LTIVG DLIIVG KY T H U A T (IVIULTIPLEX] RT-PCR/RFLP OE CHAIM DOAIM 5QIVI V A TIIVI H I E U QUAIM H E O l TRUYEIM C U A
V I R U T G A Y QEIMH A U TRUIMG T U I TREIM OIMG IVIAT
U: Quang Trung\ Dmh Quylt Tam\ TrIn Van Toln\ Truong Anh Tuan', Tang Thi Phuang^, Dang Tit ^ h l ^ Phiing Hfiu Chinh' T6MTAT
Ky thuat da thanh phan (multiplex) RT-PCR/RFLPs da va dang dugc ung dung rong rai de nghien cii'u vi nit au tning tui (SBV) gay hai nghiem trong tren ong ehau A Apis cerana va ong chau Au A. melltfera Trong nghien ciiu nay, ky thuat tren dugc img dung de tim ra ehi thi phan tii chan doan som va tim hieu mdi quan he di truyen ciia cac chiing SBV tren ong A. mellifera (AmSBV), SBV Tmng Quoc (AeCSBV) va SBV Thai Lan (AcTSBV) tren o n g ^ . cerana. Cac cap moi dugc thiet ke bang each so sanh eae trinh tu ciing Vl tri tren he gien eua 3 chung SBV da dugc cong bd tren Ngan hang gien; cac trinh tu nay eiia SBV thu tren ong A. mellifera a Due, Ao, Anh, An Dp, Trung Quoc, Ne-pan va Viet Nam, Moi phan iing multiplex RT-PCR dugc chay bang 10-20 ng ARN cua SBV tach tu au tning gia cua 5 dan ong A. cerana chua co bieu hien lam sang ve benh aiu triing tiii va 3 moi nhan. San pham RT-PCR duge cat bang enzym han che Pstl, giai trinh tu va so sanh vai trinh tu chi thi cua cac chung SBV khac da cong bd tren Ngan hang gien. Ket qua phan tich eho thay 3 trong 5 dan ong A. cerana Hoa Binh bi AcCSBV ky smb. Da hinh ve trinh tu' eiia eac chi thi nay con cho thay SBV tren ong A. mellifera a An Do va Ne Pan eo the tien hoa tir AcCSBV, Da hinh chieu dai eua san pham RT-PCR va san pham cat b i n g enzym ban che trong nghien cuu nay la ehi thi phan tir de chan doan som 3 chung SBV. Ket qua nghien cuu nay con cho thay ongyl. mellifera co the bi ca AmSBV va AeCSBV ky sinh. Ky thuat multiplex RT-PCR/RFLP eo the ap dung de chan doan som eac chung SVB tren ong A. mellifera dang nuoi o' nuae ta.
Tir khoa: Vi nit aiu trung tui (SBV), Apis cerana, A. mellifera, da thanh phdn sao chep lai trdi nguac- phdn ung ddy triing hap/Gidi han hien tuang nhieu dang ve chieu ddi cdc doan vi rut au triing tiii (SBV).
1. DAT VAni DE
T r o n g sd 18 loai vi n i t gay hai c h o o n g m a t da dugc nghien cim phan lap (Allen va Ball, 1996), Morator aetatulae (vi n i t l u t r i m g tui hoac sacbrood vi nit, vilt t i t la SBV) la tac n h a n gay chef h a n g loat l u triing tudi ldn va giam tudi t h g o n g t r u d n g t h i n h cua c l ong chau A (Apis cerana) va o n g chau Au (A.
mellifera). Au triing o n g chef do b e n h nay cd hinh tui chiia dich va SBV b e n t r o n g n e n ggi la b e n h au t r u n g tui (Bailey et ai, 1964). SBV t h u g c h g Pieornaviridae va CO toi 3 chiing da dugc n g h i e n cun dinh loai, bao gdm SBV tren o n g A. mellifera (AmSBV) (Bailey et al, 1964), SBV Thai Lan t r e n o n g A. cerana (AcTSBV), va SBV T r u n g Q u d c tren o n g A. cerana (AcCSBV) (Bailey, 1982; Z h a n g et al, 2001, Rana v l Rana, 2007). SBV co the tdn tai tren d i n o n g tii n a m n l y qua nam khac (Bailey, 1969, 1982; Bailey va Fernando, 1972). Kha n a n g lay n h i l m cua b e n h rat cao va CO t h i b u n g phat t h a n h dich (Bailey, 1969).
