进境玉米种子中玉米褪绿斑驳病毒的检测鉴定

Detection and identification of Maize chlorotic mottle virus from imported maize seeds

2011年3月,黑龙江出入境检验检疫局技术中心植检室先后接收两批进境的玉米种子,标明来自德国,报验号分别为:11201100021、11201100022,要求检测玉米褪绿斑驳病毒等检疫性有害生物。分别选取300粒种子进行两组室内盆栽试植试验,11201100021组观察到两株具有典型褪绿斑驳症状的病苗;11201100022组观察到褪绿斑驳和褪绿畸形不同症状的两株病苗。对11201100021组病苗进行RT-PCR检测,扩增出约711bp的特异性片断,通过正反向引物测PCR产物的序列,拼接后可知扩增的目的基因片断大小为711bp,在NCBIBLAST中进行核苷酸序列比对,可知该目的片断是玉米褪绿斑驳病毒的CP全长基因,进一步验证了RT-PCR扩增结果。核苷酸序列分析结果表明,核苷酸序列与美国GenBank上登录号为GU594293的MCMV外壳蛋白序列完全相同。与其他分离物外壳蛋白基因核苷酸序列同源性为97%～99.8%对11201100022组病苗进行了针对MCMV的ELISA检测,结果1株为阳性,另1株为弱阳性。根据以上检测鉴定结果判断这两批进境玉米种子携带玉米褪绿斑驳病毒。

Two lots of maize seeds from Germany were sent to Technical Center of Inspection and Quarantine of Heilongjiang Entry-Exit Inspection and Quarantine Bureau to test Maize chlorotic mottle virus （MCMV） and other quarantine pests in March 2011, and marked No.11201100021 and No.11201100022, respectively. Two lots of imported maize seeds were planted in a quarantine labora- tory, respectively. Two seedlings of No.11201100021 lot with chlorotic mottle symptoms were observed. One seedling of No.11201100022 lot with chlorotic deformation symptoms were observed and another seedling with chlorotic mottle symptoms were observed. The samples with symptoms of No.11201100021 lot were detected by RT-PCR method and the 711 bp size target bands were amplified specificatly. The nucleotide sequence of both RT-PCR product was the same as the capsid protein sequence of American isolate of MCMV （GU594293 on GenBank）. The similarity range of nucleotide sequence of both RT-PCR product and six others MCMV coat protein genes was 97%-99.8%. The samples with symptoms of No.11201100022 lot were detected by ELISA. The results showed that the chlorotic deformation seedling reacted positively with antibody against MCMV. Another positive result was weak. Based on the above results, MCMV was carried by the two lots of imported maize seeds.