nghiên cứu sản xuất kit, chẩn đoán nhanh virus gây bệnh vàng lùn, lùn xoăn lá trên lúa

jlKWiinKiuMi* witii **i'iMi>,<i niiiiit iiiViiiii ilpi'iinjicj 2010 O NONG NGtllfeF VA PHAT TKMN NONG TH6N rKINII T'ROMG mim PHAT 1'RI£N VA (fNG DVNG CNSH TUOHG Kmiy Huml^p \k mwY rmin NONU TH6N GIAI DOAN imi»2o:ie iKtm.«.m4 KET QUA KHOA HOC CONCS N(JHJ& DE TM 't¥h:a¥.TAl: T.iT-f'ri. -^ii. t CIIU ir\ tliacm : 'Tnmg imn Nghien cim bao ve site khoe c&y trong m. vat mM (Lleis hiep c^c hot ICIi$liT VN : Plidf^g 3t)9 xM% B, Tnrtmg BH Ndog iiglilep If a. Not ; #438766491 E - mall vtITlall.20§6@¥a^soo,€oal*¥l1 n'Vv ? „ r' „ '"'/ 'k ?•.; HANOI:-6/2010 tSSWlSSWSSStSWtMsgt^^ BO NONG NGHIEP vA PHAT TRIEN NONG THON CHUOTVG TRINH TRQNG DIEM PHAT TRIEN VA UNG DUNG CNSH TRONG LiNH VUC NONG NGHIEP VA PHAT TRIEN NONG THON GIAI DOAN 2007-2010 k X \ BAO CAO TONG KET KET QUA KHOA HOC CONG NGHE DE TAI NGHIEN ClTU SAN XUAx KIT CHAN DOAN NHANH VIRUS GAY BENH VANG LUN, LUN XOAN LA TREN LUA Chu nhigm de tai. GS.TS. VQ Trieu Man Cc quan chu tri. Trung tam Nghien ciru bao ve sue khoe cay trong va vat nuoi (Lien hiep cac hoi KH$KT VN) Dia chi. Phong 309 nha B, Tru-ong DH Nong nghiep Ha Noi Dien thoai. 0438766491 E-mail. vtman2006@yahoo.com.vn HA NOI, 6/2010 Z>f f T (^ ^^ Bdo cao tong ket de tai san xuat KIT-ELISA virus RGSV & RRSV Loi cam an Chu nhiem de tai xin chan thanh cam on Ban Chii nhiem va Van phong Chirong trinh trong di^m phat trien va ung dung CNSH trong ITnh vyc Nong nghiep va phat tri8n nong thon giai doan 2007 - 2010, Bo Khoa hoc cong nghe, Bo Nong nghiep va Phat trien Nong thon da quan tam giup do va tao dieu kien cho chuang trinh nghien ciiu. Chiing toi cQng cam on Vien Bao ve thirc vat, Truong Dai hoc Can Tha va Vien nghien ciiu cay bong va phat trien nong nghiep Nha Ho, Cue Bao ve thuc vat va cac Trung tam, cac Chi Cue da giiip da dh tai trong chuang trinh nay. CO QUAN CHU TRI TRUNG TAM NGHIEN CUtJ SUC KH6E CT&VN CHU NHIEM DE TAI GS.TS. Vu Trieu Man w; IS.c^^^^^ Thai gian thirc hien. 2007 - 2009 Bdo cao tong ket de tai san xudt KIT-ELISA virus RGSV & RRSV PHAN 1: MO DAU Theri gian thuc hi6n. 2007 2009 Bdo cdo t6ng kit dl tdi sdn xudt KIT-ELISA virus RGSV & RRSV AMV - RT AS Bp BVTV CP cDNA DNA dNTP dsRNA DBSCL EDTA ELISA FAO HSP HVDT IgG IRRI KHT Kam Kb LB LC LXL PBS PBS-T PBS-T- PVP- PEG RNA RGSV PVP RRSV RTBV RT - RCR RTSV TAE VL CAC CHLT VIET TAT Avian myeloblastosis virus Reverse transcrptase Ammonium sulfate Base pair Bao ve thye vat Coat protein Complementary DNA Deoxyribonucleic acide Deoxy Nucleotide triphosphate Double stranded RNA Dong bang song Cmi Long Ethylene diaminetetra acetate Enzyme linked immuno sorbent assay Food and Agriculture Organization Heat