| 摘要: |
| 【目的】对新城疫（ND）国标RT-PCR诊断方法进行改进和优化，建立一种灵敏度高、特异性好、能直接从组织病料中进行目的基因检测的NDV套式RT-PCR(nest RT-PCR，RT-nPCR)方法。【方法】根据GenBank上发表的新城疫病毒基因序列，设计并合成了2对引物，通过筛选最佳RT nPCR条件，建立了NDV RT-nPCR检测方法。通过灵敏性试验、特异性试验、病料检测对比试验等，验证了RT-nPCR方法的可靠性和适用性。【结果】 建立的NDV RT-nPCR方法灵敏度较高，检测的极限为5.6×10-5 ng/L；特异性较好，仅能从NDV中扩增到目的条带。从6份疑似组织病料中能直接检测出4份阳性结果，而国标方法均未检出，病毒分离鉴定结果与优化后的RT-nPCR方法符合率达100%。【结论】建立了一种灵敏度高、特异性好、可直接从组织病料中快速检测NDV核酸的RT-nPCR方法。 |
| 关键词: 新城疫 诊断方法 RT-PCR |
| DOI： |
| 分类号: |
| 基金项目:咸阳市2011年科技计划项目(2011K04-12) |
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| Optimization and application of national standard based RT-PCR diagnostic technique for Newcastle disease |
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WU Xu-jin,ZHU Xiao fu
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| Abstract: |
| 【Objective】Based on improvement and optimization of RT-PCR diagnostic technique for Newcastle disease,this study established a modified RT-PCR diagnostic technique (RT-nPCR),which was sensitive and specific for direct test of NDV from clinical tissue samples.【Method】According to published GenBank NDV gene sequences,two pairs of primers were synthesized to detect NDV.The best conditions for the establishment of NDV RT-nPCR detection were obtained and sensitivity test,specificity test,direct tissue samples test and virus isolation test were carried out to verify the applicability and superiority of the RT-nPCR method.【Result】The established method had high sensitivity with the detection limit of 5.6 ×10-5 ng/L,and the objective band could be amplified only from NDV template.Using the RT-nPCR method,4 out 6 suspicious samples were detected positive,which was 100% in compliance with virus isolation method.On the contrary,the national standard RT-PCR did not identify any positive samples.【Conclusion】A sensitive and specific RT-nPCR method for rapid NDV detection from clinical tissue samples was successfully established. |
| Key words: newcastle disease diagnosis RT-PCR |
