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

Abstract

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.

CLC Number:

S858.315.3

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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References

［1］CALNEK B.Pathogenesis of Marek’s disease virus infection［M］.Marek’s Disease. Berlin：Springer，2001：25-55.
［2］FADLY A M，WITTER R L，LEE L F.Effects of chemically or virus-induced immunodepression on response of chickens to avian leukosis virus［J］.Avian Dis，1985，29(1)：12-25.
［3］ENRIETTO P J，WYKE J A.The pathogenesis of oncogenic avian retroviruses［J］.Adv Cancer Res，1983，39：269-314.
［4］ROESSLER D E.Studies on the pathogenicity and persistence of avian reovirus pathotypes in relation to age resistance and immunosuppression［M］.Delaware：University of Delaware，1987.
［5］SMYTH J A，MOFFETT D A，CONNOR T J，et al.Chicken anaemia virus inoculated by the oral route causes lymphocyte depletion in the thymus in 3-week-old and 6-week-old chickens［J］.Avian Pathol，2006，35(3)：254-259.
［6］SHARMA J M，KIM I J，RAUTENSCHLEIN S，et al.Infectious bursal disease virus of chickens：pathogenesis and immunosuppression［J］.Dev Comp Immunol，2000，24(2-3)：223-235.
［7］佟美姣，高雪丽，李博韬，等.传染性法氏囊病毒对 SPF 雏鸡免疫器官细胞凋亡及 bcl-2 mRNA 表达的影响［J］.畜牧兽医学报，2013，44(4)：617-621.
TONG M J，GAO X L，LI B T，et al.The apoptosis and expression of bcl-2 mRNA in the immune organs of SPF chicks infected with infectious bursal disease virus［J］. Acta Veterinaria et Zootechnica Sinica，2013，44(4)：617-621.(in Chinese)
［8］CUI Z Z，SUN S，WANG J.Reduced serologic response to Newcastle disease virus in broiler chickens exposed to a Chinese field strain of subgroup J avian leukosis virus［J］.Avian Dis，2006，50(2)：191-195.
［9］FARAGHER J T，ALLAN W H，WYETH P J.Immunosuppressive effect of infectious bursal agent on vaccination against Newcastle disease［J］.Vet Rec，1974，95(17)：385-388.
［10］MCCONNELL C D，ADAIR B M，MCNULTY M S.Effects of chicken anemia virus on cell-mediated immune function in chickens exposed to the virus by a natural route［J］.Avian Dis，1993，37(2)：366-374.
［11］张洪海，刘青，邱波，等.地方柴鸡中 J 亚群禽白血病与马立克氏病的混合感染［J］.畜牧兽医学报，2009，40(8)：1215-1221.
ZHANG H H，LIU Q，QIU B，et al.Mixed infection of ALV-J and MDV in a flock of Shandong free range chickens［J］. Acta Veterinaria et Zootechnica Sinica，2009，40(8)：1215-1221.(in Chinese)
［12］秦立廷，高玉龙，潘伟，等.我国部分地区蛋鸡群ALV- J 及与REV、MDV、CAV 混合感染检测［J］.中国预防兽医学报，2010，32(2)：90-93.
QIN L T，GAO Y L，PAN W，et al.Investigation of co-infection of ALV-J with REV，MDV，CAV in layer chicken flocks in some regions of China［J］.Chinese Journal of Preventive Veterinary Medicine，2010，32(2)：90-93.(in Chinese)
［13］DREW J E，MAYER C D，FARQUHARSON A J，et al.Custom design of a GeXP multiplexed assay used to assess expression profiles of inflammatory gene targets in normal colon，polyp，and tumor tissue［J］. J Mol Diagn，2011，13(2)：233-242.
［14］YANG M J，LUO L，NIE K，et al.Genotyping of 11 human papillomaviruses by multiplex PCR with a GeXP analyzer［J］.J Med Virol，2012，84(6)：957-963.
