HQI NGHj KHOA HOC TOAN QUOC v £ SINH THAI VA TAI NGUYEN SINH VAT L A N T H C 5
ITNG D U N G K Y T H U A T R E A L T I M E - P C R
TRONG CHAN DOAN VIRUS TUI (SACBROOD VIRUS) NHIEM TREN D A N O N G MAT NUOI TAX HA NQI
PHAM HONG THAI, TRINH THI THU THUY, NGUYEN THI LAN, NGUYEN VAN CU*CfNG, NGUYEN VAN GIANG, HA VIET CU'dNG, O^NG HU^ONG LAN Tric&ng Dai hgc Nong nghiep Ha Noi Viet Nam nam trong vung c6 khi hau nhiet dai gio mua nen c6 nguon hoa va ngu6n mat phong phu phu hgp cho sy phat trien nghS nuoi ong mat. Do do, nghe nuoi ong l^y mat tir lau da va dang dong mot vai tro quan trong trong doi song con ngu-ai bai vi: No mang lai Igi nhuan kinh te to Ian cho nguai nuoi ong, c6 gia trj chua benh, giai tri va giir can b^ng sinh thai (Pliimg Huu Chi'nh. 2012; Crane, 199!).
Cling nhir cac dong vgt khac ong mat de bj tan cong bdi mot so vi sinh vat. Trong do, nguy hiem han ca la benh do virus, vi benh do virus khong the dilu tri b^ng thu6c khang sinh dugc, Trong so han 18 loEii virus gay liai cho ong mat (Bakonyi el a!. 2002; Aubert el al.
2007; Nielsen el al., 2008, Thai el ai, 2011) thi Sacbrood virus gay chet ku trung, la mot trong nhirng benh virus nghiem trong nhat (Ball, 1999; Liu el al, 2010; Neumann and Careck, 2010). Benh lam chet au trung chii yeu a giai doan sau vit nap va tien nhong Kha nang lay nhiem cua virus nay la rat Ion, chi can mot au triing benh c6 the lay nhiem cho 3000 dan ong khoe (Bailey and Ball, 1991). Viec phat hien sam virus nay la v6 cung quan trong de dira ra phuang phap phong tri kjp tlioi va ngan ngira dirge ton du khang sinh trong mat ong (Mascher el al, 1996). Trong so cac phuang phap chan doan hien dai thi ky thuat Realtime-PCR (hay con goi la quantitative PCR/qPCR) dugc cho la nhanh va cliinh .\ac han ca (Fleige and Michael, 2006; Siede et ai. 2008). Muc tieu cua nghien ciru nay nham danh gia do nhay ciia ky thuat Realtime-PCR so vai ky thuat RT-PCR (Reverse Transcription PCR) va kha nang phat hien virus a nong do thap.
I. DOI TUQNG VA PHUONG PHAP NGHIEN CUtJ
Doi li(p?ig: Mau au triing ong dugc thu thap a Gia Lam va bao quan trong con (etlianol 90") 6 dieu kien phong thi nghiem cho den khi tdch chiet RNA.
Phicmigphap Idch chiet RNA long so: RNA long so dirge tach chiet tir 18 man au triing ong b5ng TRIzol-LS {InvitrogenJ. Truoc khi tien hanh thirc hien phan ling PCR, san pham tacli chiet dirge kiem tra ham Iirgng va do tinh sach bang may NanoDrop.
Cap moi sic cliing: Trong ca hai ky thuat RT-PCR va Realtime-PCR deu sir dung cap moi SBIf-SB2r (Grabensteiner et ai. 2001), moi m6i bao g6m 20 nucleotid sau day. SBlf (ACC AAC C G A T T C C T C A C T AG), SB2r (CCT TGG AAC TCT GCT GTG TA)
Qiiy Innh RT-PCR: One step RT-PCR. vai thanh phan phan Crng va chu trinh nhiet nlur sau:
HOI NGHI KHbA HQC TOAN QUOC VS SINH T H A I V A TAI N G U Y S N SINH VAT LAN THC S Thanh phan phan ijng {20pl)
H2O
Dream Taq Master Mix 2X Moi SB 1 f MOI SB 2 r Reverse aid RNA
7,5pl lO.Oiil 0,5M O.SlJl 0,5lJl I.OiJl
Chu trinh nhi^t Nhi#t dp (°C)
42 95 94 54 72
Thai gian 30 phut
5 phut 45 giay 30 giay 1 phut
Chulty 1x 1x 35x
Dipt di san pham RT-PCR: Sau khi chay xong phan ling RT-PCR, san phSm khuech dai dugc dien di tren gel agarose (1%) a hieu dien the lOOV trong thai gian 20 phut. Ban gel sau khi dien di dugc doc ket qua tren he thong U V geldoc-Biorad.
