应用多重GeXP-PCR同时检测6种鸡免疫抑制病病毒的研究

doi:10.11843/j.issn.0366-6964.2016.06.015

摘要/Abstract

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100 copies•20 μL^-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。

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 efficiency of the GeXP-multiplex PCR assay.The assay specificity for each immunosuppressive viral target was individually and simultaneously tested with a mixture of 8 sets of chimeric primers in a multiplex PCR assay.Plasmid DNA and transcribed ssRNA were diluted to a final concentration ranging from 105 copies•20 μL^-1 to 1 copy•20 μL^-1 and then subjected to the GeXP-multiplex PCR assay with 8 sets 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 6 viruses at a sensitivity of 100 copies•20 μL^-1 when all 6 viruses 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 of 6 immunosuppressive viruses that can be used for differential diagnosis and molecular epidemiologic surveys.

中图分类号:

S858.315.3

曾婷婷，谢芝勋，谢丽基，邓显文，谢志勤，罗思思，黄莉，黄娇玲. 应用多重GeXP-PCR同时检测6种鸡免疫抑制病病毒的研究[J]. 畜牧兽医学报, 2016, 47(6): 1198-1208.

ZENG Ting-ting，XIE Zhi-xun，XIE Li-ji，DENG Xian-wen，XIE Zhi-qin，LUO Si-si，HUANG Li，HUANG Jiao-ling. Simultaneous Detecting Six Immunosuppressive Chicken Viruses by a GeXP Analyser-based Multiplex PCR Assay[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(6): 1198-1208.

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链接本文: https://www.xmsyxb.com/CN/10.11843/j.issn.0366-6964.2016.06.015

https://www.xmsyxb.com/CN/Y2016/V47/I6/1198

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