Trung tam Nghien ciiu Phat trien Ong: " Vien Sinh thai Tai nguyen Sinh vat
N a m 1990 An D o da m a t h a n g trieu dan ong do SBV;
d nuoc ta, ttr 1974-1975 SBV da h u y diet 7000 tren t d n g sd 74000 dan o n g A. cerana ( P h u n g Hun Chinh, 1990). T r o n g n h u n g n a m g a n day, SBV con lam gilm 40-80% n a n g s u i t m a t cua o n g A. mellifera nuoi d n u d e ta ( P h u n g H u u Chinh, 2008). R l t k h o d e chan doan b e n h khi d a n o n g mdi bi n h i e m SBV vi au tning hi b e n h t h u o n g d u g c o n g t h g g I p ra khdi td (Bailey, 1969; Bailey va F e r n a n d o , 1972). D e n nay chua cd loai t h u d c nao tri sach b e n h au t r u n g tiii khi da phat hien dugc b e n h v l m a t lam s i n g : nhin thay au trung tui trong 16 td. H o n n u a , SBV con co t h i t i l n boa d l thich nghi tren d a n o n g d cac vung dia ly khac nhau
( G r a b e n s t e i n e r et al, 2001). Vi vay, nghien cun chan doan s o m va chinh xac t u n g chiing vi rut hai ong cung nhu quan he di truyin cua chung nhlm dua ra bien phap phong trir thich hgp va bieu qua dong vai trd sdng cdn cua nganh ong.
Chan doln SBV thuong dua vao nhung phuong phap truyin thdng nhu kinh hien vi dien tu', huylt thanh hgc, mien dich hgc... Nhung phan tich nay cd do nhay va tinh dac thu thap, nhilu khi khong thyc
34 NONG NGHIEP VA PHAT TRIEN NONG THON - SO 1 - THANG 1/2010
hien dugc do khong the tach va nhan vi nit in vitro (Anderson, 1984; Grabensteiner et ai, 2001). Mot han che nua la kho co fhl phan biet dugc cac chimg SBV khac nhau trong cung loai sir dyng cac phuong phap tren. Gin day, ky thuat da thanh phan RT- PCR/RFLP bao gdm nhan doan gien tren ARN cua vi rut bing nhieu cap mdi (multiplex RT-PCR), sau dd d t sin pham RT-PCR bing enzym han e h l (RFLP) dang dugc ung dung hieu q u i de chan doan som benh tren dan ong do vi nit gay ra cting nhu de nhan biet mdi quan he di truyin gifia elc loai vi nit vl giua cac chimg 'vi nit trong loai (Tanaka et al., 2001;
Barros et al, 2004) Ky thuat nay cung dang duoe ap dung thanh cong de chan doan sdm va xac dinh khac bict cung nhu quan he di truyen cac chiing SBV tren ong mat chi d n mot lugng nhd 10-20 ng ARN/phIn img (Zhang et al, 2001; Davision et ai, 2003; Topley et ai, 2005; Grabenstemer et ai, 2001, 2007).
6 nude ta, SBV duoc phat hien gay hai tren ong A. cerana tir 1974 (Tnnh Dinh U, 1983; Pham Nggc Vien, 1984) va tren ong A. mellifera cudi nam 2006 (Phung Hun Chinh, 2008). Cho den nay, •viec chin doan SBV chu ylu bang quan sat lam sang. CQng co mot vai nghien cun ung dung phuong phap truyen
Bing 1. Trinh tu doan cADN tr^n hS gien cua SBV dugc su dung d l thilt k l c l e cap moi v l phan tich quan h$
di truyin cle chung SBV. AcTSBV: vi rut au trung tui Thai Lan; AcCSBV: vi nit lu trung tiii Trung Qudc; A.c:
Apis cerana; A. tn: Apis mellifera; AF092924: ma dang nhap he gien SBV tren ong A. mellifera a Anh
thdng (P Van Nggc et al., 2004) hoac ky thuat phan fir (Le et ai, 2003) d l xlc dinh SBV tren ong A.
