Nghiên cứu phát triển quy trình real time pcr phát hiện bệnh do vi bào tử trùng enterocytozoon hepatopenaei ehp gây ra trên tôm

BAN QUẢN LÝ KHU NÔNG NGHIỆP CÔNG NGHỆ CAO TP HCM TRUNG TÂM NGHIÊN CỨU VÀ PHÁT TRIỂN NÔNG NGHIỆP CÔNG NGHỆ CAO BÁO CÁO NGHIỆM THU NGHIÊN CỨU PHÁT TRIỂN QUY TRÌNH REAL-TIME PCR PHÁT HIỆN BỆNH DO VI BÀO TỬ TRÙNG Enterocytozoon hepatopenaei (EHP) GÂY RA TRÊN TÔM ThS Nguyễn Phạm Trúc Phương Thành phố Hồ Chí Minh, tháng 01/2018 BAN QUẢN LÝ KHU NÔNG NGHIỆP CÔNG NGHỆ CAO TP HCM TRUNG TÂM NGHIÊN CỨU VÀ PHÁT TRIỂN NÔNG NGHIỆP CÔNG NGHỆ CAO BÁO CÁO NGHIỆM THU (Đã chỉnh sửa theo góp ý Hội đờng nghiệm thu) NGHIÊN CỨU PHÁT TRIỂN QUY TRÌNH REAL-TIME PCR PHÁT HIỆN BỆNH DO VI BÀO TỬ TRÙNG Enterocytozoon hepatopenaei (EHP) GÂY RA TRÊN TƠM CƠ QUAN CHỦ TRÌ (Ký tên, đóng dấu xác nhận) CHỦ NHIỆM ĐỀ TÀI (Ký tên) Thành phố Hồ Chí Minh, tháng 01/2018 T Enterocytozoon phát hepatopenaei (EHP) SWP2F/SWP2R 300nM/ SWP 200nm 950 C giây; 650C 30 giây X Enterocytozoon -time PCR hepatopenaei 50 copies n (100%), g bi n thiên n i ph n ng c a quy trình real-time PCR EHP DNA có %CV t bi n thiên liên ph n ng th p nh t 0,582%, h s bi n thiên liên ph n ng cao nh t 1,427% Enterocytozoon hepatopenaei, EHP, SWP, real-time PCR, ABSTRACT A TaqMan probe real-time PCR assay for rapid detection of Enterocytozoon hepatopenaei is based on a gene encoding a spore wall protein (SWP) of EHP, which has appeared on shrimp in recent years The optimum concentration of primers SWP2 F, SWP2-R were 300nM, 200nM, respectively; and that of the TaqMan probe was 400 nM in the TaqMan-based EHP qPCR amplification mixture PCR amplification was performed for at 95°C, followed by 40 cycles for 5s at 95°C, 30s at 65°C The detection limit of the qPCR method was as low as 50 copies/ reaction and analytical specificity was 100% The intra-assay coefficient of variation were 0,612% to 1,202% The inter-assay coefficient of variation were 0,582% (minimum) and 1,427% (maximum) Enterocytozoon hepatopenaei, EHP, SWP, real-time PCR, ii Trang i tài ii iii vii viii xi 1.1 1.2 Microsporidia 15 1.2.1 15 1.2.2 17 1.3 18 1.4 19 24 24 24 24 24 2.2.3 Thi 24 25 2.3.1 Thi t k primer probe c hi phát hi n vi bào 25 t trùng Enterocytozoon hepatopenaei (EHP) 25 2.3.1.2 25 iii 2.3.1.3 Ki m tra c p m i probe phát hi n EHP 25 a 25 in silicon b c Ki ho d Ki m tra c hi u c a m i probe 28 ng th c t c a m i probe 28 28 2.3.1.4 Ch tiêu theo dõi 2.3.2 28 : Xây d ng t -time PCR phát 28 hi n vi bào t trùng Enterocytozoon hepatopenaei (EHP) gây tơm 2.3.2 28 thí 28 a Ly trích DNA 29 b 29 c 31 d 32 e 33 2.3.3 N i dung 3: nh thơng s k thu t c a quy trình real-time 34 PCR phát hi n vi bào t trùng Enterocytozoon