shock protein Hien vi dien tii Immunoglobulin gama Intemational Rice Research Institute Khang huyet thanh Kanamycine Kilobase Luria-Bertani Liia CO Liin xodn la Phosphate buffer saline Phosphate buffer saline -with 0,05% Tween 20 O Phosphate buffer saline - Tween - Polyvinylpyrolindon Ovalbumin Polyethylene glycol Ribonucle acid Rice Grassy Stunt Vims Polyvinyl pyrrolindone Rice Ragged Stunt vims Rice Tungro Bacilliform vims Reverse transcriptase - PCR Rice Tungro Spherical Vims Tris acetate EDTA Vang liin Thai gian thuc hifn. 2007 - 2009 Bdo cdo tong ket dl tdi sdn xudt KIT-ELISA virus RGSV & RRSV MUC LUC • • Lai cam an i PHAN LMdDAu ii CAC CHU'VIET TAT iii MUC LUC iv DANH MUC BANG ix DANH MUC HINH xiv NHITNG NGUCil THAM GIA THUC HIEN DE TAI xviii BAO CAO THONG KE KET QUA THU'C HIEN DE TAI/DU' AN SXTN xix L THONG TIN CHUNG xix II. TINH HINH THU'C HIEN xx III. SAN PHAM KH&CN CUA DE TAI, DU AN xxvii DATVANDE 2 CHUONG 1 TONG QUAN TAI LIEU 5 I.I. Giai thieu chung ve benh vang lun va lun xoan laaliia 5 1.1.1. Benh vang lun va virus gay benh vang lun 5 Pham vi ki chii, trieu chung benh 6 Thiet hai do Rice grassy stunt virus (RGSV) 9 Cac dong virus, cau tnic th§ virus va ciu tnic phan tu bo gen RGSV 9 ciu tnic phan tu va dac tinh ly hoc 10 Clutnic bo gen 11 1.1.2. Giai thieu v^ benh liia lun xoan la va virus Rice ragged stunt oryzavirus (RRSV) 13 Lich su phat hien va phan b6 13 Ca chi truyen benh 14 ciu tnic phan tu ciia virus RRSV 15 Thai gian thuc hifn. 2007 2009 iv Bdo cdo tong ket de tai sdn xudt KIT-ELISA virus RGSV & RRSV Cac phuang phap chin doan 18 1.1.3.Tinhhinhbenh vang lun va benh liia lun xoin la a Viet Nam 19 Benh lun xoan la va vai tro cua virus RRSV 21 Mot so giai phap ciia Viet Nam tru6c thuc trang dich benh vang liin va lun xoan la 24 1.2. Nhiing nghien cuu ve phuang phap huyet thanh hoc trong chan doan virus thuc vat 25 1.2.1. Nguyen ly ciia phuang phap huyet thanh hoc va iing dung 25 1.2.2. Chi tao khang huylt thanh da dong thuc vat 26 CHUONG 2 MUC TIEU, NOI DUNG VA PHUONG PHAP NGHIEN Ciru 28 2.1. Muc tieu 28 2.2. Npi dung nghien cuu 28 2.3. Phuang phap nghien cuu 28 2.3.1. Cac phuang phap dieu tra thu thap mlu 28 2.3.2. Cac phuang phap phat hien benh 29 2.3.3. Phuang phap tinh chiet phan tu virus (Theo phuang phap dang dugc su dung tai IRRI) 32 2.3.4. Phuang phap tinh chiet protein NCP ciia RGSV (Miranda va Koganezawa, 1995) 33 2.3.5. Chilt tach RNA tdng s6 34 2.3.6. RT - PCR, PCR va giai trinh tu 35 2.3.7. Phan tich trinh tu 35 2.3.8.Phuang phap bien nap vao te bao vi khuan E.coU 36 2.3.9. Phuang phap bilu hien protem trong te bao vi khuin E.coli BL21 (DE3) 37 2.3.10. Phuang phap tao khang huylt thanh 44 2.3.11. Phuang phap