Objective To investigate the detection of pathogen from children with hand-foot-mouth disease (HFMD) in Kunming, in order to provide evidence for HFMD prevention and dynamic monitoring. Methods From April to August in 2011, 83 specimens including 73 throat swabs and 10 vesicle fluid were collected from 73 patients with HFMD in Kunming. CoxA16 and EV71 were detected by real-time RT-PCR. Three specimens randomly sampled from the positive amplified products of EV71 were bi-directly amplified. Nucleotide sequence of the products was analyzed and compared with reference virus strain of Gen-Bank. The phylogenetic analysis was based on VP1 gene of enterovirus. Meanwhile, epidemiological data were collected and analyzed. Results The positive detection rates of throat swab and vesicle fluid specimens were 80.82% (59/73) and 100% (10/10), respectively; the difference was statistically significant (χ2=6.997, P＜0.01). The viral loads of throat swab and vesicle samples were (1.42±2.71)×104 and (1.24±1.57)×104, respectively, and there was no significant difference. In 73 patients, the positive rates of CoxA16 and EV71 were 80.82% (59/73) and 20.55% (15/73), respectively, and the rate of mixed infection with CoxA16 and EV71 was 12.33% (9/73). VPl-based phylogenetic analysis showed that three isolated strains of EV71 have a high degree (94.0% - 98.4%) of homology with subgenogroup C4 and belong to the same branch as the 11th strain of subgenogroup C4. Among 73 children with HFMD, there were 50 (68.49%) mild cases including 40 (80%) infected with CoxA16 and 1 mixedly infected with CoxA16 and EV71, and 23 severe cases including 9 infected with CoxA16, 5 infected with EV71, and 9 mixedly infected with CoxA16 and EV71. Conclusions CoxA16 and EV71 were the important pathogens of HFMD epidemic in Kunming during 2011; the positive detection rate of virus from vesicle fluid was higher than from throat swabs.%目的 了解手足口病患儿不同标本中病原检出情况,为手足口病的综合防治和肠道病毒的动态监测提供依据.方法 2011年4月-8月,收集昆明地区73例手足口病患儿的咽拭子标本73份,疱疹液标本10份.采用实时荧光定量PCR方法进行CoxA16和EV71病毒核酸的定性和定量检测；并随机抽取3份EV71阳性标本进行VP1基因节段的双向扩增,测定和分析核苷酸序列,并与GenBank中的EV71参考毒株进行比较,依据VP1片段构建基因亲缘性关系树；同时收集患儿的临床流行病学资料,进行临床特征分析.结果 73份咽拭子标本中检出阳性标本59份(80 82％),10份疱疹液标本中检出阳性标本10份(100％),差异有统计学意义(x2=6.997,P＜0.01)；咽拭子标本的病毒载量拷贝数为(1.42±2.71)× 104,疱疹液标本为(1.24± 1.57)×104,差异无统计学意义.CoxA16阳性59例(80.82％),EV71阳性15例(20.55％)；CoxA16和EV71均为阳性9例,混合感染率12.33％.3株EV71病毒株的VP1节段核苷酸序列与C基因型中的C4亚型代表株的核苷酸同源性高达94.0％～98.4％；与C4亚型的第11株毒株同属一个分支.73例HFMD患儿中,普通病例50例(68.49％),其中40例(80％)为CoxA16感染,1例为EV71和CoxA16混合感染,其余9例患儿未检测出EV71和CoxA16病毒；重症病例23例(31.51％),其中9例 (39.13％) CoxA16感染,5例(21.74％) EV71感染,9例(39.13％) EV71和CoxA16混合感染.结论 CoxA16和EV71是2011年昆明地区手足口病的重要致病病原；疱疹液标本的病毒检出率高.
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