| 禽白血病病毒和鸡传染性贫血病毒二重荧光LAMP检测方法的建立及应用 |
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| 引用本文： | 张民秀,谢芝勋,谢志勤,谢丽基,张艳芳,邓显文,曾婷婷,范晴,罗思思,黄娇玲.禽白血病病毒和鸡传染性贫血病毒二重荧光LAMP检测方法的建立及应用[J].南方农业学报,2021,52(7):2007-2014. |
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| 作者姓名： | 张民秀 谢芝勋 谢志勤 谢丽基 张艳芳 邓显文 曾婷婷 范晴 罗思思 黄娇玲 |
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| 作者单位： | 广西兽医研究所/广西兽医生物技术重点实验室, 南宁 530001 |
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| 基金项目： | 广西科技基地和人才专项（桂科AD17195083）；广西科技重大专项（桂科AA17204057）；广西八桂学者岗位专项（2019-79）；国家万人计划科技创新领军人才专项（W02060083） |
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| 摘 要： | 【目的】建立同时鉴别ALV和CIAV的二重荧光环介导等温扩增（LAMP）检测方法，为临床上快速诊断及有效防控ALV和CIAV的单一或混合感染提供技术支持。【方法】参考ALV （A亚群、B亚群、C亚群、D亚群和J亚群）的pol基因和CIAV的VP2基因保守序列，设计2套用于LAMP的特异性引物，并在ALV和CIAV的F1c和B1c覆盖区域分别设计双标记探针ALV-Probe （5'端标记FAM荧光基团，3'端标记BHQ3淬灭基团）和CIAV-Probe （5'端标记CY5荧光基团，3'端标记BHQ3淬灭基团）]。在Loopamp LA-320C实时浊度仪中反应结束后，将反应管置于多色荧光系统内进行观察分析；并通过特异性试验、灵敏度试验及临床样品检测验证二重荧光LAMP检测方法的适用性和可靠性。【结果】优化后的二重荧光LAMP反应体系20.0μL:DNA/cDNA模板2.0μL，2×Reaction Mix 10.0μL，Bst DNA聚合酶0.8μL，内引物ALV-FIP、ALV-BIP、CIAV-FIP和CIAV-BIP （工作浓度40.0μmol/L）各0.8μL，外引物ALV-F3、ALV-B3、CIAV-F3和CIAV-B3（工作浓度5.0μmol/L）各0.4μL，ALV-Probe （工作浓度0.5μmol/L）0.4μL，CIAV-Probe （工作浓度0.5μmol/L）0.8μL，以ddH2O补足至20.0μL。扩增程序:62℃反应60 min，80℃灭活5 min。建立的二重荧光LAMP检测方法能特异性同时检测ALV和CIAV，对其他禽类病原体无特异性扩增；检测CIAV和ALV单一模板的下限均为102拷贝/μL，检测混合模板时ALV的检测下限为102拷贝/μL、CIAV的检测下限为103拷贝/μL。应用建立的二重荧光LAMP检测方法对13份咽喉和泄殖腔棉拭子样品进行检测，其检测结果与常规PCR检测结果的吻合率达100%。【结论】建立的二重荧光LAMP检测方法能实现在同一反应管内鉴别诊断ALV和CIAV，具有特异性好、敏感性高及污染风险小等优点，且检测结果可通过多色荧光系统进行肉眼观察，适用于ALV和CIAV的临床快速筛查。
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| 关 键 词： | 禽白血病病毒(ALV) 鸡传染性贫血病毒(CIAV) 二重荧光LAMP 探针 特异性 敏感性 |
| 收稿时间： | 2020-07-31 |
A duplex fluorescence LAMP assay for identification of avian leukosis virus and chicken infectious anemia virus |
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| ZHANG Min-xiu,XIE Zhi-xun,XIE Zhi-qin,XIE Li-ji,ZHANG Yan-fang,DENG Xian-wen,CENG Ting-ting,FAN Qing,LUO Si-si,HUANG Jiao-ling.A duplex fluorescence LAMP assay for identification of avian leukosis virus and chicken infectious anemia virus[J].Journal of Southern Agriculture,2021,52(7):2007-2014. |
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| Authors: | ZHANG Min-xiu XIE Zhi-xun XIE Zhi-qin XIE Li-ji ZHANG Yan-fang DENG Xian-wen CENG Ting-ting FAN Qing LUO Si-si HUANG Jiao-ling |
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| Affiliation: | Guangxi Veterinary Research Institute/Guangxi Key Laboratory of Veterinary Biotechnology, Nanning 530001, China |
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| Abstract: | 【Objective】 To establish a duplex fluorescence loop-mediated isothermal amplification(LAMP) method for simultaneous identification of avian leukosis virus(ALV) and chicken infectious anemia virus(CIAV), and provide technical support for clinical rapid diagnosis and effective prevention and control of single or mixed infections of ALV and CIAV.【Method】 According to the conserved sequences of the ALV(A, B, C, D and J subgroups) pol gene and the CIAV VP2 gene, two sets of specific primers were designed for LAMP, and a double labeled probe was designed in the areas covered by F1c and B1c of ALV and CIAV sequences, respectivelyALV-probe(5' end was labeled with a FAM fluorescent dye, 3' end was labeled with a BHQ3 quencher dye) and CIAV-probe(5' end was labeled with a CY5 fluorescent dye, 3' end was labeled with BHQ3 quencher dye) ]. After the reaction in the Loopamp LA-320 C turbidimeter, the results could be observed in the polychromatic fluorescence system. The applicability and reliability of the duplex fluorescence LAMP test method were validated by specificity testing, sensitivity testing, and clinical sample testing.【Result】 The duplex fluorescence LAMP was optimized in 20 μL volume containing DNA/cDNA template 2.0 μL, 2×Reaction Mix 10.0μL, Bst DNA polymerase 0.8 μL, internal primers ALV-FIP, ALV-BIP, CIAV-FIP and CIAV-BIP(working concentration 40.0 μmol/L) 0.8 μL each. External primers were ALV-F3, ALV-B3, CIAV-F3 and CIAV-B3(working concentration 5.0μmol/L) 0.4 μL each. ALV-Probe(working concentration 0.5 μmol/L) 0.4 μL, CIAV-Probe(working concentration 0.5) μmol/L) 0.8 μL, making up to 20.0 μL volume with ddH2 O. Amplification procedure: the reaction mixture was incubated at 62 ℃ for 60 min and inactivation at 80 ℃ for 5 min. The results showed that duplex LAMP detection method could simultaneously detect ALV and CIAV, and had no specific amplification for other avian pathogens;the minimum detection limit of single template of CIAV and ALV in sensitivity test were 102 copies/μL. The minimum detection limit of mixed template in sensitivity test was 102 copies/μL for ALV and 103 copies/μL for CIAV;13 throat and cloacal swabs were detected by the duplex fluorescence LAMP. The results were consistent with those of ALV and CIAV by single PCR methods, and the coincidence rate was 100%.【Conclusion】 The duplex fluorescence LAMP method for simultaneous detection of ALV and CIAV in this study has the advantages of good specificity, high sensitivity and low pollution. The detection results can be observed by naked eyes in the polychromatic fluorescence system, which is applicable for rapid clinical screening of ALV and CIAV. |
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