kit tiia khang thi tu khang huylt thanh bing ammonium Thai gian thuc hien. 2007 - 2009 V Bdo cdo tong ket de tdi sdn xuat KIT-ELISA virus RGSV & RRSV sulfate (AS) 44 2.3.12. Tinh chilt protein cua cay va hap thu cheo 45 CHUONG III KET QUA NGHIEN CU'U vA THAO LUAN 47 3.1. KET QUA PHAT HIEN BENH 47 3.1.1. Phat hien benh bang phuang phap ELISA 48 3.1.2. Phat hien benh bang phuong phap kinh hien vi dien tu 53 3.2. NGHIEN CUU TAO KHANG HUYET THANH BANG PHAN TU' VIRUS VA PROTEIN VIRUS TU'NHIEN 54 3.2.1. Kilm tra su co mat cua virus tao vat lieu khai dau 54 3.2.2. Thu nghiem tao khdng huyet thanh RGSV dua tren phan tu virus (virion) va protein phi ciu tnic NCP 55 3.2.3. Thu nghiem tao khang huylt thanh RRSV dua tren phan tii virus (virion) 66 3.2.4. So sanh chit lugng khang huylt thanh phan tir RGSV ciia Viet Nam vai khang thi dac hieu RGSV ciia IRRI 78 3.3. NGHIEN CUU TAO KHANG HUYET THANH BANG PROTEIN TAI TO HOP 80 3.3.1. Lua chgn gen 80 3.3.2. RT-PCR 81 3.3.3. Dong hoa gen P6, PC5 (RGSV) va P9 (RRSV) bing vector pGEM-T- Easy 83 3.3.4. Dong hoa gen PC5 (RGSV) va P9 (RRSV) vao vector bilu hien pET28a 86 3.3.5. Cam ung bilu hien P9 va PC5 90 3.4. CAC NGHIEN CUU PHAN TlT VE RRSV vA RGSV 94 3.4.1. Vat lieu va phuang phap 94 3.4.2. Kit qua 95 3.4.3. Thao luan 100 Theri gian thirc hien. 2007 2009 vi oao cao tong ket de tdi sdn xudt KIT-ELISA vims RGSV & RRSV 3.5. KET QUA DIEU TRA PHAT HIEN BENH VIRUS HAI LUA QUA CACTHir NGHIEM KIT ELISA 103 3.5.1. Kit qua dilu tra benh virus hai liia tai VTnh Long nam 2007 104 3.5.2. Tai tinh Hau Giang nam 2007 106 3.5.3. Tai tinh Soc Trang nam 2007 108 3.5.4. Tai tinh Ba Ria-Vung Tau nam 2007 109 3.5.5. Tai tinh Binh Thuan nam 2007 110 3.5.6. Tai tinh Ninh Thuan nam 2007 111 3.5.7. Tai tinh Daklak nam 2007 112 3.5.8. Tai tinh nSm tinh Tien Giang, Ben Tre, Long An, An Giang va Dong Thap 113 3.5.9. Tai tinh Vmh Long (Vu Dong xuan 2008) 114 3.5.10. Tai tinh Hau Giang (Vu Dong xuan 2008) 116 3.5.11. Tai tinh Soc Trang (Vu Dong xuan 2008) 118 3.5.12. Tai thanh ph6 Cin Tha (Vu Dong xuan 2008) 119 3.5.13. Tai tinh Vmh Long (vu He Thu 2008) 121 3.5.14. Tai tinh Hau Giang (vu He Thu 2008) 123 3.5.15. Tai tinh Soc Trang (vu He Thu 2008) 124 3.5.16. Tai Thanh pho Cin Tha (vu He Thu 2008) 126 3.5.17. Ket qua chung ve tinh hinh benh lun xoan la, vang liin va dang liia co a Vu He Thu 2008 tai cac tinh Vinh Long, Hau Giang, Soc Trang va Thanh ph6 cin Thathugc DBSCL 128 3.5.18. Tai tinh Ba Ria-Vung Tau 129 3.5.19. Tai tinh Binh Thuan 130 3.5.20. Tai tinh Daklak 130 3.5.21. Ty le benh virus liia tai 5 tinh Tiln Giang, Bin Tre, Long An, An Giang, D6ng Thap 131 3.5.22. Tai tinh Vinh Long 133 ThM gian thuc hien. 2007 - 2009 vii Bdo cdo