CN113897461B - Alv、rev、ibdv、ciav、arv和ghv的六重pcr检测引物组和试剂盒 - Google Patents
Alv、rev、ibdv、ciav、arv和ghv的六重pcr检测引物组和试剂盒 Download PDFInfo
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Abstract
本发明涉及禽白血病病毒、禽网状内皮增生症病毒、鸡传染性法氏囊病病毒、鸡传染性贫血病毒、禽呼肠孤病毒和鸡马立克氏病病毒的六重PCR检测引物组和试剂盒，属于病毒检测技术领域。本发明所述所述引物组包括检测禽白血病病毒的引物，检测禽网状内皮组织增殖病病毒的引物，检测鸡传染性法氏囊病病毒的引物，检测鸡传染性贫血病毒的引物，检测禽呼肠孤病毒的引物，和检测鸡马立克氏病病毒的引物。本发明的引物组能够通过一次PCR同时检测该六种病原体，能够大大节约检测的时间，对于饲养场实际生产、防治具有重要意义。
Description
技术领域
本发明涉及病毒检测技术领域，具体涉及禽白血病病毒、禽网状内皮增生症病毒、鸡传染性法氏囊病病毒、鸡传染性贫血病毒、禽呼肠孤病毒和鸡马立克氏病病毒的六重PCR检测引物组和试剂盒。
背景技术
禽白血病/肉瘤病毒群病毒(Avian Leukosis Virus，ALV)是由禽C型反录病毒群的病毒引起的禽类多种肿瘤性疾病的统称，主要是淋巴细胞性白血病，其次是成红细胞性白血病、成髓细胞性白血病。此外还可引起骨髓细胞瘤、结缔组织瘤、上皮肿瘤、内皮肿瘤等。大多数肿瘤侵害造血系统，少数侵害其他组织。禽白血病病毒的多数毒株能在11～12日龄鸡胚中良好生长，可在绒毛尿囊膜产生增生性痘斑。腹腔或其他途径接种1～14日龄易感雏鸡，可引起鸡发病。禽白血病/肉瘤病毒对脂溶剂和去污剂敏感，对热的抵抗力弱。本病在自然情况下只有鸡能感染。不同品种或品系的鸡对病毒感染和肿瘤发生的抵抗力差异很大。母鸡的易感性比公鸡高，多发生在18周龄以上的鸡。传染源是病鸡和带毒鸡。先天性感染的雏鸡常有免疫耐受现象，它不产生抗肿瘤病毒抗体，长期带毒排毒，成为重要传染源。自然条件下，本病主要以垂直传播方式进行传播，也可水平传播，但比较缓慢。饲料中维生素缺乏、内分泌失调等因素可促进本病的发生。
禽网状内皮组织增殖病病毒(Reticuloendotheliosis virus，REV)是禽类的一种肿瘤性传染病。病鸡表现为贫血、消瘦，生长缓慢等。病理解剖特点是肝脏、肠道、心脏和其他器管有淋巴瘤，胸腺和法氏囊萎缩，腺胃炎。本病能侵害机体的免疫系统，可导致机体免疫机能下降继发其它疾病。该病为免疫抑制性疾病，发病日龄多在80日龄左右，本病毒是低温病毒，高温季节不易发病，鸡群中的发病率和死亡率不高，呈慢性死亡，死亡周期约为10周。患病家禽是本病的主要传染源，可从口、眼分泌物及粪便中排出病毒，通过水平传播使易患鸡感染。本病亦可通过种蛋垂直传播，但传播能力较弱。本病能侵害机体的免疫系统，可导致机体免疫机能下降继发其它疾病。
鸡传染性法氏囊病病毒(Infectious bursal disease virus，IBDV)引起的鸡传染性法氏囊病(IBD)又称甘波罗病，是一种急性、高度传染性疾病。传染性法氏囊病毒属于双股RNA病毒科禽双股RNA病毒属，其对现化因素抵抗力较强。病毒在自然界存活时间较长，在病鸡舍中的病毒可存活122天。本病的危害主要是病毒侵害鸡的体游人免疫中枢——法氏囊，使鸡的法氏囊的淋巴细胞产生受到破坏，降低或不能产生免疫球蛋白，导致免疫机能障碍。以法氏囊肿胀、出血、坏死，胸肌、腿肌出血，腺胃、肌胃交界处条状出血为特征。主要感染2～16周龄鸡，3～6周龄时最易感。本病一年四季都能发生，但以5～7月份发病较多。蛋雏鸡比肉仔鸡病情严重，死亡率较高，若无继发感染，死亡率一般不超过5％。传染性法氏囊炎病毒的自然宿主主要限于鸡和火鸡。
