【作者】 何闪闪；

【导师】 薛长湖；

【作者基本信息】 中国海洋大学 ， 水产品加工与贮藏， 2004， 硕士

【摘要】 致病微生物的快速检测与分离技术是致病微生物研究的热点之一。PCR能够快速检测食品中存在的致病微生物，是一种有效的快速检测手段。探针原位杂交技术可以有效的检测和快速分离食品中存在的致病微生物。本论文采用PCR和探针原位杂交技术分离贝类中的副溶血弧菌和创伤弧菌。 应用tlh探针和tdh探针检测分离存在于贝类样品中的副溶血弧菌，在近一个月的实验中有两次tlh探针杂交结果呈阳性，共分离出了六株tlh阳性菌株，但tdh探针的检测结果都为阴性，用PCR对分离出来的六株tlh阳性菌株进行检验，结果都为tlh阳性。用PCR及tlh探针和tdh探针分析从青岛市疾病控制中心得到的六株副溶血弧菌，发现这六株菌都为tlh阳性菌株，其中五株为tdh阳性菌株，且都不是新O3：K6菌株，PCR结果与探针杂交结果相一致。应用PCR技术检测贝类(主要是蛤蜊)样品中的创伤弧菌，在近四个月的检测中在蛤蜊样品中检到了三次创伤弧菌，用碱磷酶标记的VVAP探针分离创伤弧菌，分离出了两株创伤弧菌。 运用RAPD技术对六株tdh阳性菌株进行基因分型，结果表明虽然这六株菌都为tdh阳性菌株，但在遗传物质上存在差异，其中菌株VPS与其它五株从青岛分离出来的副溶血弧菌在基因上有较大的差异，引物oligo17获得的多态性条带最多，为9条。 由于国内无法将碱性磷酸酶标记到寡核苷酸探针上，采用生物素标记的探针来代替碱性磷酸酶标记的探针，经过多次的实验，得到了与碱磷酶标记的探针相近的杂交效果。水产贝类中副溶血弧菌与创伤弧菌的基因检测与分析 对从青岛分离出来的ulh阳性菌株VP3的tdh基因进行序列分析，发现VP3与WPI的tdh基因序列完全相同，与VPS的同源性为99.5%。对分离到的两株创伤弧菌的vvhA基因进行克隆，经BLASTn分析表明这两株创伤弧菌的。hA基因与菌株L一180的核昔酸同源性最高，VVI与菌株L一180的核昔酸同源性为99.57%，VVZ与菌株L一180的核营酸同源性99.72%。更多还原

【Abstract】 Techniques of quick detection and isolation for pathogenic microorganisms have been concentrated on recent years. Polymerase chain reaction (PCR) is an effective detection technology, which can be used to quickly detect pathogenic microorganisms. Probe in situ hybridization is a valid approach as well. In this paper, PCR and tlh and tdh probe were used to isolate Vibrio parahaemolyticus and Vibrio vulnificus strains in seashells.Six Vibrio parahaemolyticus strains have been isolated from seashells by using tlh and tdh probe. Two results of probe tlh hybridization were positive and all results of probe tdh hybridization were negative in about one month. It was demonstrated that all the six isolated tlh positive strains were tlh positive indeed by the PCR assay. The six Vibrio parahaemolyticus strains obtained from the Disease Control Center of Qingdao were examined by PCR assay and probe hybridization. All of the six strains were tlh positive and five of them were tdh positive, and non-new O3 :K6 Vibrio parahaemolyticus strain was found. The Vibrio vulnificus strains have been detected for three times in seashells(mainly in clams) in 4 months by PCR assay and two Vibrio vulnificus strains have been isolated with WAP probe labled with alkaline phosphatase(AP).The gene type of the six positive tdh strains were analyzed by random amplified polymorphic DNA (RAPD) technology. The results showed that although the six strains were all tdh positive, they were different in genome. The strain of VPS remarkably differed from the other five Vibrio parahaemoliticus isolated from Qingdao. The maximum polymorphic strips of nine were got in primer oligol?.Now oligo nucleotide can’t be labeled with alkaline phosphatase in China. So probe labeled with biotin was used instead of AP, and similar cross bred effect was obtained by numerous investigations.The tdh gene of VPS isolated from Qingdao was cloned and sequence analysis showed a homology of 100% between the VP3’s tdh gene and the reference strain WPl’s tdh gene available in Genbank. The homology between VPS’s tdh gene and VPS’s tdh gene was 99.5%. The vvhA genes of two isolated Vibrio vulnificus were cloned and analyzed with BLASTn. The data showed that the whA gene sequences of these two strains have the highest homology with L-180. The homology between VV1 and L-180 was 99.52%, and the homology between VV2 and L-180 was 99.72%.更多还原