cerana khi dan ong da bi benh. Tuy nhien, chua co nghien cun ung dung ky thuat phan tii de chan doan som va nghien cun quan he di truyen cua cac chung SBV tren ong A. cerana va A. mellifera D l budc diu li'ng dyng ky thuat phan tir d l tim hieu SBV tren dan ong nuoi d nude ta, nghien cun nay nhlm 1) xac dinh chi thi phan tu' ap dyng ky thuat da thanh phan RT- PCR/RFLP de chan doan som benh au trung tui: 2) so sanh trinh ty RT-PCR trong nghien cun nay voi mot sd nghien cun trong va ngoai nuoe co lien quan de phan tich quan he di truyin cua cac ch-iing SBV tren d 2 loai ong ung dung mot sd phln mem chuyen dyng.
II. niGUYEni UEU VA PHUOniG PHAP
1. Nguyen li^n
Benh pham dugc thu tii 5 din ong A. cerana cua Trung tam Nghien cuu va Phat triln Ong c6 nguon goc tii Hoa Binh va chua eo bieu hien lam sang cua benh lu tning fur Mdi dan chgn ngau nhien 5 au tning vit nip va giii d -56"C. ARN cua SBV dugc tach theo phuong phap cua Grabensteiner va ddng tac gia (2001).
STT 1-5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Viing lly miu Hoa Binb, VN Ha Tay, VN Hai Hung, VN An Do Trung Qudc An Do Ne pan Ne pan Anh Anh Ao Diic Diic Diie Dire
Vat chu A. c A. c A. c Ac Ac A. m A. m A. m A. m A. m A. m A. m A. m A. m A. m
Ma kybi^u HoaBinhVNl-5 HaTayVN HaiHungVN AcTSBV AcCSBV India Nepali Nepal3 UK AF092924 Austria Germanyl Germany6 Germany?
GermanyS
Ma trSn mang Chua eo AY216794 AY311387 EU156753 AF469603 AF284660 AF284658 AF284659 AF284648 AF092924 AF284649 AF284651 AF284655 AF284656 AF284657
Tac gia
Nghien ciiu nay he etal., 200^
Le et al. 2003 Rana va Rana, 2007 Zhang ff a/., 2001 Grabensteiner et al, 2001 Grabensteiner et al, 2001 Grabensteiner et al, 2001 Grabensteiner et al, 2001 Ghosh rf a/.,1999
Grabensteiner et al, 2001 Grabensteiner et al, 2001 Grabensteiner et al, 2001 Grabensteiner et al, 2001 Grabensteiner et al, 2001 2. Thilt k l cic cap mdi
Hai cap mdi nhan dugc thilt k l dya vao trinh tir he gien cua SBV ky sinh tren ong A. mellifera
(AmSBV), SBV Trung qudc tren ong A. cerana (AcCSBV), va ti-inh ty d c doan cua cung vi tri
(locus) da dugc dang ky tren Ngan hang gien (Bing 1) sir dung chuong trinh DNAMAN 4.0. Mdi nhan ban tir diu 5' (SBVF) co trinh ty 5'- AGGGAAATTACTAATATACCTTGCC-3' duoc dung chung cho d AmSBV, AcCSVB vl AcTSBV. Mdi tu
NONG NGHIEP VA PHAT TRIEN NONG THON-SO 1 -THANG 1/2010 35
diu 3' (CSBR) CO ti-inh tir 5'-TGGGTGTTCGTTCACT phlm RT-PCR diu khoing 600 bp. Nhung dan nly TGTTGAAAG-3' diing chung cho AmSBV vl khong bi vi rut au ti-iing tiii Thai Lan (AcTSBV) ky AcCSBV; con mdi (TSBVR) cd ti-inh tu sinh vi khong co sin pham RT-PCR vdi kich thuoc 5'CCTCCCGCCAGGCA AAATAG CAACC-3' ehi dl khoing 500 bp. Khong cd sin phlm RT-PCR tii 2 nhan bin AcTSBV tir dau 3', Chieu dai sin pham RT- trong 5 dan ong A. cerana Hoa Bmh cho thay cac PCR cua AmSBV vl AcCSBV khoing 600 bp vl eua
AcTSBV khoing 500 bp.
