Tgp chi Khoa hoc - Cong nghe Thuy sdn So 3/2013
THONG BAO KHOA HOC
DIEU TRA TINH HINH NHIEM VIRUT LAEM SINGH BANG KY THUAT RT-PCR TREN TOM SU NUOI {Penaeus monodon) 6 KHANH HOA
SURVEY ON LAEM SINGH VIRUS IN FARMED BLACK TIGER SHIRMP (Penaeus monodon) IN KHANH HOA PROVINCE USING RT-PCR
Bang Thuy Binh', Nguyen Viit Diing', Nguyin ThiAnh Thti^, Van Hdng Cam'' Ngay nhiin bai: 10/01/2013; Ngay phan bien thongqua 20/5/2013; Ngay duyet dang 10/9/2013
T O M T A T
Trong nghien cuu ndy chung toi sH di^ngphuang phdp RT-PCR (Reverse Transcriptase Polymerase chain reaction) de khdo sdt tinh hinh nhiem virut Laem-Singh (LSNV) trin tom su pong vd thuang phdm tai dja bdn tinh Khdnh Hda 13 mdu (16,25%) trong tong so 80 mdu torn (thu ngdu nhien tir 11/2011 - 5/2012 tgi cdc cha, cdc trai nuoi torn a khu vuc huy4n Vgn Ninh, Ninh Hoa, Cam Ranh vd Nha Trang - tinh Khdnh Hoa) dupc xdc dinh nhiem LSNV. Trong do. tom su thuang phdm co ddu hteu chdm ldn nhiim LSNV Id 36,67% (11/30). 8% (2/25) torn su thuang phdm nhiim LSNV tuy be ngodi khong co bieu hiin chdm lan. Nghiin cim chua phdt hiin co s\f hien dien cua LSNV tren tdm su giong (0 /25 mdu torn giong).
Tir khoa: RT-PCR. LSNV. MSGS, Penaeus monodon. Khdnh Hda A B S T R A C T
In this research, RT-PCR (Reverse Transcriptase Polymerase chain reaction) method was used to identify the proportion of Laem-Singh virus infection in post-larvae and marketed shnmp in Khanh Hoa province A survey of 80 P. monodon collected from 11/2011 to 5/2012 from markets, farms around Van Ninh. Ninh Hoa. Cam Ranh and Nha Trang (all belonged lo Khanh Hoa Province) revealed that 16.25% (13 of 80) was infected by Laem-Singh virus (LSNV).
Among these. 36,67% market shrimps with slow growth syndrome gave positive result with LSNV. In addition, 8% (2/25) commercial shrimp got LSNV infection without signal associated with disease or slow growth. There was no evidence of LSNV infection in post-larvae.
Keywords: RT-PCR. LSNV. MSGS, Penaeus monodon, Khdnh Hoa
I. OAT V A N D £ cdc m l u thu tai tinh Soe Trang (7/2008) va Kien Giang Vims Laem-Singh (LSNV) lan dau tien dugc (3/2008) la rat cao chilm 63% so mau phan tieh phdt hign tren tdm sCi Penaeus monodoti cd dau hipu Tom benh MSGS cd 3 trong so 5 dgc dilm sau:
didm Idn d tinh Laem Singh, Thdi Lan nam 2003. tdm t i i sam mdu bat thudng, kdm an, hogt dpng Sittidilokratna vd cs (2009) dilu tra tinh hinh nhiem yeu, cd dim vdng sang bat thudng tren thdn tdm, LSNV trdn tdm su thu tu 1998 den 2007. Ket qua dot byng tdm co dang than dot tre; rau tdm de bi dut eho thay cd den 46/81 mlu bi nhilm LSNV d Andhra gay; toe dp tang trudng trung binh < 0,1g/ngay trong Prades An DO; d Malaysia vd Indonesia ehi cd 1/6 4 thdng Idm kdo ddi vu thu hogch ao tdm nufii.
m l u nhilm LSNV; d Vigt Nam Id 2/6 vd d Thai Lan Nghien cuu ndy dp dyng phuang phap RT-PCR 39/40 mau tdm su nudi nhilm LSNV. Theo Nguyin mpt budc d l xac dmh su hign dien cua LSNV trdn Vilt Dung vd cs (2011) t i n xult bat gap sy hipn dien tdm su d khu vyc tinh Khdnh Hoa nham budc dau cua LSNV tren edc mau tdm cd bilu hien chgm Idn d khdo sat tinh hinh nhiim LSNV tren dja ban tinh.