鸡传染性贫血病(Chicken Infectious Anemia，CIA)是由鸡传染性贫血病毒(Chicken Infectious Anemia Virus，CIAV)引起雏鸡的以再生障碍性贫血和全身性淋巴组织萎缩为特征的一种免疫抑制性疾病，经常合并、继发和加重病毒、细菌和真菌性感染，危害很大。鸡是传染性贫血病毒的唯一宿主。各种年龄的鸡均可感染，自然感染常见于2～4周龄的雏鸡，不同品种的雏鸡都可感染发病。随着日龄的增加，鸡对该病的易感性迅速下降，肉鸡比蛋鸡易感，公鸡比母鸡易感。鸡传染性贫血病毒可通过垂直传播和水平传播。本病的唯一特征性症状是贫血。一般在感染后10天发病，14～16天达到高峰。病鸡表现为精神沉郁，虚弱，行动迟缓，羽毛松乱，喙、肉髯、面部皮肤和可视粘膜苍白，生长不良，体重下降；临死前还可见到拉稀。
马立克氏病病毒(Marek’s Disease Virus，MDV)属于疱疹病毒科、α-疱疹病毒(Gallid alphaherpesvirus，GHV)亚科。该病原引起的马立克氏病(Marek’s Disease，MD)又名神经淋巴瘤病(neurolymphomatosis)，是一种鸡的淋巴组织增生性疾病，由细胞结合性疱疹病毒引起外周神经、性腺、虹膜、各种内脏器官、肌肉和皮肤的单个或多个组织器官发生单核细胞浸润，导致器官和组织形成传染性肿瘤病。病鸡常见消瘦、肢体麻痹，并常有急性死亡。在病原学上可以与鸡的其他淋巴样肿瘤病相区别。鸡是主要的MD自然宿主。鹌鹑、火鸡和山鸡可发生自然感染MD，但不出现疾病。乌鸡(竹丝鸡)也可自然感染，而且易感性强，死亡率很高。大多数鸡群开始暴发本病是从8～9周龄开始，12～20周龄是高峰期。但也有3～4周龄的幼鸡群和60周龄的鸡群暴发本病的事例。感染MD的病鸡，大部分为终生带毒，病毒不断从脱落的羽毛囊皮屑中排出有传染性的MDV，这就是MD的传播难于控制的带有根本性的原因。MD的症状被分为三个型：神经型(古典型)、内脏型(急性型)和眼型。各型混合发生也时有出现。
禽呼肠孤病毒(Avian reovirus，ARV)是鸡病毒性关节炎和腱鞘炎的主要病原之一，属于呼肠孤病毒科、正呼肠孤病毒属。禽呼肠孤病毒流行于鸡、火鸡、鸭、鹦鹉和其它禽类，可水平传递亦可经卵垂直传递。已由多种疾病分离出该类病毒，包括病毒性关节炎、腱鞘炎、矮小综合征、呼吸道病、肠病、吸收障碍综合征和骨质疏松征等。患禽生长受阻，发生心包炎、心肌炎、心包积水、肠炎、肝炎，腔上囊和胸腺萎缩，骨短粗或出现急慢性呼吸道病。病毒性关节炎、腱鞘炎：发生于4～7周龄肉鸡，但也可能发生于14～16周龄。主要症状为跗关节上方胫骨和腱束双侧肿大，腱移动受限，表现不同程度的跛行。继而出现腓肠肌腱破裂。1～7日龄雏鸡可能见到肝炎、心肌炎。发病率可达100％，但死亡率低于2％。病鸡可能在1～3周内由急性期恢复，但也可能变为慢性。
禽白血病病毒、禽呼长孤病毒、鸡传染性贫血病毒、鸡马立克病毒、鸡传染性法氏囊病毒和网状内皮组织增殖病毒感染均为鸡免疫抑制病，除各自特征性病征外，均可破坏免疫系统、造成鸡免疫力低下，加重、并发或继发其他病毒、细菌和真菌性感染，对养殖业造成巨大经济损失。以上六种病毒同时感染或交叉感染，因有相同病征，对于临床诊断具有极大的困难，目前对这六种病原的单重、双重或四重PCR诊断方法均已有报道，但同时检测这六种病原的诊断方法还未见报道。
发明内容
本发明的目的在于提供禽白血病病毒、禽网状内皮增生症病毒、鸡传染性法氏囊病病毒、鸡传染性贫血病毒、禽呼肠孤病毒和鸡马立克氏病病毒。本发明所述引物组通过一次PCR能够诊断六种疾病，可以大大减少临床诊断的时间，对于饲养场实际生产、防治具有重要意义。