3. Da thinh phln RT-PCR
Phln ling da thinh phln RT-PCR dugc thue hien theo qui ti-inh cua Grabensteiner vl ddng tie gil
(2001) sir dyng Qiagien One Step RT-PCR Kit (Qiagien). Sin pham RT-PCR dugc kilm ti-a tren thach agaroza 1,2%, nhudm bing ethidium bromide IX ti-ong 30 phiit va quan sit dudi den UV. Chilu dli cua sin pham RT-PCR dugc udc lugng bing thang IKb-ADN cua hang Inviti-ogen™.
Sin phlm RT-PCR dugc tinh sach bing QIAquiek PCR Purification Kit (Qiagen) vl elf bing enzym ban ehl Pstl b 37"C trong 2 gid dl tim chi thi dac thii cho AmSBV vl AcCSBV. Xlc dinh vi ti-i d t cua enzym ban ehl dua tren phan tich d c trinh ty chi thi phan tir cua tiing chung (Bing 1) bing phln mlm DNAMAN 4.0.1.1 (Lynnon Biosoft) vl Mega3.1 (Kumar e^/., 2004).
4. Giai trinh tuvl phln tich trinh tu
Sin pham RT-PCR dugc giii ti-inh tir theo qui ti-inh cua Trung vl ddng tac gil (2008). CIc ti-inh ty lien quan cua AmSBV, AcCSBV vl AcTSBV (Bing 1) dugc tli tir Ngan hang gien vl so sinh vdi d c trinh tu cua nghien cim nly bing d c phln mlm Mega3.1 dl: 1) khang dinh chinh xlc trinh tu cua d c chi thi phan tir cho tiing chung; 2) tim hiiu quan he chung loai vl khoing d c h di truyin gitra elc chung. KhIc
^ biet giiia d c chung duge danh gil bang gil tri bin than mdi nhdm (Bootstrap) theo phuong phip phan tich Neighbor Joining tren phln mlm Mega3.1.
III. KET QUA NGHIEN CUU
1. Xlc dinh chi thi phln tCt cua 3 chiing vi nit lu trung tiii
Phan tich sin pham RT-PCR tren thach agroza cho fhly trong 5 miu SBV tren ong A. cerana cd ngudn gdc Hoi Binh (HoaBinhVNl-5, bing 1) chi cd 3 miu Hoa BinhVNl-3 cho san pham vdi chilu dli khoing 600 bp, chinh xac la 597 bp sau khi giii trinh ty. Kit qui RT-PCR cho thay 3 din ong nay bi cac chung SBV Trung Qudc (AcCSBV) hoac SBV ti-en ong A.mellifera (AmSBV) ky sinh -vi kich thudc san
miu cua 2 din nay khong bi SBV ky sinh.
Dya vao phan tich tiinh tir ADN cua cac chiing SBV d bing 1 cho thay chi thi phan tii cua AmSBV khong cd vi til d t ban ehl cua Pstl, ti-ong khi do AcCSBV cd vi ti-i nly va phan thinh 2 doan 411 bp va 186 bp. Trong thi nghiem khi nghien cim, sin pham RT-PCR ciia 3 mau HBl-3 da hi Pstl cat thanh 2 doan co ehilu dli ti-en (Hinh 1), vi the SBV ti-en d c mau nay thugc chung AcCSBV. Kit qua vl da hinh cua chilu dai sin pham RT-PCR (500 bp cho AcTSBV vl 597 bp cho AmSBV) vl sin pham d t bing enzym han chi (hi 597 bp thinh 411 bp vl 186 bp cho AcCSBV) la chi thi phan tir dl xlc dinh 3 chung SBV nly.
600bp- -597bp
Hinh 1. Sin phlm RT-PCR cua 3 miu SBV thu trfin ong A cerana Hoi Binh dugc cat bang enzym han chi Pstl. HBl-3, HoaBinhVNl-3: hing bang 597 bp II sin pham RT-PCR cua 3 miu chua d t hit; hai hing bang 411 bp vl 186 bp la san pham d t bang Pstl. L, thang chuan ADN Gnviti-ogen™, GeneRuler).