tong ket de tdi sdn xuat KIT-ELISA vims RGSV & RRSV 3.5.23. Tai tinh Hau Giang 135 3.5.24. Tai tinh Soc Trang (Vu Dong Xuan 2009) 137 3.5.25. Tai tinh Soc Trang (Vu He Thu 2009) 139 3.5.26. Tai tinh VTnh Long (Vu Thu Dong 2009) 140 3.5.27. Tai tinh Hau Giang (Vu Thu Dong 2009) 141 3.5.28. Tai tinh S6c Trang (Vu Thu Dong 2009) 143 r 3.5.29. Ket qua chung cila tinh VTnh Long, Hau Giang, Soc Trang a Vu liia He Thu 2009 144 3.5.30. Ket qua phat hien benh Tungro d Viet Nam 147 3.6. MQT SO KET QUA THU' ELISA DUNG KHANG HUYET THANH SAN XUAT THl/NGHIEM TAI MQT SO CO S6 148 3.6.l.Kit qua thu nghiemKit ELISA nam 2008 149 3.6.2. Kit qua thu nghiem Kit ELISA nam 2009 158 CHUONG 4 KET LUAN vA KIEN NGHI 164 4.1. Kit luan 164 4.2.Dlnghi 166 TAI LIEU THAM KHAO 167 Thcri gian thuc hien. 2007 - 2009 viii [...]... qua kiem tra ELISA cdc mau liia cd trieu chiing virus Tungro 148 Bang 3.56: Ket qua so sdnh antiserum tai trung tam BVTV phia nam 149 Bang 3.57 Kit qua kilm tra Kit ELISA phdt hien virus RGSV 150 Bang 3.58 Kit qua kilm tra Kit ELISA phdt hien virus RRSV 152 Bang 3.59 Kit qua kilm tra Kit ELISA virus RRSV vd RGSV tren riy 153 Bang 3.60 Kit qua thu nghiem Virus RRSV 155 Bang 3.61 Kit qua thii nghiem khdng..."Ido'cdo tdng kit dl tdi sdn xudt KIT-ELISA vims RGSV & RRSV DANH MUC BANG • Bang 1.1 Cac loai virus trong chi Tenuivirus [ngudn ICTVdB ] 5 Bang 1.2 Cac doan RNA va protein cua virus lun xoin la (RRSV) 18 Bang 1.3 Cac virus gay benh dugc xdc dinh tren liia/riy nau dugc thu thap tai mgt s6 dia phuang thugc miln nam Viet Nam (ngu6n CLRRI) 23 Bang 3.1 Kit qua... Bang 3.60 Kit qua thu nghiem Virus RRSV 155 Bang 3.61 Kit qua thii nghiem khdng huylt thanh RGSV 156 Bang 3.62: Kit qua kilm tra virus liia cd vd virus virus lun xoan Id tai Tnmg tam Bao ve thuc vat mien Trung 157 Bang 3.63: Kit qua so sdnh muc do phan ung cua kit ELISA virus RRSV va RGSV do phdng thi nghiem san xuit vdi Kit ELISSA ciia IRRI ThM gian thuc hi8n 2007 2009 159 xii tjao cao tong Ket ae... 3.65: Kit qua kilm tra kit ELISA virus RRSV vd RGSV tren liia d Tien Giang 160 Bang 3.66: Ket qua kilm tra kit ELISA virus RRSV vd RGSV tren liia d cdc tinh Binh Thuan (M), Quang Nam (N), 6 Mdn - Cin Tha (O), Xuan Ldc Bien Hda (X), Binh Duang (Y) 161 Bang 3.67: Kit qua kilm tra kit ELISA virus RRSV vd RGSV tren liia d c i n Tho 162 Bang 3.68: Kit qua kilm tra kit ELISA virus RRSV vd RGSV tren liia d... Hinh tren riy due cdnh dai; Hinh dudi riy edi cdnh ngin (Hibino, 1982) 8 Hinh 1.3 Anh ciia phan tu virus RGSV trong kinh hien vi dien tii, xem trong dung dich uranyl acetate 1% (Hibino, 1986) 11 Hinh 1.4 Bd gen ciia virus RGSV 12 Hinh 1.5 Trieu chung benh RRSV tren liia Cin Tho nam 2008 14 Hinh 