Quy trinh Realtime-PCR v d i thanh phan phan irng va chu trinh nhiet nhir sau:
Thanh phan phan i>ng (20pl) H2O
QuantiFast SYBR Green RT-PCR Moi SB 1 f
Moi SB 2 r QuantiFast RT Mix RNA
8.2ul 10,0pl 0,3ul 0,3ul 0,2ul 1.0pl
Chu trinh nhi^t Nhi«t d9 (°C)
50 95 95 54 72 95
Thcri gian 10 phut 5 phut 10 giay 30 giay 1 phut Ogiay
Chuli}
1x 1x 40x 1x Xac dinh nong do virus c6 the phat hien tren mdu benh: Pha loang cac mau RNA tir 10"' den 10"' vathuc hien phan irng Realtime - PCR (theo quy trinh n h u n h u m o t a a t r e n ) .
U. KET QUA NGHIEN CUXl VA THAO LUAN
Tat ca cac mau RNA tach chiet dugc deu c6 ham lugng va dg tinh sach can thiet cho phan ling PCR. K6t qua phan tich dugc trinh bay a bang 1 va hinh 1 cho thay: Trong khi ky thuat RT-PCR chi phat hien dugc 6/18 m l u duong tinh (33,33%) thi vai ky thuat Realtime-PCR da phat hien hau h i t cac mau m i u duang tinh, 17/18 (94,44%). R6 rang ky thuat Realtime-PCR CO dg nhay cao han nhieu so vai ky thuat RT-PCR.
TT 1 2 3 4 5 6 7 8 9
S6 hi^u m^u MIS M19 M20 M26 M27 M28 M29 M30 M32
K e t qua PCR
*
+
+ + +
kiem t r a ciia RT-PCR
•f
+ + + + + +
*
2 k j t h u a t P C R TT 10 11 12 13 14 15 16 17 18
Sd hi#u m l u M33 M34 M49 M50 M51 M52 M53 M54 M55
PCR
+
RT-PCR + + + + +
•t
+ + +
HOI NGH| KHOA HQC TOAN QUOC Vg SINH THAI VA TAI NGUYEN SINH VAT LAN THLf 5
l^-^jffr.
lism-
Hinh I. Ty le chdn doan ciia haiphirongphap RT-PCR vd Realtime-PCR Hinh 2 cho thay nong do RNA cua virus trong cac mau cho duang cong khuech dai khac nhau, Nhirng duong xuat hien sam la cac mau c6 nong do virus cao hon va ngugc lai la mau c6 nong dg thap hoac am tinh (mlu d6i chirng am). XEM BONG 1592, 1593
1 - 1 . DC I - U M 3 4
2 me - 1 3 , hM9
- 3 - M 1 9 - - 1 4 M50 -
Amplification Curves - 4 M20 - 5 M26 - 6 M27 - 7. M2a
15 M51 - 1 6 M52 - 1 7 M53 - 1 6 M54 - B M2S - 1 9 - M S 5
- 9 lrf3D - 1 Q M 3 2 - 11,M33 1
1 2 3 4 5 6 7 8 9 1
Hinh 2. Dirang cong khuech dgi san pham RNA ciia 18 mdu virus
De lam sang to dg nhay cua phan irng Realtime-PCR, chinig toi tiln hanh pha loang RNA
long s6 ciia mHu M50 (mau dirang tinli trong bang 1) tir 10'' din 10"'. Phan ung Realtime-PCR
dugc thirc hien \a ket qua dirge trinh bay tai bang 2 va hinh 3 thi den nong do 10"' khong thi
pliai hien dugc tren may.
HOI NGHI KHOA HQC TOAN QUOC VS SINH THAI VA TAl NGUYEN SINH VAT l-AN THlJ 5
Chu TT
1 2 3 4 5 6 7 8
k y ngirong va nhiet do nong chay cua cac m a u pha loang 86 hi^u m l u
DC(-) M50-1 M50-2 M50-3 M50-4 M50-5 M50-e M50-7
Chu ky ngipong
> 35.00 23.97 24 87 28,50 30 65 32 56 34.26
> 35.00
Nhi^t d9 nong chay 75.63 81.17 81.68 81.79 81.89 81 95 82 00 76.18
Bdng2
A m p f i f i c a l i o n C u r v « s
^•3 - S M50-1 - & M50 i - 7 MSqe - 9' MSO-Z |
26 27 28 29 30 31 33 33 34 35 36 37 38 39 40
Hinh 3. Dumg cong khuech dgi ciia cdc mdu pha hang fW -10') Ket qua thi nghiem xac djnh chu ky nguang va nhiet do nong chay cua cac mau pha loang (bang 2) cung cho thay: MSu cang loang dan thi chu ky nguang cang cao va khi nong dg pha loang tai 10"^ thi Reaitime-PCR khong phat hien dugc (mlu am tinh). Trong khi do theo Bailey and Ball (1991) thi mot in triing chet do benh nay c6 chira klioang lO'' phiin tir virus. Tron^
thuc te thi nghiem nay, chung toi chi Igy '/i ca th6 au triing bi benh, do do khi pha loang tai 10 thi gan nhu khong con kha nang phat hien virus trong mau. Dieu nay cung cht'rng to rang chi mot lugng nho virus c6 trong au triing ong benh van c6 the phat hien dugc.