• TS. B|ng Thuy Binh, ^ Nguyen Vi^t Dung, ^ Nguyen Thj Anh Thu, * Van Hong Ckm- Vi|n Cong ngh# Sinh hgc vS Mfli trudng - Trudng Dgi hpe Nha Trang
TRUONG DAI HQC NHA TRANG • 7
Tgp chi Khoa hoc - Cong nghe Thuy sdn So 3/2013
II. VAT LIEU VA PHUONG P H A P NGHIEN CUU 1. Doi tirgng vd phuong phdp thu m l u
Mdu tdm su (Penaeus monodon) gom: 25 mdu tdm gilng, 30 mdu tdm thuang phim cd dau higu Cham Idn (Monodon Slow Growth Syndrome - MSGS) vd 25 m l u tdm thuang pham khoe manh dugc thu nglu nhien tai edc chg thdnh phd Nha Trang, cae trgi nudi d khu vyc huyen Vgn Ninh, Ninh Hda vd thdnh p h i Cam Ranh - tinh Khdnh Hda, tip thdng 11/2011 tdi thdng 5/2012. Mau tdm dugc thu sing va bao quan trong thiing da, van chuyen nhanh v l phdng thi nghiem vd bao quan d -40°C.
Tdm ed dau hipu chgm Idn dugc thu d giai doan tu 16 den 104 ngdy tuoi (trpng lugng tmng binh 5,5 ± 0,3g vd chilu dai trung binh 5,5 ± 0,5cm, n = 30). NhUng m l u tdm ndy d i u cd dau hieu gidm lugng tieu thy thue an, bai vdo bd bat thudng.
Tdm phdt triln binh thudng eiing dugc thu d giai dogn tCf 16 din 104 ngdy tuoi (trpng lugng tnjng binh 6,8 ± 0,3g vd chieu ddi tmng binh 7,4 ± 0,5em, n = 25).
Mlu ehung duang LSNV dugc eung cap bdi Vi#n Nghidn cuu Nudi trong Thuy san 2 (RIA2).
2. Tdch chiet RNA t i n g so
RNA ting s i cOa tdm su P. monodon dugc tdch chilt dya tren phuang phdp cua Chomczynski va Saechi (2011). Mlu tdm (100-150mg tu mang eua tdm thuang phim hogc 30-50 cd the tdm giong dugc nghiin nhuyln vdi cdc tdc nhan biin tinh mgnh (Guanidine 14M vd phenol). Sau khi da bo sung dung djch: phenol ehlorofomi, ly tam d l loai bd cdc thdnh phan khong mong muon trong m l u dong thdi d l tdch RNA hda tan trong pha nude ra khdi pha phenol. Tilp dd, RNA dugc kit tOa bdi isopropanol vd n>a hai lan vdi ethanol 75%. Cuoi ciing hda tan RNA vdi lOOpI nude tinh khilt (DEPC-Diethyl pyrocarbonate). Trudc khi tien hanh phdn ung RT-PCR, dung d|Ch RNA tong s i dugc biln tinh d 70°C trong 10 phut vd Idm Ignh nhanh trong dd.
3. Phat hi#n virut LSNV bang phircng phap RT-PCR
Phdn ung RT-PCR phdt hien vinit LSNV dugc tiln hdnh theo Sritunyalucksana vd cs (2006) vd dugc hi^u chinh bdi Nguyen Vilt Dung vd es (2011).
