CN113416796A - 一种家禽白血病病毒检测方法 - Google Patents
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Abstract
本发明属于分子生物学技术领域,公开了一种家禽白血病病毒检测方法,包括检测家禽白血病病毒A亚群、B亚群和J亚群的检测引物,同时,公开了多重PCR的反应体系和反应条件;应用包括以下步骤:(1)提取家禽全基因组DNA,作为多重PCR的模板;(2)以家禽白血病病毒亚群A检测引物、家禽白血病病毒亚群B检测引物和家禽白血病病毒亚群J检测引物为3对检测引物,进行多重PCR;(3)取步骤(2)中反应液进行琼脂糖凝胶电泳检测。本发明的成本相比之下非常低廉,效率也相当高;在灵敏度方面,本方法灵敏度和特异性都非常高,能够检出的模板最小拷贝数是1×103拷贝/μL。本发明所提出的检测引物具有快速、准确、廉价的优点。
Description
技术领域
本发明属于分子生物学技术领域,尤其涉及一种家禽白血病病毒检测方法。
背景技术
目前白血病病毒(Avain leukosis virus,以下简称ALV)是以引起家禽各种造血细胞肿瘤性增生为特征的一类反转录病毒群,能感染鸡的可分为A、B、C、D、E和J等6个亚型,其中A、B、C、D和J亚型属于外源性ALV,临床上常见A和J亚型,B、C、D亚型相对少见,E亚型属于内源性ALV,本身不具有致病特征。近几年,外源性ALV尤其是ALV-J已在我国的蛋鸡和地方品种鸡中广泛传播,造成了巨大的经济损失。并且宿主范围的扩大、ALV本身的变异以及与其他病毒的混合感染等都给禽白血病的防制提出了新的挑战。由于目前没有商业化疫苗可用,只能从源头上阻断病毒的传播,因此,必须加强外源性ALV检测方法的研究,及时检测并淘汰感染或者疑似感染鸡,才能有效控制外源性ALV。
自1908年首次报道并分离到ALV以来,禽白血病已分布于世界各地,我国也有该病的流行和发生。近年来,湖北、山东等地鸡白血病疑似病例频发,重庆、广西等省份也有相关报道,鸡群ALV的感染率高达60%,病死率高达50%以上。我国每年需要对进口的约120万套种鸡中0.5%-1%个体进行病原学抽样检测,保证进口种鸡的洁净度;并对全国10-15个市场占有量最大的自繁自养种鸡场的核心群开展彻底的净化程序(每个鸡场每年需病原学检测1-1.5万只鸡,每代鸡2-3次),至少持续4-5年;同时,特别需要对农业部国家地方鸡种基因库禽白血病的净化,每个品种核心群至少500只,该基因库现保存有31个我国重要的纯地方品种鸡,承担国家的保种任务。因此在实际生产实践中迫切需要一种快速、准确、廉价的检测方法。
通过上述分析,现有技术存在的问题及缺陷为:
传统的各种诊断方法都存在各自的不足之处,如病毒的分离鉴定法很难确保鸡体内不存在潜伏感染的病毒,更不能排除鸡发生再感染的可能;琼脂双向扩散试验法对大型集约化祖代鸡场来讲,要实现全群净化,实际操作非常困难;荧光抗体实验需要荧光显微镜,所以有一定的局限性;现有的PCR方法成本较高、操作较复杂耗时、对人员素质要求较高,这些都限制其在生产中的实际应用等。
发明内容
针对现有技术存在的问题,本发明提供了一种家禽白血病病毒检测方法。
本发明是这样实现的,一种家禽白血病病毒检测方法,包括针对家禽白血病病毒A、B、J亚群的3对特异性寡核苷酸引物序列:
家禽白血病病毒亚群A检测引物:
上游引物A3:5'-GGTTGGTCTAGACAGGA-3'
下游引物A4:5'-CATTGCCACAGCGGTAC-3'
家禽白血病病毒亚群B检测引物:
上游引物B3:5'-CATACGATAGTCCGGCTG-3'
下游引物B4:5'-CCCCACACATCCTGACA-3'
家禽白血病病毒亚群J检测引物:
上游引物J3:5'-GGAGTTCATCTATTGCAA-3'
下游引物J4:5'-GCGCCTGCTACGGTGGT-3'。
进一步地,所述的一种家禽白血病病毒检测方法在家禽白血病病毒检测中的应用。
进一步地,所述的应用,包括以下步骤:
(1)提取家禽全基因组DNA,作为多重PCR的模板;
(2)以家禽白血病病毒亚群A检测引物、家禽白血病病毒亚群B检测引物和家禽白血病病毒亚群J检测引物为3对检测引物,进行多重PCR;
(3)取步骤(2)中反应液进行琼脂糖凝胶电泳检测。
进一步地,所述多重PCR的反应条件为:
反应体系为25μL:三个亚群A、B、J的上下游每条引物2μL;模板3μL;预混酶13μL;水补足至25μL;
预变性:95℃ 5min;
变性:94℃ 50s;
退火:56℃ 50s;
延伸:72℃ 1min;
循环数:40;
循环末延伸:72℃ 5min;
结合上述的所有技术方案,本发明所具备的优点及积极效果为:
(1)本发明所提出的家禽白血病病毒检测引用物实现了在一次实验中快速检测出三个家禽白血病病毒亚群的目的,相当于传统的三次PCR反应。
(2)本发明的成本相比之下非常低廉,效率也相当高;在灵敏度方面,本方法灵敏度和特异性都非常高,能够检出的模板最小拷贝数是1×103拷贝/μL。