本发明提供了禽白血病病毒、禽网状内皮增生症病毒、鸡传染性法氏囊病病毒、鸡传染性贫血病毒、禽呼肠孤病毒和鸡马立克氏病病毒的六重PCR检测用引物组，所述引物组包括检测禽白血病病毒的引物ALV-F和ALV-R，检测禽网状内皮组织增殖病病毒的引物REV-F和REV-R，检测鸡传染性法氏囊病病毒的引物IBDV-F和IBDV-R，检测鸡传染性贫血病毒的引物CIAV-F和CIAV-R，检测禽呼肠孤病毒的引物ARV-F和ARV-R，和检测鸡马立克氏病病毒的引物GHV-F和GHV-R；
所述ALV-F的核苷酸序列如SEQ ID NO.1所示，所述ALV-R的核苷酸序列如SEQ IDNO.2所示；
所述REV-F的核苷酸序列如SEQ ID NO.3所示，所述REV-R的核苷酸序列如SEQ IDNO.4所示；
所述IBDV-F的核苷酸序列如SEQ ID NO.5所示，所述IBDV-R的核苷酸序列如SEQID NO.6所示；
所述CIAV-F的核苷酸序列如SEQ ID NO.7所示，所述CIAV-R的核苷酸序列如SEQID NO.8所示；
所述ARV-F的核苷酸序列如SEQ ID NO.9所示，所述ARV-R的核苷酸序列如SEQ IDNO.10所示；
所述GHV-F的核苷酸序列如SEQ ID NO.11所示，所述GHV-R的核苷酸序列如SEQ IDNO.12所示。
本发明还提供了一种禽白血病病毒、禽网状内皮增生症病毒、鸡传染性法氏囊病病毒、鸡传染性贫血病毒、禽呼肠孤病毒和鸡马立克氏病病毒的六重PCR诊断试剂盒，所述试剂盒包括上述技术方案所述的引物组和反应混合液。
优选的是，所述试剂盒每25μL的反应体系包括：ARV-F和ARV-R各0.25μL，CIAV-F和CIAV-R各0.25μL，GHV-F各0.25μL，IBDV-F和IBDV-R各0.3μL，REV-F和REV-R各0.3μL，ALV-F和ALV-R各0.3μL，反应混合液12.625μL，水6.875μL，ARV待测样品、CIAV待测样品、GHV待测样品和IBDV待测样品各0.3μL，REV待测样品和ALV待测样品各0.5μL。
在本发明中，所述引物组中各引物的浓度优选均为10μM。
优选的是，所述试剂盒的反应程序包括：95℃5min；95℃1min，55℃1min，72℃1min，30个循环；72℃10min。
本发明提供了ALV、REV、IBDV、CIAV、ARV和GHV的六重PCR检测引物组和试剂盒。目前尚无能够同时检测禽白血病病毒，禽网状内皮增生症病毒、鸡传染性法氏囊病病毒、鸡传染性贫血病毒、禽呼肠孤病毒和鸡马立克氏病病毒六重PCR诊断方法，本发明的引物组能够通过一次PCR同时检测该六种病原体，能够大大节约检测的时间，对于饲养场实际生产、防治具有重要意义。
附图说明
图1为本发明提供的六重PCR的特异性验证结果图；
图2为本发明提供的六重PCR的敏感性验证结果图。
具体实施方式
本发明提供了禽白血病病毒、禽网状内皮增生症病毒、鸡传染性法氏囊病病毒、鸡传染性贫血病毒、禽呼肠孤病毒和鸡马立克氏病病毒的六重PCR检测用引物组，所述引物组包括检测禽白血病病毒的引物ALV-F和ALV-R，检测禽网状内皮组织增殖病病毒的引物REV-F和REV-R，检测鸡传染性法氏囊病病毒的引物IBDV-F和IBDV-R，检测鸡传染性贫血病毒的引物CIAV-F和CIAV-R，检测禽呼肠孤病毒的引物ARV-F和ARV-R，和检测鸡马立克氏病病毒的引物GHV-F和GHV-R；
所述ALV-F的核苷酸序列如SEQ ID NO.1所示，所述ALV-R的核苷酸序列如SEQ IDNO.2所示；
所述REV-F的核苷酸序列如SEQ ID NO.3所示，所述REV-R的核苷酸序列如SEQ IDNO.4所示；
所述IBDV-F的核苷酸序列如SEQ ID NO.5所示，所述IBDV-R的核苷酸序列如SEQID NO.6所示；
所述CIAV-F的核苷酸序列如SEQ ID NO.7所示，所述CIAV-R的核苷酸序列如SEQID NO.8所示；