Dung dieh tdch chilt ARN da biln tinh dugc diing cho phan dng RT-PCR mdt budc de khulch dai dogn gen RdRp (RNA dependent RNA polymerase) su dyng egp m l i 20AF (5- TTGCCTTCTCCC- GAGTGGTC - 3') va 20AR (5'- CCGGGTGAGG- TAGCTGCTTG - 3') (Sntunyalueksana vd es, 2006) Phdn irng dugc Bin hanh vdi tong the tich 25pl (gim 2pl khudn ARN, 35pmol m l i moi),
dem Coloriess GoTaq® Flexi IX, 0,2 mM dNTPs, 2mM MgClj, 5U AMV Reverse trancriptase, 1,25U Gotaq® Hotstart Polymerase (Promega, My). Phdn ung dugc chgy tren mdy ludn nhigt Icycler (Bio-rad) theo chuang trinh nhi#t dp: Budc ting hgp eONA tai 44°C tmng 40 phut; t i p theo Id Id budc biln tinh DNA tgi 95''C trong 3 phut, sau dd Id 35 ehu kJ ting hgp DNA bat dau d 94"^ trong 40 giay, 60''C frong 20 gidy, 72''C trong 30 gidy, euli cCing Id budc keo ddi tgi 72"C trong 10 phCrt.
San phim cua phdn ung PCR duge di$n di kiem tra tren gel agarose 1,5% nhupm ethidium bromide. K i t qud dugc ghi nhgn sir dyng h$ thing ghi anh gel ty ddng Geldoc va phin mIm Quantity One (Bio-rad).
III. K^T QUA NGHIEN CUU VA T H A O L U A N 1. D i u hi$u b$nh ii
M i u tdm su thuang phim cd d i u hi#u ch^m Idn (30 mau), trong dd 8/30 ed dau hi#u Idng v6 (LSS - loose shell syndrome); 5/30 m l u tdm bj b§nh d i m trang (WSSV - White Spot Syndrome Virus);
3/30 mau tdm bj b$nh hogi tir ea quan tgo mdu vd ea quan bilu md (IHHNV - infectious Hypodermal and Hematopoietic Necrosis Virus). Tdm khde mgnh cd trpng lugng vd chilu ddi trung binh ldn han tdm cd dau higu chdm Idn (Trpng lugng cCia tdm khde mgnh: 7,4 ± 0,5cm vd trpng lugng cOa tdm chdm Idn: 6,8 ± 0,3g, n = 25; Chilu ddi cua tdm khde manh: 5,5 ± 0,5cm vd chilu ddi cCia tdm chdm tdn:
5,5 ± 0,3g, n = 30). Dau higu b#nh 1^ vd sy khdc bi$t kich thudc duge mc ta a hinh 1.
•fBMM''
Hinh 1. Tom sii tha nuoi sau 104 ngiy Sayc thu tir cCiDg mfl ao nuoi torn v la Van Thanh, huyfn Vfn Ninh
1 Tom khoe mifnh. B Tom CO ddu hiiu c i a m Ion, C. Tom c6 dau hiru chfin ldn vi
> TRUONG OAI HOC NHA TRANG
Tgp chi Khoa hoc - Cong nghe Thuy sdn So 3/2013
2. Khuech dai sdn pham PCR
San p h i m PCR khulch dgi bdi moi 20AF va 20AR cd kich thudc 197 bp phO hgp vdi tinh todn ly thuylt (hinh 2). K i t qud eung phd hgp vdi nghien cCru cOa Sritunyalucksana vd es (2006) vd Nguyin Viet Dung vd es(2011).
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1000 b p ^
SOObp-
200 b p -
Hinh 2. Kit qua dif n di sin pham cda quy trinh RT-PCR chin dodn b^nh LSNV tren tom su GlagM OyagdniSnDNAlOObptGBieRuler^J.gungl 2-MdudumgiiahLSNyinnldmkhoemwil, pag3.4 miudunngnnhISNVIratl6mMSGS
pagS.S. mdu ±img tilth LSNV lrinl6mcadJuhifu long io.g,^ 7 S mdu duang linh LSNV irai idm bi bath dom trdng, gieng 9 JO hfdu dmmg Imh \m LSHViren lorn bibiKhlHHNV;gamgtl, 12-Miu dm linh Irert tout gidng. giaig IS Dotchaagdm girng 14 E)di chmg duong
3. Tinh hinh nhiem LSNV d* Khanh Hda
K i t qud phan tich cho thay cd 13/80 mau tdm sii thuang pham bj nhiem virus LSNV, khdng phdt hien mau tdm giong nhiim LSNV. K i t qua phan tieh ty Id nhiim fren cac mau tdm sCi thu thap duge the hign 6 bang 1.