附图说明
为了更清楚地说明本申请实施例的技术方案,下面将对本申请实施例中所需要使用的附图做简单的介绍,显而易见地,下面所描述的附图仅仅是本申请的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下还可以根据这些附图获得其他的附图。
图1是本发明实施例提供的实验步骤流程图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
针对现有技术存在的问题,本发明提供了一种家禽白血病病毒检测方法,下面结合附图对本发明作详细的描述。
实施例
一种家禽白血病病毒检测方法,包括针对家禽白血病病毒A、B、J亚群的3对特异性寡核苷酸引物序列:
家禽白血病病毒亚群A检测引物(SEQ ID NO:1):
上游引物A3:5'-GGTTGGTCTAGACAGGA-3'
下游引物A4:5'-CATTGCCACAGCGGTAC-3'
家禽白血病病毒亚群B检测引物(SEQ ID NO:2):
上游引物B3:5'-CATACGATAGTCCGGCTG-3'
下游引物B4:5'-CCCCACACATCCTGACA-3'
家禽白血病病毒亚群J检测引物(SEQ ID NO:3):
上游引物J3:5'-GGAGTTCATCTATTGCAA-3'
下游引物J4:5'-GCGCCTGCTACGGTGGT-3'。
进一步地,所述的一种家禽白血病病毒检测方法在家禽白血病病毒检测中的应用。
进一步地,所述的应用,包括以下步骤:
(1)提取家禽全基因组DNA,作为多重PCR的模板;
(2)以家禽白血病病毒亚群A检测引物、家禽白血病病毒亚群B检测引物和家禽白血病病毒亚群J检测引物为3对检测引物,进行多重PCR;
(3)取步骤(2)中反应液进行琼脂糖凝胶电泳检测。
进一步地,所述多重PCR的反应条件为:
反应体系为25μL:三个亚群A、B、J的上下游每条引物2μL;模板3μL;预混酶13μL;水补足至25μL;
预变性:95℃ 5min;
变性:94℃ 50s;
退火:56℃ 50s;
延伸:72℃ 1min;
循环数:40;
循环末延伸:72℃ 5min;
以上所述,仅为本发明较优的具体的实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,都应涵盖在本发明的保护范围之内。
<110>贵州省畜牧兽医研究所
<120>一种家禽白血病病毒检测方法
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ggttggtctagacagga
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catacgatagtccggctg
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ggagttcatctattgcaa
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Claims (4)
1.一种家禽白血病病毒检测方法,包括针对家禽白血病病毒A、B、J亚群的3对特异性寡核苷酸引物序列:
家禽白血病病毒亚群A检测引物:
上游引物A3:5'-GGTTGGTCTAGACAGGA-3'
下游引物A4:5'-CATTGCCACAGCGGTAC-3'
家禽白血病病毒亚群B检测引物:
上游引物B3:5'-CATACGATAGTCCGGCTG-3'
下游引物B4:5'-CCCCACACATCCTGACA-3'
家禽白血病病毒亚群J检测引物:
上游引物J3:5'-GGAGTTCATCTATTGCAA-3'
下游引物J4:5'-GCGCCTGCTACGGTGGT-3'。
2.权利要求1所述的一种家禽白血病病毒检测方法在家禽白血病病毒检测中的应用。
3.根据权利要求2所述的应用,包括以下步骤:
(1)提取家禽全基因组DNA,作为多重PCR的模板;
(2)以家禽白血病病毒亚群A检测引物、家禽白血病病毒亚群B检测引物和家禽白血病病毒亚群J检测引物为3对检测引物,进行多重PCR;
(3)取步骤(2)中反应液进行琼脂糖凝胶电泳检测。
4.所述多重PCR的反应条件为:
反应体系为25μL:三个亚群A、B、J的上下游每条引物2μL;模板3μL;预混酶13μL;水补足至25μL;
预变性:95℃5min;
变性:94℃50s;
退火:56℃50s;
延伸:72℃1min;
循环数:40;
循环末延伸:72℃5min。
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ROBERT; F. SILVA等: "区分ALV亚群PCR方法的建立:污染商品马立克氏病疫苗ALV的检测", 中国畜牧兽医学会禽病学分会鸡淋巴白血病防控技术研讨会论文集, pages 294 - 299 * |
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