2. Quan h$ di truyin ciia cle chung vi nit lu trung tiii
Phan tich Neighbor-Joining dya vio da hinh vl trinh tir cua d c ehi fhf phan tii cho fhly ba chirng AmSBV, AcCSBV, vl AcTSBV rlt khIe biet vl di truyin. Cay phlt sinh chung loai chia d c chung SBV nly thinh 4 nhdm (Hinh 2). Nhdm AmSBVl gdm d c miu SBV tren ong A. mellifera b Dtic (Germanyl, -6, -7), Ao (Austi-ia), Nepal (Nepal3), Anh (UK AF092924). Trinh tu ADN cua nhdm nay tuong ddng vdi trinh ty he gien cua AmSBV tren ong A. mellifera b Anh (AF092924). Nhdm AcCSBV bao gdm cle dai dien SBV tren ong A. cerana a mot sd tinh cua nude fa. Trinh tir ADN cua nhdm nay ciing nhanh voi he gien cua chung SBV Trung Qudc AcCSBV. Khac biet di truyin ciia AmSBVl va AcCSBV rlt cao voi gia tri 36
NONG NGHIEP VA PHAT TRIEN NONG THON - S6 1 - THANG 1/2010
Bootsti-ap toi 99-100%. Nhdm AmSBV2 gdm dai dien ky sinh ti-en ong A. mellifera d An Do (India) va d Nepal (Nepali). Nhom AcTSBV chi co 1 dai dien vdi khoing each di truyin rat xa voi ba nhdm cdn lai.
Ciing voi kit qui d t bing enzym ban ehl sin phlm RT-PCR, viec phan tich quan he chung loai dya vao ti-inh tir ADN ciia cac miu SBV trong nghien cun nay va cac trinh tir chi fhf phan tii cua cac chimg SBV da dugc cong bd tren Ngan hang gien tham khao d bang 1 cd fhl khIng dfnh ring cac miu Hoa Binh VN 1-3 thuoc chiing SBV Trung Qudc (AcCSBV).
AmSBVl
AmSBV2
Hinh 2. Quan hi di truyin cua cle chiing vi nit gay b$nh lu trimg tiii trIn ong A mellifera vl A cerana theo cic vimg dia ly khIc nhau. AmSBVl:
nhdm SBVl ti-en ong A. mellifera; AmSBV2: nhdm SBV2 tren ong A. mellifera; AcCSBV: nhdm SBV Trung Qudc; AcTSBV: nhdm SBV Thai Lan. Sd dudi cac nhlnh cua cay chung loai la gia tri Bootsti-ap. Chi tilt ky hieu noi thu miu xem bing 1.
Trong khi hai nhdm AcCSBV vl nhom AmSBVl cd khoing elch tin cay vl di truyin (gia tri bootstrap 91-100%, hinh 2), hai dai dien cua AmSBV2 co fhl coi II nhdm tiling gian giira hai nhdm ti-en, nhtmg cd quan he chiing loai gin vdi AcCSBV hon (gia tri bootstrap chi cd 67%, hinh 3A) so vdi AmSBVl (gia ti-i Boosti-ap toi 91%, hinh 3B). Them vao dd ti-inh tir chi thi phan tii cua cle dai dien trong nhdm AmSBV2 vl h-ong nhdm AcCSBV ciing ed vi ti-i d t bang enzym ban chi Pstl (Hinh 3C) cdn ti-inh tir cac dai dien cua AmSBVl khong co dilm d t nly (Hinh 3D).
Dilu do chting td nhdm AmSBV2 cd quan he chung loai gin voi nhdm AcCSBV hon la voi nhdm AmSBVl. Voi kit qui phan tich tren, cd the goi nhdm AmSBV2 II AcCSBV-like (gidng voi AcCSBV).