所述ARV-F的核苷酸序列如SEQ ID NO.9所示，所述ARV-R的核苷酸序列如SEQ IDNO.10所示；
所述GHV-F的核苷酸序列如SEQ ID NO.11所示，所述GHV-R的核苷酸序列如SEQ IDNO.12所示。本申请所述引物组的具体核苷酸序列见表1。本发明所述引物组能够通过一次PCR同时且高效地检测该六种病原体，能够大大节约检测的时间。
表1六重PCR引物序列
本发明还提供了一种禽白血病病毒、禽网状内皮增生症病毒、鸡传染性法氏囊病病毒、鸡传染性贫血病毒、禽呼肠孤病毒和鸡马立克氏病病毒的六重PCR诊断试剂盒，所述试剂盒包括上述技术方案所述的引物组和反应混合液。在本发明中，所述反应混合液优选包括宝日医生物技术(北京)有限公司Multiplex PCR Assay Kit Ver.2。
在本发明中，所述试剂盒每25μL的反应体系优选包括：ARV-F和ARV-R各0.25μL，CIAV-F和CIAV-R各0.25μL，GHV-F各0.25μL，IBDV-F和IBDV-R各0.3μL，REV-F和REV-R各0.3μL，ALV-F和ALV-R各0.3μL，反应混合液12.625μL，水6.875μL，ARV待测样品、CIAV待测样品、GHV待测样品和IBDV待测样品各0.3μL，REV待测样品和ALV待测样品各0.5μL。实施例结果表明，其他相同条件下，本发明通过4组不同引物比例的实验进行比较，a、b和c组引物扩增出的六重条带亮度不均一、不稳定，甚至不能完全扩增出6个条带；而d组(本发明)比例的引物，能够稳定的扩增出六条亮度相近的条带。
在本发明中，所述试剂盒的反应程序优选包括：95℃5min；95℃1min，55℃1min，72℃1min，30个循环；72℃10min。本发明的反应程序能够保证六种病毒的cDNA均可特异性较好的扩增出条带。
下面结合具体实施例对本发明所述的禽白血病病毒、禽网状内皮增生症病毒、鸡传染性法氏囊病病毒、鸡传染性贫血病毒、禽呼肠孤病毒和鸡马立克氏病病毒做进一步详细的介绍，本发明的技术方案包括但不限于以下实施例。
实施例1
1.主要试剂
Premix TaqTM(TaKaRa TaqTM Version 2.0plus dye)、Multiplex PCR Assay KitVer.2、MiniBEST Viral RNA/DNA Extraction Kit Ver.4.0和PrimeScriptTM RT reagentKit with gDNA EraARVr(Perfect Real Time)购自宝日医生物技术(北京)有限公司，D2000 DNA Marker购自天根生化科技(北京)有限公司。
2.引物设计与合成
根据Genbank公布的ARV(s113)s1 mrna基因序列、ALV isolate js16jh10(env)基因序列、CIAV capsid protein(vp1)基因序列、IBDV vp2基因序列、REV strain r-cs1701(env)基因序列和GHV isolate lyc(hlj06i)(meq)基因序列，利用PrimerPlex 2多重PCR引物设计软件设计6对引物。引物由生工生物工程(上海)股份有限公司合成。
现设计多重PCR引物的方法多采用单重PCR引物设计软件设计或者文献公开的特异性引物进行逐一验证。多重试验中，扩增时将多对引物一起加入体系，需要通过长时间的筛选和摸索，才有可能筛选出合适的引物，单重引物设计软件仅能够就该对一对引物的Tm值、GC卡数量，自身二聚体、发卡结构等引物设计关键因素进行评估，多对引物之间的评估常常要靠实验人员的经验、手动计算或大量实验进行效果进行评估，效率低下且结果并不理想。而公开的特异性序列需要大量实验进行验证及组合筛选，效率低下。本发明中的六对引物能够确保特定和有效的扩增，经大量实验筛选，为最优引物组合，可以同时、高效的对目的片段进行扩增。
