摘要
利用pH9.5的甘氨酸缓冲液性处理太平洋牡蛎(Crassostrea gigas)匀浆液,PEG沉淀病毒粒子的方法对未知的是否污染有诺瓦克样病毒(Norwalk-like Virus,NLVs)的牡蛎样品进行处理,并提取病毒RNA,进行RT-PCR,凝胶电泳检测扩增结果。初步建立了牡蛎中NVLs的检测方法。纯化所得的特异PCR产物,克隆到T-载体中,筛选转化子,提取质粒,并进行PCR电泳鉴定。重组质粒序列测定结果在NCBI上进行BLASTn显示所得产物与已发表的诺瓦克样病毒的相应序列具有极高的同源性(>95％);根据Genebee在线分析系统AliBee-Multiple Alignment进行对中国的诺瓦克样病毒粪便分离株和牡蛎分离株的扩增序列进行相似性分析,结果从我国养殖的牡蛎样品中和胃肠炎病人粪便中分离出2个的诺瓦克样病毒之间无显著的差异。
Thirty-seven experimental oyster came from three farms of China.Beijing Precautionary Institute provided the positive virus(Norwalk-like viruses, NLVs) separated from gastroenteritis patients. The oysters were shucked and homogenized at high speed. About 25 g homogenization with 7 volume Glycine buffer (pH 9.5) shook at 250 r/min at room temperatura,and centrifuged at 3 000 g for 30 min at 4 ℃ ;the peliet was discarded and the supernatant was adjusted to pH 7.5 then 16% PEG6000 was added to a final concentra-tion of 8% . Precipitation at 4 ℃ for 4 h,then centrifuged at 3 000 g for 30 min.Discarded the supernatant and suspended the peliet in 0.15 mol/L Na2HPO4. Stirred at 100 r/min for 20 min at room temperature and then centrifuged at 10 000 g for 30 min at 4 ℃. The supernatant was adjusted to pH 7. 4 and stored at -20 ℃ for use.300 μL solution taken from the upper steps was used for RNA extraction with TRIzol(Sangon,Shang-hai). The pellet was dissolved with DEPC H2O after died and stored at - 20 ℃ .250 μL stool solution was dealt with the same method as above.A set of nested primers N1, N2, N3 was used for PCR. cDNA was synthesized from primer N1 by the M-MLVRT system(Sangon,shanghai). The PCR product amplified by N1 and N2 was about 237 bp;the PCR product amplified by N1 and N3 was about 326 bp. Among 37 samples six samples were detected positive and most were collected in winter. Four of the six samples could not be amplified by primers N1 and N3. This is due to the fact that many shellfish samples contained a mixture of strains with a few samples containing up to two different strains with both genogroups represented. The amplified 237 bp fragments were electrophoresed on 2.0% agarose gel,purified with the Wizard PCR Preps DNA Purification System (Promega, A7170) ,and ligated with pGEM-T Easy vector by T4 DNA ligase for l h at 16 ℃. JM109 was transformed with the ligation products. Luria-Bertani ( LB) agar plates containing 100 μg/mL ampicillin, 100 μg/mL IPTG and 200 μg/mL X-Gal was used to screen the recombinant colonies. Insertion of the PCR product was verified by PCR and agarose gel electrophorese.Blastning the sequenced results online (NCBI) showed that these cloned fragments were related to NLVs RNA-dependent RNA polymerase gene. Compared the two cloned sequences with online alignment software of Genebee,it showed that the homology of the two sequences is 98.0%.According to the results, winter is the primary season in which oysters are easily contaminated with NLVs. The genotype of strains in China was close to that found in Japan and Korea according to Blastn ( NCBI) . Their homology percentage was higher than 95% , which indicates that NLVs in close geographical locations commonly are homologous.
出处
《中国水产科学》
CAS
CSCD
北大核心
2004年第6期525-530,共6页
Journal of Fishery Sciences of China
基金
宁波市博士科学基金.