A) 4^|-HoaBinhVnr
f-JlHaTayVI) HaiHungVU HoaBmhVNI HoaEinhVM2 CSBV India
Nepali
— TSB'/
AcCSBV
T A mSBV2
B)
~y AcTSBV
- A m S B V l
A m S B V 2 AcTSBV
cr
CSBV
HoaBinhVNl GTGGCGG
HoaBinliVN2 GTGGCGGCTGCAGjGTTTA HoaBiiihVn3
HaiHung'VK HaTayVN I n d i a N e p a l i
h *( tt tr *
GTGGCAGCTGCAGGTTTA-
GTGGCGG
G T G G C G G G T G G C A G
ilr * Ar Ar A ' *
C T G C A G G T T T A C T G C A G G T T T A G T G G C G G C T G C A G j G T T T A
AcCSBV
CTGCAGiGTTTA C T G C A G G T T T A l G T G G C G G C T G C A G G T T T A J
AmSBVZ
Didm cat cOa Pstl _)
UK
D)
AF092924 Nepal3 Germanyl Germany6 Germany?
GermanyS Austria India Nepali
G T G G C G G C T G C G G G T T T A -|
G T G G C G G C T G C G G G T T T A G T G G C G G C T G C G G G T T T A G T G G C G G C A G C A G G T T T A G T G G C G G C A G C A G G T T T A G T G G C G G C A G C A G G T T T A G T G G C G G C A G C G G G T T T A G T G G C G G C A G C A G G T T T A G T G G C A G
G T G G C G G
AmSBVl
C T G C A G G T T T A
C T G C A G G T T T A
}
AmSBV2BiSm cat cOa Pstl
Hinh 3. So sinh c3p vl quan h6 di truyin giiia 3 nhdm vi nit lu trung tiii AmSBV2, AcCSBV vl AmSBVl. A) Cay phlt sinh chung loai so sanh ti-inh tir cua nhdm AmSBV2 voi AcCSBV tren ong A.
cerana; B) So sinh AmSBV2 vdi AmSBVl tren ong A. mellifera; C) Pstl ti-en ti-inh tir cua AmSBV2 va AcCSBV; D) Pstl chi co ti-en ti-inh ty cua AmSBV2 khong cd ti-en AmSBVl. Chi tilt ky hieu noi thu miu xem bang 1.
NONG NGHIEP VA PHAT TRIEN NONG THON - SO 1 - THANG 1/2010 37
III. THAO LUAN
1. Phlt triln chi thi phln tu d l x l c dinh sdm SBV ky sinh trfin ong
Kit qui cua nghien cun nay da khIng dinh tinh img dung cao cua ky thuat da thinh phan RT- PCR/RFLP de chan doan sdm benh au ti-ung tui ky sinh tren ong mat. Chi thi phan fir phat trien fir ky thuat RT-PCR/RFLPs cho p h i p phan biet giira chung SBV tren ong A. mellifera (AmSBV) voi SBV Trung Qudc ti-en ong A. cerana (AcCSBV) d giai doan sdm, khi chua quan sat dugc frieu chiing ciia benh ve lam sang, dya vao phan tich da hinh vl chieu dai cua san phlm RT-PCR dugc d t b i n g enzym ban e h l Pstl.
Nhieu nghien cim ve chan doan ciing nhu tim hieu quan he di truyin d mue do loai va duoi loai cua vi nit da dang dugc thyc hien ung dung ky thuat nay.
Tren thyc tl, ky thuat RT-PCR/RFLPs da dugc dung rong rai trong y hgc d l chan doan d c benh do vi nit, dac biet nhirng vi rut gay benh tren ngudi (Tanaka et al, 2001; Saito et ai. 2002; Banos et al, 2004).
Khong chi dimg lai d vai tro chan doln som benh au triing tui, trinh ty san pham RT-PCR do ky thuat nly cdn giup kham pha ngudn gdc phat sinh ciia vi rut tren ong d cac vung dia ly each biet (Grabensteiner et al, 2001, 2007). Trong nghien cun nly, ung dyng ky thuat multiplex RT-PCR/RFLP, SBV ti-en 3 din ong A. cerana duge phan tich thugc chung AcCSBV va SBV tren ong A. mellifera a Ne Pan, An Do dugc phat hien cd quan he g i n vl di truyin vdi chung AcCSBV tren ong A. cerana hon II voi chung AmSBV tren o n g ^ . mellifera.