ARV最佳退火温度的确定：
方法：
Mix 12.5μL，ARV-F 0.5μL，ARV-R 0.5μL，H2O 10.5μL，DNA1μL，总体积25μL。
扩增条件：95℃5min；95℃1min，(退火温度分别是：49℃、51℃、53℃、55℃、57℃、59℃)1min，72℃1min，30个循环；72℃10min；产物经2％琼脂糖凝胶电泳检测。
CIAV最佳退火温度的确定：
方法：
Mix 12.5μL，CIAV-F 0.5μL，CIAV-R 0.5μL，H2O10.5μL，DNA 1μL，总体积25μL。
扩增条件：95℃5min；95℃1min，(退火温度分别是：49℃、51℃、53℃、55℃、57℃、59℃)1min，72℃1min，30个循环；72℃10min；产物经2％琼脂糖凝胶电泳检测。
GHV最佳退火温度的确定：
Mix 12.5μL，GHV-F 0.5μL，GHV-R 0.5μL，H2O 10.5μL，DNA 1μL，总体积25μL。
扩增条件：95℃5min；95℃1min，(退火温度分别是：49℃、51℃、53℃、55℃、57℃、59℃)1min，72℃1min，30个循环；72℃10min；产物经2％琼脂糖凝胶电泳检测。
IBDV最佳退火温度的确定：
Mix 12.5μL，IBDV-F 0.5μL，IBDV-R 0.5μL，H2O 10.5μL，DNA 1μL，总体积25μL。
扩增条件：95℃5min；95℃1min，(退火温度分别是：49℃、51℃、53℃、55℃、57℃、59℃)1min，72℃1min，30个循环；72℃10min；产物经2％琼脂糖凝胶电泳检测。
REV最佳退火温度的确定：
Mix 12.5μL，REV-F 0.5μL，REV-R 0.5μL，H2O 10.5μL，DNA 1μL，总体积25μL。
扩增条件：95℃5min；95℃1min，(退火温度分别是：49℃、51℃、53℃、55℃、57℃、59℃)1min，72℃1min，30个循环；72℃10min；产物经2％琼脂糖凝胶电泳检测。
ALV最佳退火温度的确定：
方法：
Mix 12.5μL，ALV-F 0.5μL，ALV-R 0.5μL，H2O 10.5μL，DNA1μL，总体积25μL。
扩增条件：95℃5min；95℃1min，(退火温度分别是：49℃、51℃、53℃、55℃、57℃、59℃)1min，72℃1min，30个循环；72℃10min；产物经2％琼脂糖凝胶电泳检测。
综上，通过不同退火温度实验检测，最终确定ARV、CIAV、GHV、IBDV、REV和ALV六重PCR的最佳退火温度为55℃。
不同引物比例
方法：ARV-F和ARV-R的浓度独立为10μM，CIAV-F和CIAV-R的浓度独立为10μM，GHV-F和CIAV-R的浓度独立为10μM，IBDV-F和IBDV-R的浓度独立为10μM，REV-F和REV-R的浓度独立为10μM，ALV-F和ALV-R的浓度独立为10μM。
a：引物ARV-F/R各0.5μL，CIAV-F/R各0.5μL，GHV-F/R各0.5μL，IBDV-F/R各0.5μL，REV-F/R各0.5μL，ALV-F/R各0.5μL；
b：引物ARV-F/R各0.3μL，CIAV-F/R各0.3μL，GHV-F/R各0.3μL，IBDV-F/R各0.3μL，REV-F/R各0.3μL，ALV-F/R各0.3μL；
c：引物ARV-F/R各0.25μL，CIAV-F/R各0.25μL，GHV-F/R各0.25μL，IBDV-F/R各0.25μL，REV-F/R各0.25μL，ALV-F/R各0.25μL；
d：引物ARV-F/R各0.25μL，CIAV-F/R各0.25μL，GHV-F/R各0.25μL，IBDV-F/R各0.3μL，REV-F/R各0.3μL；ALV-F/R各0.3μL.
