosphate preferentially precipitated manganese, resulting in a complex and visible calcein-magnesium as bright green fluorescence. So a positive reaction was observed by a colour change from orange to green with the naked eyes after completion at 60°C for at least 30 min or under UV light detection. As the signal recognition is highly sensitive, this system enables visual discrimination of results without costly specialized equipment. This detection method reveals a great potential in basic research on medicine and pharmacy, environmental hygiene, point-of-care testing etc.

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