［15］HU X，ZHANG Y，ZHOU X，et al.Simultaneously typing nine serotypes of enteroviruses associated with hand，foot，and mouth disease by a GeXP analyzer-based multiplex reverse transcription-PCR assay［J］.J Clin Microbiol，2012，50(2)：288-293.
［16］RAI A J，KAMATH R M，GERALD W，et al.Analytical validation of the GeXP analyzer and design of a workflow for cancer-biomarker discovery using multiplexed gene-expression profiling［J］.Anal Bioanal Chem，2009，393(5)：1505-1511.
［17］LIU Y，XU Z，ZHANG Q，et al.Simultaneous detection of seven enteric viruses associated with acute gastroenteritis by a multiplexed Luminex-based assay［J］.J Clin Microbiol，2012，50(7)：2384-2389.
［18］QIN M，WANG D Y，HUANG F，et al.Detection of pandemic influenza A H1N1 virus by multiplex reverse transcription-PCR with a GeXP analyzer［J］.J Virol Methods，2010，168(1-2)：255-258.
［19］XIE Z，LUO S，XIE L，et al.Simultaneous typing of nine avian respiratory pathogens using a novel GeXP analyzer-based multiplex PCR assay［J］.J Virol Methods，2014，207：188-195.
［20］TELESNITSKY A，GOFF S P.Reverse transcriptase and the generation of retroviral DNA［M］.Retroviruses.Cold Spring Harbor (NY)：Cold Spring Harbor Laboratory Press，1997.
［21］崔治中，孟姗姗，姜世金，等.我国白羽肉用型鸡群中CAV、REV和REOV感染状况的血清学调查［J］.畜牧兽医学报，2006，37(2)：152-157.
CUI Z Z，MENG S S，JIANG S J，et al.Serological surveys of chicken anemia virus，avian reticuloendotheliosis virus and avian reovirus infections in white meat-type chickens in China［J］.Acta Veterinaria et Zootechnica Sinica，2006，37(2)：152-157.(in Chinese)
［22］罗思思，谢芝勋，谢丽基，等.同时鉴别6种鸡病毒性呼吸道病GeXP检测方法的建立［J］.病毒学报，2013，29(3)：250-257.
LUO S S，XIE Z X，XIE L J，et al.Development of a GeXP assay for simultaneous differentiation of six chicken respiratory viruses［J］.Chinese Journal of Virology，2013，29(3)：250-257.(in Chinese)
［23］ZHANG M X，XIE Z X，XIE L J，et al.Simultaneous detection of eight swine reproductive and respiratory pathogens using a novel GeXP analyser-based multiplex PCR assay［J］.J Virol Methods，2015，224：9-15.
［24］李瑾，毛乃颖，秦萌，等.GeXP多重PCR技术同时检测12种常见呼吸道病毒［J］.病毒学报，2011，27(6)：526-532.
LI J，MAO N Y，QIN M，et al.A GeXP based multiplex RT-PCR assay for simultaneous detection of twelve human respiratory viruses［J］. Chinese Journal of Virology，2011，27(6)：526-532.(in Chinese)
［25］ZHANG Y F，XIE Z X，XIE L J，et al.GeXP analyzer-based multiplex reverse-transcription PCR assay for the simultaneous detection and differentiation of eleven duck viruses［J］.BMC Microbiol，2015，15：247.
［26］XIE Z X，XIE L J，PANG Y S，et al.Development of a real-time multiplex PCR assay for detection of viral pathogens of penaeid shrimp［J］.Arch Virol，2008，153(12)：2245-2251.
［27］何玮玲，张驰，杨静，等.食品中 4 种肉类成分多重 PCR 的快速鉴别方法［J］.中国农业科学，2012，45(9)：1873-1880.
HE W L，ZHANG C，YANG J，et al.A quick multiplex PCR method for the identification of four meat ingredients in food products［J］.Scientia Agricultura Sinica，2012，45(9)：1873-1880.(in Chinese)

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Simultaneous Detecting Six Immunosuppressive Chicken Viruses by a GeXP Analyser-based Multiplex PCR Assay

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