棕熊蛔虫ITS rDNA的PCR扩增与序列分析

CLONING AND SEQUENCE ANALYSIS OF ITS RDNA OF ASCARID NEMATODES FROM URSUS ARCTOS

目的以核糖体DNA（rDNA）的第一与第二内转录间隔区序列（ITS-1及ITS-2）鉴定从广州动物园棕熊分离的蛔虫种类。方法应用PCR方法以保守引物NC5和NC2扩增蛔虫样本的ITS及5．8SrDNA序列，将PCR扩增出的片段纯化后克隆至pGEM-TEsay载体，用菌落PCR及酶切鉴定阳性菌落，对阳性菌落进行测序，并与Gen．Bank“公布的人蛔虫（Ascaris lumbricoides）、猪蛔虫（Ascariis suum）、浣熊贝利斯蛔虫（Baylisascaris procyonh）及狮弓蛔虫（Toxascaris leonina）ITS序列比较。结果从棕熊分离的2个蛔虫样本的ITS及5．8SrDNA总长为859—861bp，种内相似性为99．7％，与GenBank^TM公布的人蛔虫、猪蛔虫、浣熊贝利斯蛔虫及狮弓蛔虫的相似性分别为84．6％、84．5％、89．3％及72．3％，序列差异明显。结论棕熊蛔虫不同于上述种类蛔虫，可能为Baylisacaris transfuga.

Objective To determine the specific status of the ascarid samples isolated from a brown bear （ Ursus arctos） in Guangzhou Zoo, China, according to the sequences of the first and second internal transcribed spacers （ITS） of ribosomal DNA （rDNA）. Methods With a pair of conserved primers （ NC2/NC5 ） , the ITS rDNA was amplified by PCR from the ascarid samples. The amplicons were purified and cloned into pGEM-T Easy vector, and, after being identified by PCR amplification and restriction digestion, the positive clones were sequenced and the sequences were compared with those of Baylisascaris procyonis, Ascaris lumbricoides, Ascaris suum, and Toxascaris leonina publicated in GenBankTM. Results The results revealed that the two ascarid specimens from the brown bear had different ITS and 5, 8S rDNA sequences, the length of which lengths were 859-861bp and the similarity were 99.7%. The ITS sequences of the ascarid nermatodes from brown bear were significantly different from that of B. procyonis, A. lumbricoides, A. suum and T. leonina and the similarity was 89.3% , 84.6% , 84.5% and 72.3%, respectively. Conclusion The ascarid nematodes collected from the brown bear were different from the above species, which might be Baylisascaris transfuga.