2. Vi nit lu trimg tiii trfin ong A cerana Vi$t Nam ed ngudn goc ttr AcCSBV
Vi rut au tning tiii Trung Qudc tren ong A.
cerana (AcCSBV) dugc phat hien diu tien d Quang Dong, Trung Qudc nam 1972. Cho din nay, AcCSBV da cd mat tren ong A. cerana a k h i p Tmng Qudc vl cac nude Dong Nam A (Zhang et al, 2001). Theo kit qua phan tich cua nghien cim nly, 3 mau SBV dugc thu tren ong A. cerana cd ngudn gdc Hoi Binh va hai mau da nghien ciru truoc day tren ong A. cerana Ha Tay va HIi Hung (Le et al., 2003) deu thugc chiing AcCSBV. Kit q u i nay phu hgp vdi hch sir lan truyen ciia chimg AcCSBV len ong A. cerana a Viet Nam.
Theo Trinh Dinh U (1983) vl Pham Nggc Viln (1984), AcCSBV dugc phat hien dau tien d Viet Nam tren nhirng dan ong A cerana nhap tir Bac Kinh, Trung Qudc vao nam 1974. Sau do benh lan tran ra
khap cac tinh ddng bing, trung du, mien nui phia Bic va cac tinh Ble Trung bg (Phung Hun Chinh, 1990; 2008). Nhu vay, SBV tren dan ong A. cerana thuoc dai dien cua 3 tinh phia Bic (Hoa Binh, HIi Hung va Ha Tay cu) co ngudn gdc tu AcCSBV nhap tir Trung Qudc vao Viet Nam, tu' dau nhung nam 70 cua t h i ky truoc la hoan toan co co so Ngoai ra, theo Phung Huu Chinh (1990), ben canh AcCSBV, SBV Thai Lan tren ong A. cerana (AcTSBV) cung da dugc phat hien tren ong A cerana a miln Nam Viet Nam vao nam 1989. Tuy nhien, nghien ciin nay chua phat hien dugc AcTSBV vi thilu dai dien SBV tren ong A.
cerana nuoi d cac tinh phia Nam.
3. AmSBV v l AcCSBV cd fhl cung ky sinh trfen ong A mellifera nudi d Vi$t Nam
Theo phan tich so sanh khi nghien cim, nhdm AmSBV2 hoac AcCSBVdike ky sinh tren ong A.
mellifera b Ne Pan va An Do (Nepali, India. Hinh 2) cd fhl CO tiln hda tir chimg AcCSBV d l ky sinh tren ong A. mellifera vi chung cd quan he di truyen gan voi cac dai dien cua chiing AcCSBV ti-en ong A.
cerana hon la voi AmSBVl tren ong A. mellifera chau Au (Hinh 2, 3), Kit qua cua nghien citn nay tning voi Grabensteiner vl ddng tic gia (2001). Cac tac gil nay cho ring 4 dai dien ciia SBV tren ong A., mellifera d An Do va Ne Pan tach biet thanh nhanh rieng khdi nhom SBV tren ong A. mellifera a cac nuoc chau Au trong cay chiing loai (Hinh 4). Do khong so sinh trinh ty eua ehi thi phan tir dac trung chiing AcCSBV nhu trong thi nghiem cua nghien cun nay da lam cd fhl la nguyen nhan din tdi viec Grabensteiner va ddng tac gia (2001) dy doan ring 4 dai dien SBV tren ong A. mellifera d An Do va Ne Pan cd ngudn gdc tu ong A. mellifera chau Au vl nhdm nay co khoang each di truyen tin cay so voi chung gdc tir chau Au la do q u i trinh tiln h o i d l phii hgp voi dilu kien moi tru'dng moi. Nghien cim nly da dua ra bing chimg cho thay SBV tren ong A mellifera An Do va tren mot sd dan d Ne Pan co ngudn gdc tir AcCSBV. Mot cau hdi khac dugc dat ra trong nghien cim ciia Grabensteiner va ddng tac gil (2001) la tai sao mdt mau SBV tren ong A. mellifera cua Ne Pan (Nepal3) lai khong tiln boa theo kien moi trudng cua nuoc nay nhu cac dai dien Nepali, -2, -4 (Hinh 4), Cau hdi nay CO fhl dugc giai thich theo k i t qua nghien cun nly la ong A. mellifera ciia Ne Pan da hi ky sinh boi c l hai chung AmSBV va AcCSBV, 6 nuoe ta. AcCSBV duoc phat hien tu' gan 40 nam truoc tren ong A. cerana.
