| 周钧,陶开华,李越希,钱万红,张锦海,王勇,张兆松.聚合酶链反应及基因芯片技术检测日本血吸虫的研究[J].Chinese journal of Epidemiology,2004,25(2):154-157 |
| 聚合酶链反应及基因芯片技术检测日本血吸虫的研究 |
| Detection of Schistosomia japonicum 5D gene by polymerase chain reaction and genechip technique |
| Received:April 08, 2003 |
| DOI： |
| KeyWord: 日本血吸虫 基因芯片 钉螺 |
| English Key Word: Schistosoma japonicum Genechip Oncomelania |
| FundProject:全军“十五”重点课题资助项目(01L006) |
| Author Name | Affiliation | | ZHOUJun | Department of Pathogenic Biology, Nanjing Medical University, Nanjing 210002, China | | TAO Kai-hua | Department of Pathogenic Biology, Nanjing Medical University, Nanjing 210002, China | | LI Yue-xi | Department of Pathogenic Biology, Nanjing Medical University, Nanjing 210002, China | | QIAN Wan-hong | Department of Pathogenic Biology, Nanjing Medical University, Nanjing 210002, China | | ZHANG Jin-hai | Department of Pathogenic Biology, Nanjing Medical University, Nanjing 210002, China | | WANG Yong | 南京医科大学病原生物学系 | | ZHANG Zhao-song | 南京医科大学病原生物学系 |
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| Abstract: |
| 目的 探索研制检测日本血吸虫的基因芯片。方法 依据日本血吸虫高度保守的编码免疫原性毛蚴抗原的5D基因,筛选、设计聚合酶链反应(PCR)的引物和基因探针,制成日本血吸虫基因芯片。检测时首先抽提尾蚴、成虫、虫卵、感染性钉螺的DNA,同时利用对照华支睾吸虫、姜片虫、卫氏并殖吸虫的DNA分别进行PCR扩增及不对称PCR荧光标记,然后用荧光标记后的靶序列与制备的检测芯片进行杂交,杂交结果用ScanArray 3000扫描仪扫描。结果 研制的基因芯片能有效地检测出单个尾蚴、虫卵、阳性钉螺或极微量成虫组织DNA,而常见的华支睾吸虫、姜片虫、卫氏并殖吸虫等未见阳性结果。结论 成功制备了检测日本血吸虫的基因芯片,并具有快速、灵敏、特异等特点。 |
| English Abstract: |
| Objective In order to develop the diagnostic genechip for specific detection of Schistosoma japonicum (Chinese mainland strain). Methods Probe and primers were designed based on the Schistosoma japonicum 5D gene encoding an immunogenic miracidial antigen. The probe for the conservative and specific gene sequence was spotted onto the specially treated glass slides by pin-based spotting robot Pixsys 5500 and was employed to make genechips. A polylmerase chain reaction (PCR) protocol was designed to effetively amplify the 5D gene fragment containing the probe sequence from cercaria, egg, adult worm and infected Oncomelania DNA as well as other flukes DNA, respectively. After 35 cycles by PCR, the products were then labeled with fluorescent Cy3-labeled primer, using dissymmetrical PCR. The labeled PCR products of the target genes were hybridized to the diagnostic genechips for detection of Schistosoma japonicum and a fluorescent scanner (ScanArray 3000) was used to observe and record the hybridization signals. Results The result obtained from the study showed that a 262 bp DNA fragment was amplified from cercaria, egg and adult worm with the designed primers and enable the genechip be applied to detect a single cercaria, egg and adult worm. When the genechip was used to detect Clonorchis sinensis, Fasciolopsis busk, and Paragonimus westermani DNA, the results showed negative, indicating that the genchip had good specificity. Conclusion The genchip technique for detection of Schistosoma japonicum was estabished successfully and having the charcteristics of high sensitivity and specificity. |
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