【摘要】 拟建立一种基于GeXP多基因表达分析系统的多重PCR方法,同时检测6种鸡免疫抑制病病毒——鸡马立克病毒、禽白血病病毒（包括A、B和J亚群）、禽网状内皮组织增生症病毒、禽呼肠孤病毒、鸡传染性贫血病毒和传染性法氏囊炎病毒。多重PCR反应使用嵌合引物和通用引物组合的反应体系,经过GeXP多基因表达分析系统进行毛细管电泳,鉴别PCR产物片段;通过调整嵌合引物浓度、Mg²⁺浓度、Taq酶浓度优化反应体系及调整退火温度和时间优化反应条件。应用建立的多重GeXP-PCR,分别单独和同时检测6种鸡免疫抑制病病毒的8个目的基因,同时以其他常见禽类病毒和鸡组织器官核酸为对照,验证其特异性;检测8个目的基因不同浓度的克隆质粒,验证其敏感性;应用建立的多重PCR检测300份临床样品,并同时用荧光定量PCR和测序验证其检测结果的准确性。结果表明建立的多重GeXP-PCR方法能够分别扩增出6种病毒,8个特异性目的片段,不能扩增其他常见禽类病毒和鸡组织器官的基因;在低至100copies·20μL^-1的水平能同时特异性地检测出6种鸡免疫抑制病病毒核酸;检测300份临床病死鸡样品,190份样品显示为阳性,PCR和测序结果与多重GeXP-PCR结果相符。本研究建立的多重GeXP-PCR方法可特异、敏感、高通量地检测6种鸡免疫抑制性病毒,可用于临床鉴别诊断和分子流行病学调查,具有很高的临床应用价值。更多还原

【Abstract】 The objective of this research was to develop a novel,high-throughput Genome Lab Gene Expression Profiler Analyser-based multiplex PCR method（GeXP-multiplex PCR）for simultaneous detection of 6 immunosuppressive chicken viruses.Using chimeric primers,6 immunosuppressive chicken viruses,including Marek’s disease virus（MDV）,three subgroups of avian leucosis virus（ALV-A/B/J）,reticuloendotheliosis virus（REV）,infectious bursal disease virus（IBDV）,chicken infectious anaemia virus（CIAV）and avian reovirus（ARV）were amplified and identified by their respective amplicon sizes.The concentration of the chimeric primers,Mg²⁺and Taq,and annealing temperature,and time were optimised according to the amplification efficiencyof the GeXP-multiplex PCR assay.The assay specificity for each immunosuppressive viral target was individually and simultaneously tested with a mixture of 8sets of chimeric primers in a multiplex PCR assay.Plasmid DNA and transcribed ssRNA were diluted to a final concentration ranging from 105 copies·20μL^-1 to 1copy·20μL·（-1） and then subjected to the GeXP-multiplex PCR assay with 8sets of chimeric primers,both individually and in pre-mixed solutions.A total of 300 clinic specimens of disease chicken were detected by using GeXP-multiplex PCR.The results were confirmed using independent real-time PCR and sequencing to determine true positives.The specificity and sensitivity of the optimised GeXP-multiplex PCR assay were evaluated,and the data demonstrated that this technique could selectively amplify these 6viruses at a sensitivity of 100copies·20μL^-1 when all 6viruses were present.Of the 300 examined clinical specimens,190 were positive for immunosuppressive viruses according to the GeXP-multiplex PCR assay.The GeXP-multiplex PCR assay is a high-throughput,sensitive and specific method for the detection of6 immunosuppressive viruses that can be used for differential diagnosis and molecular epidemiologic surveys.更多还原