Bang 1. K i t qud khdo sdt nhiem LSNV mau tdm su g i l n g vd nudi t h u v n g pham tgi khu vipc tinh Khanh Hda
M l u duang tinh Tong
Tom gi6ng S 6 m l u
0 / 2 5 Tyl§
0%
Tom thuong phim MSGS SSn,lu
11/30 T»l«
36,67%
Tom UiLfcmg phim p h i t trien binli t l i i r ^ g So mau
2/25 TylS 8%
1 3 / 8 0 ( 1 6 , 2 5 % ) K i t qud nghign ciru khdng phdt hipn cd sy hi$n
dign cua LSNV tren tdm giong (0/25 m l u nghien ct>u). (y Viet Nam da phdt hign ra tdm su giong bj nhilm LSNV chilm ty l# 1%. K i t qua nghidn CLPU ndy phCi hgp vdi nghien cuu cua Nguyin Vilt Dung vd cs (2011), dieu ndy cho thay xdc xuat phdt hidn b§nh LSNV frdn tdm gilng la khd thap.
T i n suat bdt ggp cua viais LSNV trdn tdm su cd dau higu chgm Idn it khu vyc nudi tinh Khdnh Hoa Id 36,67%. Tdm thuang phim cd dau hieu cham Idn dugc ttiu d edc chg vd dia tgi Nha Trang, Ninh Hda vd Cam Ranh. Trpng dd, 3/8 mau tdm cd d i u higu chdm Idn vd Idng vd (LSS); 3/5 m i u tdm ed d i u hi$u chdm Idn vd duang tinh vdi benh dim trdng (WSSV); vd 2/3 mdu tdm cd dau hieu chgm Idn vd duang tinh vdi b^nh hogi ti> ea quan tgo mdu vd ea quan bilu md (IHHNV) d i u phdt hidn duang tinh vdi LSNV
d Thai Lan ndm 2007, 39/40 mau tdm su cd d i u higu chgm Idn da dugc phdt hign ed nhiem vinjs LSNV. b Vi§t Nam, 63% m l u phdn tieh td- tdm cd d i u hiOu chdm Idn phdt hi$n cd sy hipn dipn cda LSNV tgi tinh Sde Trdng. K i t qud nghien cuu ciia Chung tdi eho thiy fr^n cde m i u tdm cd d i u higu
cham Idn d khu vyc trong tinh cd ti Ig nhilm LSNV thap han so vdi mlu d Soc Trang - Vi0t Nam vd mdu d Thdi Lan, tuy nhien tan suit 36,67% nhiim LSNV tren ^ a ban tinh la mdt con s i ddng bdo dpng. Nghien cu-u eua Prakasha vd cs (2007) cung phat hi^n 3 mau duang tinh vdi LSNV cd dau higu Idng vd (LSS); 2 trong 3 mdu khdc duang tinh vdi LSNV cung nhiem virus dim trdng (WSSV) vd vinjs gay benh edi (MBV). Rd rang ring nhiim LSNV tren quan t h i tdm cd d i u hieu chdm Idn cd t^ le khd cao, dieu ndy cho thay cd moi quan hg gida tdm cd dau hieu chgm Idn (MSGS) vd nhilm LSNV, cung nhu t i e dp tdng tnjdng ehgm ed t h i dnh hudng bdi cdc b#nh ly khdc (nhu LSS, WSSV, MBV IHHNV hayHPV...).
Cdc m l u tdm sLJ phat tnin binh thudng fren dja bdn tinh bi nhiim LSNV Id 8%. Sy xuat hien cua vinjs LSNV tren cd tdm cd d i u higu chdm Idn vd tdm phdt trien binh thudng cung da tCrng duge ghi nhdn trud-c ddy. (3 An Dp, ti 1^ nhilm vims LSNV trdn cdc ao nudi edng nghiep Id 5,4% trong 56 mau phdn tieh ngau nhien. Nam 2007 eung tgi An Dp ty le nhilm LSNV trdn tdm khdng cd dau hi^u chgm Idn dd dugc ghi nhgn cao han (56% trong 81 m l u
TRUONG OAI HOC NHA TRANG • 9
Tgp chi Khoa hoc - Cong nghe Thiiy sdn So 3/2013
nghien ci>u) Trong khi dd, ty le nhiem LSNV trencdc m l u tdm thu ngau nhien tgi khu vuc ding bang sdng CCPU Long Id 10.6%. Ket qud nghien ciru eua ehung tdi ghi nhdn sy hien dign cua virus LSNV trong hg thong tdm nudi d khu vyc tinh Khanh Hda.