经过实验验证，确定六重PCR的最佳反应条件为：引物ARV-F/R各0.25μL，CIAV-F/R各0.25μL，GHV-F/R各0.25μL，IBDV-F/R各0.3μL，REV-F/R各0.3μL，ALV-F/R各0.3μL，Mix12.625μL，H206.875μL，DNA ARV、CIAV、GHV、IBDV各0.3μL，REV、ALV0.5μL，25μL体系。其他相同条件下，本发明通过4组不同引物比例的实验进行比较，a、b和c组引物扩增出的六重条带亮度不均一、不稳定，甚至不能完全扩增出6个条带。而d组比例的引物，能够稳定的扩增出六条亮度相近的条带。
95℃5min，95℃1min，55℃1min，72℃1min，72℃10min；30个循环。
补充说明：
(1)Premix TaqTM(TaKaRaTaqTM Version 2.0plus dye)、Multiplex PCR AssayKit Ver.2购自宝日医生物技术(北京)有限公司；退火温度筛选时使用的Mix是PremixTaqTM(TaKaRaTaqTM Version 2.0plus dye)，不同浓度引物筛选、特异性、敏感性实验使用的Mix都使用Multiplex PCR Assay Kit Ver.2。
(2)病毒总DNA/RNA提取方法按照TaKaRa MiniBEST Viral RNA/DNA ExtractionKit Ver.4.0试剂盒说明书进行，RNA反转录cDNA方法按照PrimeScriptTM RT reagent Kitwith gDNA EraARVr(Perfect Real Time)试剂盒说明书进行，购自宝日医生物技术(北京)有限公司；ARV、CIAV、GHV、IBDV、REV、ALV病毒来源均为哈尔滨兽医研究所惠赠；
(3)PCR扩增采用的仪器设备的品牌：Applied Biosystems型号：Veriti 96-WellThermal Cycler PCR仪。
六重PCR的特异性：
分别以双蒸水、ARV+CIAV+GHV+IBDV+REV+ALV混合DNA(或cDNA)，鸡毒支原体DNA、副鸡禽杆菌DNA，沙门氏菌DNA、传染性喉气管炎病毒DNA、新城疫cDNA为模板，利用已优化的六重PCR扩增条件进行PCR扩增。
A：引物ARV-F/R各0.25μL，CIAV-F/R各0.25μL，GHV-F/R各0.25μL，IBDV-F/R各0.3μL，REV-F/R各0.3μL，ALV-F/R各0.3μL，Mix12.625μL，H209.075μL；
B：引物ARV-F/R各0.25μL，CIAV-F/R各0.25μL，GHV-F/R各0.25μL，IBDV-F/R各0.3μL，REV-F/R各0.3μL，ALV-F/R各0.3μL，Mix12.625μL，H206.875μL，模板ARV、CIAV、GHV、IBDV各0.3μL，REV、ALV0.5μL；
C：引物ARV-F/R各0.25μL，CIAV-F/R各0.25μL，GHV-F/R各0.25μL，IBDV-F/R各0.3μL，REV-F/R各0.3μL，ALV-F/R各0.3μL，Mix12.625μL，H208.575μL，cDNAARV 1μL
D：引物ARV-F/R各0.25μL，CIAV-F/R各0.25μL，GHV-F/R各0.25μL，IBDV-F/R各0.3μL，REV-F/R各0.3μL，ALV-F/R各0.3μL，Mix12.625μL，H208.575μL，DNACIAV 1μL
E：引物ARV-F/R各0.25μL，CIAV-F/R各0.25μL，GHV-F/R各0.25μL，IBDV-F/R各0.3μL，REV-F/R各0.3μL，ALV-F/R各0.3μL，Mix12.625μL，H208.575μL，DNAGHV 1μL
F：引物ARV-F/R各0.25μL，CIAV-F/R各0.25μL，GHV-F/R各0.25μL，IBDV-F/R各0.3μL，REV-F/R各0.3μL，ALV-F/R各0.3μL，Mix12.625μL，H208.575μL，cDNAIBDV 1μL
G：引物ARV-F/R各0.25μL，CIAV-F/R各0.25μL，GHV-F/R各0.25μL，IBDV-F/R各0.3μL，REV-F/R各0.3μL，ALV-F/R各0.3μL，Mix12.625μL，H208.575μL，cDNAREV 1μL
H：引物ARV-F/R各0.25μL，CIAV-F/R各0.25μL，GHV-F/R各0.25μL，IBDV-F/R各0.3μL，REV-F/R各0.3μL，ALV-F/R各0.3μL，Mix12.625μL，H208.575μL，cDNAALV 1μL
I：引物ARV-F/R各0.25μL，CIAV-F/R各0.25μL，GHV-F/R各0.25μL，IBDV-F/R各0.3μL，REV-F/R各0.3μL，ALV-F/R各0.3μL，Mix12.625μL，H208.075μL，DNA鸡毒支原体1μL
J：引物ARV-F/R各0.25μL，CIAV-F/R各0.25μL，GHV-F/R各0.25μL，IBDV-F/R各0.3μL，REV-F/R各0.3μL，ALV-F/R各0.3μL，Mix12.625μL，H208.075μL，DNA副鸡禽杆菌1μL
K：引物ARV-F/R各0.25μL，CIAV-F/R各0.25μL，GHV-F/R各0.25μL，IBDV-F/R各0.3μL，REV-F/R各0.3μL，ALV-F/R各0.3μL，Mix12.625μL，H208.075μL，DNA沙门氏菌1μL
