中国豹源豚鼠气单胞菌的分子鉴定与毒力基因分析

Molecular Identification and Virulent Gene Analysis of Aeromonas Caviae from China Leopard(Panthera pardus japonensis)

从表现为急性出血性败血症的中国豹分离到一株细菌,为了鉴定这株细菌,并对其主要毒力基因进行检测分析,实验中进行了细菌的分离培养、生化鉴定及动物毒力试验;PCR扩增gyrB基因并结合系统发育树的构建和分析进行了菌种的分子鉴定;对分离菌进行了气溶素(aerolysin,aer)、热敏感细胞肠毒素(heat-Labile cytotonic enterotoxin,alt)、细胞毒性肠毒素(cytotoxic enterotoxin,act)、热敏蛋白酶(temperature-sensitive protease,eprCAI)、丝氨酸蛋白酶(serine protease,ahp)5种毒力基因的PCR扩增和测序。结果显示:根据培养特性、生化试验、小白鼠毒力试验鉴定分离菌为豚鼠气单胞菌;中国豹源细菌gyrB基因与豚鼠气单胞菌(JQ815381)的同源性为98.9%,应为豚鼠气单胞菌;具有act、alt、eprACI 3种毒力基因。该研究可为快捷、准确检测气单胞菌引起的疾病提供参考。

To identify bacteria isolated from China leopard （ Panthera pardus japonensis） which showed acute hemorrhagic septicaemia and document its pathogenicity, we amplified the gene of gyrB using PCR and constructed and analyzed the phylogenetic tree of the gyrB sequence. Five virulence genes, aerolysin （ aer）, heat - labile cytotonic enterotoxin （ alt ）, cytotoxic enterotoxin （ act）, temperature - sensitive protease（ eprCAI ） and serine protease（ ahp）, were amplified by PCR and sequenced. Molecular identification of the bacteria was confirmed using culture characteristics, biochemical identification and virulence test. The gyrB gene of the bacteria from China leopard fell within a sister group with Aeromonas punctata and the homology was 98. 9% with Aeromonas cavi- ae（JQ815381 ）. The isolated bacteria was identified as Aeromonas caviae which carried act, air and eprACI genes and had potential pathogenicity, which were confirmed from culture characteris- tics, biochemical identification and virulence test. The amplification and sequence analysis of genes gyrB, air, act, aer, ahp and eprACI provides a reference for the rapid and accurate detec- tion of the diseases caused by Aerornonas.