摘要
于1994和1995两年的7月从青岛崂山上马镇养虾场中染病的中国对虾中分离纯化出一种C型杆状病毒。为建立中国对虾病毒的鉴别和检测技术，利用随机扩增多态性DNA技术对电泳纯化的病毒DNA进行分析和研究。将病虾匀浆、经差速离心和蔗糖密度梯度离心纯化获得完整病毒粒子；从病毒中提取DNA后再进行电泳纯化，收集长度完整、均一的病毒DNA；在其他参数保持不变的情况下，对Operon生产的10碱基随机引物K试剂盒进行扩增试验。结果显示：1．病毒DNA有两种存在形式；2．K试剂盒中有2个引物能将病毒DNA扩增出长度不同的产物，其中编号为OPK—01的随机引物可产生多态性DNA片段；3．1995年中国对虾杆状病毒的RAPD分析结果与1994年相同，证实1994和1995两年度引起中国对虾疾病的病原是同一种杆状病毒。由此可以认为：随机扩增多态性DNA技术能将中国对虾C型杆状病毒扩增出多态性DNA片段而达到病毒鉴别和诊断的目的。
A kind of type C baculovirus was detected and purified from diseasedshrimps, Penaeus chinensis collectcd from Laoshan shrimp farms, Qingdao in July1994 and 1995. In order to establish the identification and detection technique,randomly amplifled polymorphic DNA(RAPD) was used to analyze the virus DNAextracted from the virus itselfMorphologically intact baculovirus and nucleocapsids were isolated and purified bydifferent centrifugations of 6 000r / min to 10 000r / min and density gradientcentrifugations on 10%-50% sugrose at 25 000 r / min from cepholothorax homogenate.Viral genomic DNA was extracted from purified virions with a TE buffer (10 mmol/LTris-HCl,100 m mol / L EDTA,pH = 8.0) containing 0.5% (W/V) SDS and 200μg /ml proteinase K, at 55℃for 1.5h, followed by phenol-chloroform extraction andethanol precipitation. The DNA was electrophoresed in 0.7% low-melting point agrosegels (Sigma) to recover the main band. The DNA concentration was estimatod byelectrophoresis.Ten base random primers of Operon kit K were testod individually under the sameexperimental conditions. Random amplification of viral DNA was performed in a 25 μlreaction mixture containing 50-100μg tomplate DNA, 10 mmol / L Tris-HCl, pH =8.3, 50 mmol / L KCl, 2 mmol / L MgCl2, 0.001% gelatin, 200 μmol / L of eachdeoxynucleotide triphosphare, 15 pmol of each primer, and 2 units of Tag DNApolymerase. The reactions were performed by 45 cycles of 94℃denaturation, 33℃annealing and 72℃polymerization, respectively, for 1 min. A complete extension wasdone by 72t for 10 min. An aliquot of 20μl of the prepared samples waselectrophoresed at 4 V / cm for 1.5 h in a 1.5% agarose gel, followed by stainingwith ethidium bromide. DNA fragment was visualized and photographed underultraviolet lightThe results show that: 1) the viral DNA extracted from the basulovirus existedin 2 forms; 2) two random primers in Operon kit K could produce different productsin length from viral DNA, and one of which, designated OPK-01 primer, couldproduce polymorphic and reproducible fingerprint; and 3) RAPD provided the sameresults for vira DNA. The front and back bands recovered from electrophoresing viralDNA respectively produced similar RAPD fingerprints corresponding to the twoseparate years. It is concluded that the baculovimses purifled from diseased P.chinensis in 1994 and 1995 are the same species. The results of this study indicatethat RAPD is a potentially powerful technique for shrimp baculovirus identification anddiagnosis because of its polymorphism and reproducibility.
出处
《海洋与湖沼》
CAS
CSCD
北大核心
1997年第4期394-398,共5页
Oceanologia Et Limnologia Sinica
基金
国家攀登计划"B"资助!PDB6-6-1