L：引物ARV-F/R各0.25μL，CIAV-F/R各0.25μL，GHV-F/R各0.25μL，IBDV-F/R各0.3μL，REV-F/R各0.3μL，ALV-F/R各0.3μL，Mix12.625μL，H208.075μL，cDNA新城疫病毒1μL
M：引物ARV-F/R各0.25μL，CIAV-F/R各0.25μL，GHV-F/R各0.25μL，IBDV-F/R各0.3μL，REV-F/R各0.3μL，ALV-F/R各0.3μL，Mix12.625μL，H208.075μL，DNA传染性喉气管炎病毒1μL
扩增条件：95℃5min，95℃1min，55℃1min，72℃1min，72℃10min；30个循环
产物经2％琼脂糖凝胶电泳检测，结果如图1所示。图1为六重PCR的特异性验证结果图，其中，M：DL2000 DNA Marker，1：体系A(阴性对照)2：体系B(ARV+CIAV+GHV+IBDV+REV+ALV)，3：体系C(IBDV)，4：体系D(ALV)，5：体系E(CIAV)，6：体系F(REV)，7：体系G(ARV)，8：体系H(GHV)，9：体系I(鸡毒支原体)，10：体系J(副鸡禽杆菌)，11：体系K(沙门氏菌)，12：体系L(新城疫病毒)，13：体系M(传染性喉气管炎)。
结果显示，PCR能够同时扩增出约1182bp、976bp、751bp、579bp、418bp、217bp六个特异性片段，鸡毒支原体、副鸡禽杆菌，沙门氏菌、传染性喉气管炎病毒、新城疫病毒和阴性对照均未扩增出目的片段，表明本实验建立的四六重PCR方法具有良好的特异性。
其它条件不变，将引物替换为通过查找文献找到的ARV、CIAV、GHV、IBDV、REV和ALV的引物序列(详见表2)，文献所述引物序列特异性不如本发明引物。
表2文献ARV、CIAV、GHV、IBDV、REV和ALV的引物序列
六重PCR的敏感性：
分别调整ARV，CIAV，GHV，IBDV，REV，ALV的DNA、cDNA初始浓度为5×101ng/μL，分别取ARV、CIAV、GHV、IBDV各60μL，REV、ALV各50μL模板混合后逐级进行10倍梯度稀释(10-1～10-7)，后每个稀释度取2.2μL混合物作为模板，按照已优化的六重PCR条件进行扩增，计算该六重PCR方法对ARV，CIAV，GHV，IBDV，REV，ALV的最低检出量，评价其敏感性。结果如图2，图2为六重PCR的敏感性验证结果图，其中，M：DL2000 Marker，1：阴性对照、2：5×100ng/μL、3：5×10-1ng/μL、4：5×10-2ng/μL、5：5×10-3、6：5×10-4、7：5×10-5ng/μL、8：5×10-6ng/μL、9：5×10-7ng/μL。
由图2可知，随着浓度的逐渐减少，扩增产物的亮度逐渐降低，泳道8中已无扩增条带，说明IBDV、ALV、CIAV、REV、ARV和GHV的最低检出量均为5×10-3ng/μL，敏感性良好。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 内蒙古自治区农牧业科学院
内蒙古农业大学
乌兰察布市农林科学研究所
乌兰察布市动物疫病预防控制中心
<120> ALV、REV、IBDV、CIAV、ARV和GHV的六重PCR检测引物组和试剂盒
<160> 24
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
tcagcgacct caccattcc 19
<210> 2
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atgccaagcg ttcaatctcc 20
<210> 3
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
caacccacct ggtcaataga tg 22
<210> 4
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
gagtgcgaga catattcctc ct 22
<210> 5
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
gagccttctg atgccaacaa c 21
<210> 6
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
atgccaagac ggtccctct 19
<210> 7
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ccgcttccaa ggagtcatct 20
<210> 8
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
accgagtgct gtgagtgtt 19
<210> 9
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
cggcggtctt gtcttatagt tc 22
<210> 10
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
attggagatg gcagtggagt t 21
<210> 11
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
cctgttaccg agccgtgta 19
<210> 12
