TẦN SỐ XUẤT HIỆN VIBRIO CHOLERAE TRÊN TÔM VÀ NHUYỄN THỂ, XÁC ĐỊNH SEROGROUP O1, O139 VÀ BIOTYP CỦA V. CHOLERAE BẰNG KỸ THUẬT M – PCR

BỘ GIÁO DỤC VÀ ĐÀO TẠO TRƯỜNG ĐẠI HỌC NÔNG LÂM TP HỒ CHÍ MINH NGUYỄN THỊ XUÂN TRANG TẦN SỐ XUẤT HIỆN VIBRIO CHOLERAE TRÊN TÔM VÀ NHUYỄN THỂ, XÁC ĐỊNH SEROGROUP O1, O139 VÀ BIOTYP CỦA V CHOLERAE BẰNG KỸ THUẬT M – PCR Chuyên ngành: Thú y Mã số: 60 62 50 LUẬN VĂN THẠC SỸ KHOA HỌC NƠNG NGHIỆP Thành phố Hồ Chí Minh - Tháng 10/2011 BỘ GIÁO DỤC VÀ ĐÀO TẠO TRƯỜNG ĐẠI HỌC NƠNG LÂM TP HỒ CHÍ MINH NGUYỄN THỊ XN TRANG TẦN SỐ XUẤT HIỆN VIBRIO CHOLERAE TRÊN TÔM VÀ NHUYỄN THỂ, XÁC ĐỊNH SEROGROUP O1, O139 VÀ BIOTYP CỦA V CHOLERAE BẰNG KỸ THUẬT M – PCR Chuyên ngành: Thú y Mã số: 60 62 50 LUẬN VĂN THẠC SỸ KHOA HỌC NÔNG NGHIỆP Hướng dẫn khoa học PGS TS NGUYỄN NGỌC TUÂN Thành phố Hồ Chí Minh - Tháng 10/2011 TẦN SỐ XUẤT HIỆN VIBRIO CHOLERAE TRÊN TÔM VÀ NHUYỄN THỂ, XÁC ĐỊNH SEROGROUP O1, O139 VÀ BIOTYP CỦA V CHOLERAE BẰNG KỸ THUẬT M- PCR NGUYỄN THỊ XUÂN TRANG Hội đồng chấm luận văn: Chủ tịch: PGS TS NGUYỄN NGỌC HẢI Đại học Nông Lâm TP Hồ Chí Minh Thư ký: TS TRẦN THỊ BÍCH LIÊN Đại học Nơng Lâm TP Hồ Chí Minh Phản biện 1: TS HỒ THỊ KIM HOA Đại học Nông Lâm TP Hồ Chí Minh Phản biện 2: TS LÊ HỒNG PHƯỚC Viện Nghiên cứu Nuôi trồng Thủy sản II Ủy viên: TS NGUYỄN BÁ THÀNH Trung tâm Kiểm tra Vệ sinh Thú y Trung ương II i LÝ LỊCH CÁ NHÂN Tôi tên Nguyễn Thị Xuân Trang sinh ngày 20 tháng 04 năm 1983 huyện Đức Hòa, tỉnh Long An Tốt nghiệp phổ thơng trung học trường THPT Nguyễn Trãi, tỉnh Bình Dương, năm 2001 Tốt nghiệp Đại học ngành Bác sỹ Thú y hệ quy Đại học Nơng Lâm, Thủ Đức, thành phố Hồ Chí Minh, năm 2007 Tháng 9/2007 – 7/2009: làm việc Nhà máy Chế biến thực phẩm Đồng Nai, chức vụ: trưởng phận thu mua nguyên liệu, phòng Kinh doanh tổng hợp Tháng 10 năm 2009 theo học Cao học ngành Thú y Đại học Nơng Lâm, Thủ Đức, thành phố Hồ Chí Minh Địa liên lạc: 760 tổ 10, ấp Tràm Lạc, xã Mỹ Hạnh Bắc, huyện Đức Hòa, tỉnh Long An Điện thoại: 0974 084454 – 0906 487126 Email: Xuantrangty09@gmail.com ii LỜI CAM ĐOAN Tôi cam đoan công bố luận văn trung thực phần đề tài cấp mã số B2010 – 12 – 95 PGS TS Nguyễn Ngọc Tuân làm chủ nhiệm.Những số liệu luận văn phép công bố với đồng ý chủ nhiệm đề tài Xác nhận Chủ nhiệm đề tài Học Viên iii LỜI CẢM ƠN Chân thành cảm tạ Ban Giám Hiệu Trường Đại Học Nơng Lâm Thành phố Hồ Chí Minh Ban chủ nhiệm tồn thể q thầy Khoa Chăn ni Thú y Trường Đại học Nông Lâm Thành phố Hồ Chí Minh tận tình truyền đạt kiến thức cho suốt thời gian học tập trường Quý Thầy Cơ cán phòng Đào tạo sau đại học Trường Đại học Nơng Lâm Thành phố Hồ Chí Minh nhiệt tình giúp đỡ hướng dẫn tơi suốt thời gian học tập q trình hồn thành luận văn Thành kính ghi ơn PGS.TS Nguyễn Ngọc Tuân tận tình giúp đỡ suốt thời gian học tập, thực đề tài hoàn thành luận văn Thầy Người ln động viên cho lời khuyên bổ ích để vuợt qua khó khăn sống Trân trọng cảm ơn Phòng thực tập Kiểm nghiệm thú sản Môi trường sức khỏe vật nuôi, Khoa Chăn nuôi Thú Y, Trường Đại học Nông Lâm Thành phố Hồ Chí Minh; Cơng ty Cổ phần cơng nghệ Việt Á tạo điều kiện tốt để thực đề tài Gửi lời cảm ơn đến Những anh chị, bạn bè lớp Cao học Thú y 2009 chia sẻ buồn, vui trình học tập, thực đề tài sống Nguyễn Thị Xuân Trang iv DANH SÁCH CÁC TỪ VIẾT TẮT ABC ATP-binding cassette Ace: Accessory cholera toxin ADP adenosine diphosphate ATP adenosine triphosphate bp base pairs cAMP cyclic adenosine monophosphate CDC: Centers for Disease Control and Prevention: trung tâm kiểm sốt phòng bệnh CT: Cholera toxin: độc tố gây tiêu chảy CTXφ Cholera toxin phages ICE integrative conjugating elements IDSA Infectious Diseases Society of America MATE multidrug and toxic compound extrusion MFS major facilitator superfamily NAD nicotiamide adenine dinucleotide ORF Open reading frame: khung đọc mở PMF proton-motive force RND resistance nodulation cell division SMR small multidrug