K i t qud khdo sat cung cho thay ty le nhiim LSNV tren tdm su co dau hi§u chgm Idn (36,67%) cao han tom su phdt tnIn binh thudng (8%). Dilu ndy chung to cd mil lien hp giira virus LSNV vd hien tugng tdm cham Idn tren tdm nudi d Vigt Nam dong thdi cung phu hgp vdi kit lugn eua Panphut vd cs (2011), vims LSNV Id (^eu ki§n can chir chua phai Id dieu kien du d l gdy ra bgnh chdm Idn tren tdm sii.
Do virus LSNV da dugc xde djnh hien dien tren tdm sd va ed kha ndng Idy nhilm d eac lodi giap xac quanh khu vyc nudi (Kumar vd es, 2011) nen cd t h i dnh hudng din su lan truyen den he thong canh
tdc. mdt khdc LSNV lidn quan din bgnh chdm Idn vi vdy nlu keo ddi thdi gian nudi se Idm gidm ndng suit, gdy thigt hgi cho ngudi nudi vi phdi kdo ddi thdi gian chdm soc, phdt smh them ehi phi thire an vd thule men cho tdm. Vi vgy, vi§c dp dyng phuang phdp RT- PCR d l gidm sdt virus LSNV Id can thilt d l ndng cao chat lugng tdm su tgi khu vye tinh Khdnh Hda.
iV. KtT LUAN VA KI^N NGHj
Vims LSNV da hign dign frong h§ thing nudi tdm su d tinh Khdnh Hda. TJ l# tdm sii nudi thuang phim cd dau higu chgm Idn nhilm LSNV cao han trdn tdm sil phdt trien binh thudng. Chua phdt hign tdm gilng nhilm LSNV. Vi vdy, can xay dung hg thing kiem sodt b$nh LSNV de canh bdo eho ngudi nudi torn nhdm ndng cao chit lugng tdm sii thuang phim.
TAI LIEU THAM KHAO Tieng Vi^t
1 Nguyin Viet Diing, Tran Thj Phucmg Tii, Pham Hung Van, Nguyen Vin H^o. Phat hien vir RT- PCR tren t6m sii nuoi (Penaeus monodon) a dong bang song Cim Long Nong nghiep v 0866-7020,2011:86-90.
IS Laem smgh bang ky Ihu^t Phat trien Nong thon, ISSN
Tiing Anh
Chomczynski P and Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analytical Biochemistry, 1987 162(1): 156-159.
Knshnan P, Makesh M, Chaudhari A, Punishothaman CS and Rajendran KV. Natural host-range and experimental transmission of Laem-Singh virus (LSNV). Diseases of Aquatic Organisms, 2011. 96(1): 21-27.
Panphut W, Senapin SS, Sriurairatana SS, Withyachumnamkul B and Flegel TW. A novel integrase-contaming element may interact wilh Laem-Singh virus (LSNV) to cause slow growth in giant tiger shnmp. BMC Veterinary Research, 2011.7(1): 18.
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Pratoomthai B, Sakaew W, Sriurairatana S, Wongprasert K and Withyachumnamkul B. Retinopathy in stunted black tiger sbraap Penaeus monodon and possible association with Laem-Singh virus (LSNV). Aquaculture, 2008. 284(1-4) 53-58.
Pratoomthai B, Wongprasert K, Flegel WT and Withyachumnamkul B Infection by Laem-Singh Virus in the fasciculated zone and organ of Beltonci of the eyes of small Penaeus monodon from Monodon slow-growth syndrome pond.
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Sittidilokratna N, Dangtip S, Sntunyalueksana K, Babu R, Pradeep B, Mohan CV, Gudkovs N and Walker PJ, Detection of Laem-Singh virus in cultured Penaeus monodon shnmp from several sites in the Indo-Pacific region. Diseases of Aquatic Organisms, 2009, 84(3): 195-200.
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