实时荧光定量RT-PCR法定量检测手足口病病原体的探讨

Pathogenic detection of hand-foot-mouth disease by real-time fluorescence quantitative RT-PCR

目的了解北京市门头沟区手足口病患儿感染病毒的型别，探索用荧光定量RT—PCR法对手足口病患者咽拭子标本中肠道病毒71型（EV71）、柯萨奇病毒A16（CoxA16）型病毒载量进行定量检测的可行性。方法采用实时荧光RT—PCR体外扩增法对81例手足口病患儿咽拭子标本提取的RNA进行检测。结果28例手足口病患者体内CoxA16病毒载量〉10^3 copies·ml-1。实时荧光定量RT—PCR法构建的标准曲线显示，样本阈值环数（Ct）值与病毒拷贝数的对数（10g10）之间的相关系数为0．9998，相关性良好。4例手足口病患者咽拭子标本中EV71型病毒载量〉10^3 copies·ml-1。实时荧光定量RT-PCR法标准曲线显示，Ct值与病毒拷贝数的对数之间的相关系数为0．9996，相关性较好。结论门头沟区手足口病病原谱以CoxA16型为主，34．6％的手足口病患儿CoxA16型病毒载量〉10^3 copies·ml-1，4．9％的患儿EV71型〉10^3 copies·ml-1。实时荧光定量RT-PCR法对咽拭子标本中CoxA16、EV71核酸定量检测比较简便快速、结果直观、稳定性强，为进一步探讨患者体内病毒载量与临床症状的关系打下了良好的基础。

Objective To identify the viral infection types of the children with hand-foot-mouth disease in Mentougou district of Beijing using real-time fluorescence quantitative reverse transcription-polymerase chain reaction （RT-PCR） for detection of EVT1 and CoxA16 . Methods RNA was extracted from the throat swab of the children with hand-foot-mouth disease. The extracted RNA was tested by real-time fluores- cence quantitative RT-PCR. Results The CoxA16 viral load of 28 samples exceeded 103 eopies-ml-l. The standard curve of the real-time fluorescence quantitative RT-PCR showed good correlation coefficient （ r = 0. 9998 ） between the cycle threshold values and logarithm of the viral copy number. The EVT1 viral load of d samples exceeded 103 copies＂ ml- 1. The standard curve of the real-time fluorescence quantitative RT-PCR showed good correlation coefficient （ r = 0. 9996） between the cycle threshold values and logarithm of the vi- ral copy number. Conclusions In this study, the main pathogenic detection was CoxAl6 of hand-foot- mouth disease in Mentougou district of Beijing, 34. 6% of the children with hand-foot-mouth disease were caused by CoxA16, 4. 9% of the children with hand-foot-mouth disease were caused by EVT1 in Mentougou district. Real-time fluorescence quantitative RT-PCR offers a rapid and simple method to detect EV71 or Cox A16 from specimens, and allow to analyze the association of the clinical symptoms and virus load.