resistance ST Heat- stable toxin: độc tố chịu nhiệt TCP Toxin-Coregulated Pilus: yếu tố kết bám liên quan đến pili TDH Thermostable direct hemolysin: yếu tố gây dung huyết trực tiếp VPI V cholerae pathogencity island WHO World Heath Orgazination: Tổ chức sức khỏe giới Zot Zonula occludens toxin v TÓM TẮT Tần số xuất Vibrio choleraetrên tôm nhuyễn thể, xác định serogroup O1, O139 biotyp V cholerae O1 kỹ thuật m – PCR Vibrio choleraelà tác nhân gây ngộ độc thực phẩm nước gây bệnh dịch tả người Lồi vi khuẩn thường tìm thấy sản phẩm thủy hải sản Đề tài nghiên cứu xuất loài vi khuẩn động vật thủy hải sản xác định serogroup biotyp chúng kỹ thuật m – PCR Đề tài thực 315 mẫu tôm nhuyễn thể thu thập Đồng Nai, TP Hồ Chí Minh Mẫu tăng sinh hai dung dịch muối pepton kiềm khơng có có bổ sung polymycin B 50 UI % NaCl DNA ly trích trực tiếp từ dung dịch tăng sinh kiểm tra phản ứng m – PCR để xác định (i) loài V cholerae (m – PCR1); (ii) serogroup O1, O139 (m – PCR2) (iii) biotyp (m – PCR3) Bên cạnh đó, gốc vi khuẩn định danh phương pháp sinh hóa xác định lại phản ứng m – PCR Một số gốc thử tính nhạy cảm với số kháng sinh Phản ứng m – PCR1 phát V cholerae 44,13% số mẫu môi trường 45,40% số mẫu môi trường Trong đó, phương pháp sinh hóa phát V choleraeở hai môi trường 41,27% 10,16% Phản ứng m – PCR2 sử dụng DNA ly trích trực tiếp từ dung dịch tăng sinh, kết phát gien từ 139 mẫu môi trường 143 mẫu môi trường mẫu dương tính đồng thời hai gien ctxA rfbO1, 11 14 mẫu dương tính đồng thời hai gien ctxA rfbO139, mẫu dương tính với rfbO1, mẫu dương tính với rfbO139, 17 24 mẫu dương tính với ctxA Ngồi ra, mơi trường tăng sinh giúp phát hai mẫu dương tính đồng thời gien ctxA, rfbO1 rfbO139 - Sử dụng DNA gốc định danh, kết m – PCR2 phát serogroup từ 130 mẫu môi trường 32 mẫu môi trường vi mẫu dương tính với V cholerae O139 sinh độc tố CT, 15 mẫu dương tính với V cholerae O1/O139 không sinh độc tố CT, mẫu dương tính với V cholerae non – O1/O139 sinh độc tố CT Kết phản ứng m – PCR3 cho thấy mẫu phát gien tcpA đặc trưng cho V cholerae O1 biotyp El Tor Các gốc V cholerae định danh đề kháng hoàn toàn với colistine, đề kháng cao với oxacillin nhạy cảm doxycycline norfloxacin vii SUMMARY Occurence of Vibrio cholerae in shrimp and shellfish, identification of O1 and O139 serogroups and biotypes of V cholerae by multiplex PCR assays Vibrio cholerae is a foodborne and waterborne pathogen causing cholera disease in human The bacteria have been also found to be associated with seafood This research was carried out to study the occurrence of this pathogen in seafood animals and their serogroups (O1 and O139) and biotypes by m-PCR A total of 315 samples of market shrimp and shellfish were collected from Dong Nai province and Ho Chi Minh City Two enrichment media were used: ASPW (alkaline salt peptone water) and ASPW supplemeted with polymycin B 50 UI and % NaCl DNA extracted from enriched samples were examined by three m – PCR reactions to identify (i) the species of V cholerae (m – PCR1); (ii) the O1 and O139 serogroups (m – PCR2); and the biotypes (m – PCR3) Besides, isolates from enriched media were examined using conventional methods (biochemical tests) and m – PCRs The isolates were studied for their antibiotic resistances By m – PCR1 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Lạc, xã Mỹ Hạnh Bắc, huyện Đức Hòa, tỉnh Long An Điện thoại: 0974 084454 – 0906 487126 Email: Xuantrangty09@gmail.com ii LỜI CAM ĐOAN Tôi cam đoan công bố luận văn trung thực phần đề tài cấp mã... human The bacteria have been also found to be associated with seafood This research was carried out to study the occurrence of this pathogen in seafood animals and their serogroups (O1 and O139)... hightly resistant to oxacillin; and were sensitive to doxycycline and norfloxacin ix MỤC LỤC Trang Trang chuẩn y i Lý lịch cá nhân ii Lời cam đoan iii