HI. KET LUAN
Ky thuat Realtime-PCR irng dung trong chan doan benh k\x triing tiii cho ty le phat hi^n duong tinh d6i vai Sacbrood virus cao han ky thuat RT-PCR nhilu I5n (94,44%).
Chan doan Sacbrood virus bang Realtime-PCR cho kha nang phat hien a nong dg pha loang
10"^ so vai nong do goc.
HOI NGHj KHOA HOC T O A N Q U O C V £ SINH T H A I V A TAI NGUYEN SINH VAT L A N THQ' 5
TAI LIEU THAM KHAO
1 Aubert M., Ball B.V., Fries 1., Moritz R., Milani N., Bernardinelli I. 2008. Virology and the honey bee. Directorate-General for Research EUR: 489 pp.
2. Bailey. L. & Ball. B.V., 1991. Honey bee pathology. Academic Press, UK.: 193 pp.
3. Bakonyi T., Grabensteiner E., Kolodziejek J., Rusvai M., Topolska G., Ritter W. & Nowotny N., 2002. Phylogenetic analysis of acute bee paralysis virus strains. Appl. and Environ. Microbiology, 68 (12): 6446-6450.
4. Ball B.V, 1999. Sacbrood. In M.E, Colin et ai (ed.). Bee disease diagnosis. Options Mediteranneennes, Zaragoza, Spain: 91-97.
5. Fleige S. & Michael W.P., 2006. RNA integrity and the effect on the real-time qRT-PCR performance. Molecular Aspects of Medicine 27 (2006): 126-139.
6. Grabensteiner E., Ritter W., Carter M. J., Davison S., Pechhacker H., Kolodziejek J., Boecking 0 . , Derakhshifar 1., Moosbeckhofer R., Licck E. & Nowony N., 2001. Sacbrood virus of the honeybee (^Apis mellifera): Rapid identification and phylogenetic analysis using reverse transcription- PCR. Clin. Diagn. Lab. Immun. 8 (I): 93-104.
7. Liu X., Zhang Y., Van X. & Han R., 2010. Prevention of Chinese Sacbrood Virus Infection in Apis cerana using RNA Interference. Current Microbiology Publisher: Springer New York.
8. Mascher A., Lavagnoli S. &. Curatolo M., 1996. Determination of residual oxyteiracycline in honey with an immunoassay kit. Apidologie (1996) 27: 229-233
9. Neumann P. & Carreck N.L., 2010. Honey bee colony losses. J. Apicultureal Research 49 (1): 1-6.
10. Nielsen S.L., Nicolaisen M. & Kryger P., 2008, Incidence of acute bee paralysis virus, black queen cell virus, chronic bee paralysis virus, deformed wing virus, Kashmir bee virus and sacbrood virus in honey bees (/I/j;.(/Hc'////i'm) in Denmark Apidologie. 39(2008)' 310-314
11. Sicdc R., Konig M., Bucliler R., Failing K., Tliiel H.J., 2008. A real-time PCR based survey on acute bee paralysis virus in German bee colonies. Apidologie, 39: 650-661.
12. Thai P.M., Cuoiig H.V., Giang N.V., Chicn T.D., Dinh N.V., Hung H.Q., 2011. Molecular detections of sacbrood and deformed wing virus infected on Apis mellifera in the northern Vietnam.
Sci. and Tech. J. Agric. and Rural Devel., 165: 33-36.
APPLICATION OF REALTIME-PCR FOR DETECTING SACBROOD VIRUS ON HONEYBEES IN HA NOI
PHAM HONG THAI. TRINH THI THU THUY. NGUYEN THI LAN.
NGUYEN VAN CUONG. NGUYEN VAN GIANG. HA VIET CUONG, DANG HUONG LAN SUMMARY
As well as other animals, honeybees are often infected by microorganism included sacbrood virus (SBV) The early infective S8V detection and diagnosis became necessary for control of SBV. In this research, the Realtime-PCR (qPCR) technique was applied in companson with RT-PCR technique in which the total RNA were extracted by TRIzol-LS. The results of examination of 18 honeybee samples that analysed with both techniques were showed with significant differences, in which qPCR was positive detection with 17/18 samples (94,44%) while RT-PCR technique was delected only 6/18 positive samples (33.33%), In addition, the experiment, qPCR was showed the high capacity for SBV detection even with the dilulalion concentration of 10"^ that is more sensirive in companson with RT-PCR technique