1.6 Cdc thi virus trong md Id quan sdt dudi kinh hien vi 16 Hinh 1.7 So dd ciu tnic bd gen ciia RRSV 17 Hinh... r r - De tai da iing dung thanh cong ley thuat san xuat khang huyet thanh da dong dac hieu Protein NCP va phan tii virus (Virion) ciia hai virus RGSV va RRSV hai Ilia - Hai khang huyet thanh nay co nguong pha loang theo phuong phap protein NCP la 1/250 va theo phuong phap dung phan tu virus la 1/500 - De tai da xay dung hai quy trinh ky thuat san xuat khang huyet thanh va Kit ELISA dl phuc vu viec... 2008, 2009 giiip cho viec phat hien benh nhanh chdng - Sach "virus liia" cac bai bao khoa hgc tai cac cudc hdi thao qudc gia va cac hdi nghi khoa hgc da giiip cdc co sd san xuit cd hudng diing trong r phdng chdng benh Cung vdi cue BVTV va cac co quan cac trudng, vien trung tam ciia bd Ndng nghiep va phat triln ndng thdn dl tai da gdp phin quan ly dich benh virus liia - Gia thanh ciia Kit ELISA do dl... VA VAT NUOI Doclap T u d o Hanh phuc Ha Noi, ngay 20 thdng 05 nam 2010 BAO CAO THONG KE KET QUA THlTC HIEN DE TAI/DUT AN SXTN I THONG TIN CHUNG 1 Ten de tai/dy an Nghien cun san xuat Kit chan doan nhanh virus gay benh vang lun v^ hin xoan la tren lua Ma sd de tai, du dn Thudc Chuang trinh {ten, ma so chuang trinh) Chuang trinh trong diem phdt trien ung dung Cong nghe sinh hoc trong linh vuc Nong nghiep... TT Ten van bSn I QD s6 3876/QDBNN-KHCN ngay 19/12/2006 Quyet dinh phe duyet td chuc, cd nhan, muc tieu, dy kiln kit qud, kinh phi vd thdi gian thirc hien dl tdi " Nghien cim sdn xudt Jiit chdn dodn nhanh virus gdy benh vdng lun vd lun xoan la tren lua"g\Q.\ doan 2007- 2010 2 OlAHD/TC-KHCN Hgfp dong trdch nhiem {Ve viec thuc hien de tdi NCKH) 3 16/SKCTVN Xin dieu chinh noi dung nghien ciiu ddi vdi de... dong Thdi gian 10/11/2007- 11/11/2007 Dja diem Vien KHKT Nong Lam Nghiep Tdy Kinh phi 17 tr dong Nguyen Dja dilm Vien KHKT Nong Lam Nghiep Tdy Nguyen 2 2008 Ndi dung Tmh hinh benh virus hgi lua a Ndi dung Tinh hinh binh virus hgi lua a Viet Nam hien nay vd nhirng kit qud phdt Viet Nam hiin nay vd nhung kit qud phdt hien benh bang phuang phdp ELISA hiin binh bdng phuang phdp ELISA Thdi gian 18/10/2008 . 1.1.1. Benh vang lun va virus gay benh vang lun 5 Pham vi ki chii, trieu chung benh 6 Thiet hai do Rice grassy stunt virus (RGSV) 9 Cac dong virus, cau tnic th§ virus va ciu tnic phan. TU' VIRUS VA PROTEIN VIRUS TU'NHIEN 54 3.2.1. Kilm tra su co mat cua virus tao vat lieu khai dau 54 3.2.2. Thu nghiem tao khdng huyet thanh RGSV dua tren phan tu virus (virion). MUC BANG • Bang 1.1. Cac loai virus trong chi Tenuivirus [ngudn. ICTVdB ] 5 Bang 1.2. Cac doan RNA va protein cua virus lun xoin la (RRSV) 18 Bang 1.3. Cac virus gay benh dugc xdc dinh