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
accagaccgt agactgagta tc 22
<210> 13
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
cgagagtggc tcgcgagatg g 21
<210> 14
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
actacatttc cccctcccta t 21
<210> 15
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
atggcgggtc tcaatccatc 20
<210> 16
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
ttaggtgtcg atgccggtac 20
<210> 17
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
gcaggggcaa gtaatttcaa 20
<210> 18
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
gccacacagc gatagagtga 20
<210> 19
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
ctgacggcct atctgaggag 20
<210> 20
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
ggaaaccacc agaccgtaga c 21
<210> 21
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
ggtcggagac ttcaacctac 20
<210> 22
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
tgagaattgg taatcatcgg 20
<210> 23
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
gaggaaatca tcaggcacag c 21
<210> 24
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
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ccgtgtaggc catgttgttc c 21
Claims (2)
1.一种禽白血病病毒、禽网状内皮增生症病毒、鸡传染性法氏囊病病毒、鸡传染性贫血病毒、禽呼肠孤病毒和鸡马立克氏病病毒的六重PCR诊断试剂盒，其特征在于，所述试剂盒包括禽白血病病毒、禽网状内皮增生症病毒、鸡传染性法氏囊病病毒、鸡传染性贫血病毒、禽呼肠孤病毒和鸡马立克氏病病毒的六重PCR检测用引物组和反应混合液；
所述引物组包括检测禽白血病病毒的引物ALV-F和ALV-R，检测禽网状内皮组织增殖病病毒的引物REV-F和REV-R，检测鸡传染性法氏囊病病毒的引物IBDV-F和IBDV-R，检测鸡传染性贫血病毒的引物CIAV-F和CIAV-R，检测禽呼肠孤病毒的引物ARV-F和ARV-R，和检测鸡马立克氏病病毒的引物GHV-F和GHV-R；
所述ALV-F的核苷酸序列如SEQ ID NO.1所示，所述ALV-R的核苷酸序列如SEQ ID NO.2所示；
所述REV-F的核苷酸序列如SEQ ID NO.3所示，所述REV-R的核苷酸序列如SEQ ID NO.4所示；
所述IBDV-F的核苷酸序列如SEQ ID NO.5所示，所述IBDV-R的核苷酸序列如SEQ IDNO.6所示；
所述CIAV-F的核苷酸序列如SEQ ID NO.7所示，所述CIAV-R的核苷酸序列如SEQ IDNO.8所示；
所述ARV-F的核苷酸序列如SEQ ID NO.9所示，所述ARV-R的核苷酸序列如SEQ IDNO.10所示；
所述GHV-F的核苷酸序列如SEQ ID NO.11所示，所述GHV-R的核苷酸序列如SEQ IDNO.12所示；
所述试剂盒每25μL的反应体系包括：ARV-F和ARV-R各0.25μL，CIAV-F和CIAV-R各0.25μL，GHV-F各0.25μL，IBDV-F和IBDV-R各0.3μL，REV-F和REV-R各0.3μL，ALV-F和ALV-R各0.3μL，反应混合液12.625μL，水6.875μL，ARV待测样品、CIAV待测样品、GHV待测样品和IBDV待测样品各0.3μL，REV待测样品和ALV待测样品各0.5μL。
2.根据权利要求1所述的试剂盒，其特征在于，所述试剂盒的反应程序包括：95℃5min；95℃1min，55℃1min，72℃1min，30个循环；72℃10min。
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| 禽呼肠孤病毒与禽白血病病毒双重RT-PCR检测方法的建立及应用;马超英;;中国畜牧兽医(02);第59-62页 * |
| 禽四种病毒多重ＰＣＲ诊断技术的建立和应用;张琳等;家畜生态学报;第32卷(第4期);第1-4页 * |
| 鸭瘟诊断方法研究进展;肖瑞琪;动物医学进展;第40卷(第9期);第84-88页 * |
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