38 NONG NGHIEP VA PHAT TRIEN NONG THON - SO 1 - THANG 1 2010
Trong khi do, A. mellifera duoc nhap vao Viet Nam tir hang nira fhl ky qua. Nhung din ong A mellifera nay co t h i hi ky sinh bdi AmSBV tren din ong nhap tu chau Au va co the con hi d AcCSBV lan sang tii- ong >1. cerana.
Nnpsl :
Hinh 4: Quan h6 di truyin cua AmSBV trIn ong A mellifera theo elc vimg dia ly khIc nhau. (theo hinh 11, Grabensteiner et al, 2001). Chi tilt ky hieu noi thu miu cua tac gil xem b i n g 1.
IV. KET LUAN
Chi thi phan tir dugc tim ra tir viec thilt k l cac cap mdi dac hieu co k h i nang chan doan som va chinh xac benh lu tiling tiii tren ong A. cerana. Chi thi nay cdn dugc sir dung de xac dinh quan he di truyin cua cac chung SBV khac nhau. Phuong phap nay co fhl dugc ap dyng de chin doln som benh au triing tui ti-en ong A. mellifera dang nuoi d nude ta, tii dd dua ra qui ti-inh phdng ti-i kip thdi va hieu qua.
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APPUCATION OF MULTIPLEX RT-PCR/RFLP FOR EARLY DETECTION AND GENETIC RELATIONSHIP RESEARCH OF SACBROOD VIRUSES ON HONEY-BEES IN VIETNAM
I^ Quang Trung\ Dinh Quyet Tam\ Tran Van Toan\ Truong Anh Tuan', Tang Thi Phuong', Dang Tat The^, Phung Huu Chinh'
Summary
Multiplex RT-PCR/RFLP has been -widely employed for early detection and research on genetic relationship of viruses including sacbrood virus (SBV), which causes serious economic losses to both Apis cerana and A. mellifera colonies. In order to apply these molecular tools on the SBVs in Vietnam, this research aims to develop markers for early detection of SBV on A. cerana and for discrimination of SBV on A. mellifera (AmSBV), Chinese SBV (AcCSBV) and Thai SBV (AcTSBV) on A. cerana with such approaches, and to understand their genetic relationship with sequence polymorphisms of the markers. To meet these objectives, multiplex RT-PCR was applied with RNA of SBV from 5 samples of old worker broods in 5 A. cerana colonies. The colonies were originated from Hoa Binh with no symptom of sacbrood disease. Primers for RT-PCR were designed by alignment between published sequences of the same locus of 3 SBV strains, which were originated from Germany, Austria, UK, India, Nepal, China, Thai Lan and Vietnam. RT-PCR products were then digested with Pstl. Sequences of DNA markers for 3 SBV strains, both from genebank and from this study were analysed using Neighbor-Joining -with bootstrap value.
Fragment length polymorphisms (FLPs) of RT-PCR and digestion products, and sequence polymorphism analyses showed that the 3 of 5 A. cerana colonies were parasitized with AcCSBV and that the FLPs could be markers to distinguish the 3 SBV strains. Sequence analysis revealed that SBV on A. mellifera in India and Nepal seemed to be evolved from AcCSBV (AcCSBVdike), and tbatyl. mellifere in Nepal was induced parasitized with both AmSBV and AcCSBV. Our findings implied that there may be both AmSBV and AcCSBV on A. mellifera in Vietnam, and suggested that the FLPs with RT-PCR/RFLP approach could be applied for early detection of SBV strains on A. mellifera in our country.
Keywords: Multiplex RT-PCR/RFLP, Sacbrood virus {S'SV),Apis cerana, Apis mellifera.
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Ngudi phln bi&n: TS. La Tuln Nghia
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