hepatopenaei (EHP) gây tôm 2.3.3.1 M c tiêu 34 2.3.3.2 Mơ t thí nghi m 34 ng chu n c hi u k thu t 34 35 nh y k thu t 35 l pl i 36 2.3.4 N i dung 4: Th nghi m quy trình real-time PCR phát hi n m u 36 tôm nhi m vi bào t trùng Enterocytozoon hepatopenaei (EHP) 2.3.4.1 M c tiêu 36 2.3.4.2 Mơ t thí nghi m 36 iv 37 3.1 Thi t k primer probe c hi phát hi n vi bào 37 phát hi n vi 37 t trùng Enterocytozoon hepatopenaei (EHP) bào t Enterocytozoon hepatopenaei (EHP) probe phát hi n vi 44 phát hi n vi bào t 50 real-time PCR phát 55 bào t trùng Enterocytozoon hepatopenaei (EHP) trùng Enterocytozoon hepatopenaei (EHP) 3.2 Enterocytozoon hepatopenaei (EHP) gây tôm 3.2.1 T m i 55 3.2.2 T probe 60 3.2.3 T lai c a m i m u dò 62 3.2.4 Kh o sát s chu k nhi t 63 3.3 N 65 nh thông s k thu t c a quy trình real-time PCR phát hi n vi bào t trùng Enterocytozoon hepatopenaei (EHP) gây tôm c hi u k thu t 3.3.3 65 nh y k thu t 68 l pl i 71 3.3.3.1 H s bi n thiên n i ph n ng 71 3.3.3.2 H s bi n thiên liên ph n ng 72 3.4 N i dung 4: Th nghi m quy trình real-time PCR phát hi n m u 74 tôm nhi m vi bào t trùng Enterocytozoon hepatopenaei (EHP) 79 79 v 79 80 xiv vi EHP Enterocytozoon hepatopenaei LOD Limit Of Detection PCR Polymerase Chain Reaction SWP Spore Wall Protein vii T Trang 2.1 Trình t c p m i cho ph n ng Real-time PCR 26 2.2 Trình t amplicon SWP beta-actin 27 2.3 T l n c cho gen SWP m i xuôi m 2.4 30 -time PCR 2.5 30 -time PCR 31 2.6 Thành ph n ph n ng real-time PCR t 31 2.7 Chu trình nhi t cho real-time PCR t 32 2.8 Thành ph n ph n ng real-time PCR t 2.9 Chu trình nhi t cho real-time PCR t 2.10 Thành ph n ph n ng real-time PCR t 2.11 Chu trình nhi t cho real-time PCR t 3.1 K t qu ki nhi 32 33 chu k nhi t nhi t c tính c a c p m i probe 3.2 33 34 38 46 SWP SWP1F/SWP1R3.3 56 tiêu gen SWP DNA plasmid 10 copies 3.4 58 tiêu gen SWP 3.5 60 SWP DNA plasmid 10 copies 3.6 61 SWP 3.7 DNA plasmid 103 copies viii 62 3.8 3.9 K t qu t chu k nhi t n 63 DNA plasmid khác 64 3.10 K t qu t chu k nhi t DNA ly trích t DNA tơm i EHP 3.11 K t qu kh n 64 khác nh y c a h m i SWP2F/ SWP2R 68 Taqman probe SWP2 3.12 - 70 tiêu 3.13 3.14 K t qu chu k 70 ng kh bi n thiên n i ph n ng c a quy trình real-time PCR EHP DNA plasmid n 3.15 71 khác K t qu chu k ng kh ng c a quy trình real-time PCR EHP bi n thiên n i ph n 72 c ly trích t i EHP 3.16 K t qu chu k ng kh bi n thiên n i ph n ng c a quy trình real-time PCR EHP DNA plasmid n 3.17 khác K t qu chu k ng kh ng c a quy trình real-time PCR EHP m 3.18 73 bi n thiên n i ph n 73 c ly trích t i EHP - 3.19 75 78 ix 4.1 Taqman vi Enterocytozoon hepatopenaei (EHP) 950 400nM C giây; 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