CN103882152B - 一组用于水禽圆环病毒可视化鉴别诊断的荧光定量引物 - Google Patents

一组用于水禽圆环病毒可视化鉴别诊断的荧光定量引物 Download PDF

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CN103882152B
CN103882152B CN201410134003.4A CN201410134003A CN103882152B CN 103882152 B CN103882152 B CN 103882152B CN 201410134003 A CN201410134003 A CN 201410134003A CN 103882152 B CN103882152 B CN 103882152B
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万春和
黄瑜
陈红梅
程龙飞
傅光华
施少华
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Institute of Animal Husbandry and Veterinary of Fujian Academy of Agricultural Sciences
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Abstract

本发明公开了一组用于鸭圆环病毒（DuCV）和鹅圆环病毒（GoCV）感染情况的可视化检测的实时荧光定量PCR引物及其方法，该方法是利用该引物扩增的鸭圆环病毒（DuCV）和鹅圆环病毒（GoCV）特异基因片段区域核苷酸GC含量的差异导致溶解曲线温度Tm值的差异来对DuCV和GoCV感染情况进行检测，该方法仅需一组基于SYBR GreenⅠ实时荧光定量PCR引物结合反应结束后自动生成的溶解曲线Tm值的差异即可特异性的对DuCV和GoCV感染情况进行可视化鉴别诊断。本发明鉴定方法简单，效率和准确率较高。

Description

一组用于水禽圆环病毒可视化鉴别诊断的荧光定量引物
技术领域
本发明属于动物分子病原学领域，具体涉及一组用于水禽圆环病毒（鸭圆环病毒和鹅圆环病毒）可视化鉴别诊断的荧光定量引物。
背景技术
鸭感染圆环病毒最早由Hattermann等于2003年（Hattermann K, Schmitt C,Soike D, et al. Cloning and sequencing of duck circovirus (DuCV) . ArchVirol, 2003, 148: 2471-2480.）报道，国内最早由本研究室傅光华等（傅光华，程龙飞，黄瑜，等．鸭圆环病毒全基因组克隆与序列分析．病毒学报，2008，24（2）：138-143．）报道有鸭圆环病毒感染。家禽感染圆环病毒的临床表现主要以生长发育迟缓、羽毛凌乱为特征，主要侵害免疫系统，会引起的病毒诱导性淋巴组织损伤类似，削弱动物的体液免疫和细胞免疫功能。感染鸭圆环病毒的鸭群的临床症状则主要表现为生长发育状况差，羽毛营养失调，在背部脊柱（的羽毛）尤其明显，见有羽毛羽轴出血。鸭只背部皮肤组织病理学检查主要表现为囊泡和围绕囊泡的组织异嗜性炎性渗出，内脏器官总的病理表现为鸭传染性浆膜炎协同感染的症状，对法氏囊组织病理学检查表明，淋巴细胞损伤、坏死、组织细胞增多。
鹅圆环病毒(GoCV)最早于1999年由德国学者Soike等（Soike D, Kohler B,Albrecht K. A circovirus-like infection in geese related to a runtingsyndrome. Avian Pathology, 1999, 28:199 -2021.）从患病鹅病理组织中观察到。病鹅主要表现发育不良、体重下降、羽毛凌乱等，病理组织学检查发现法氏囊、脾脏和胸腺的淋巴细胞减少，其中法氏囊病变最明显，甚至出现整个囊结构的破坏，并可观察到嗜碱性的包含体。余旭平等最早在中国浙江省鹅中检测到鹅圆环病毒，并对浙江省鹅圆环病毒的基因组结构和流行病学情况进行分析。
目前，已有研究报道多种鸭病毒性传染病跨种传播感染鹅的报道，如鸭肝炎病毒、番鸭呼肠孤病毒和黄病毒；也有鹅病毒性传染病感染跨种传播感染鸭的报道，如鹅细小病毒和鹅出血性多瘤病毒感染番鸭和半番鸭等报道。
目前，有报道DuCV和GoCV的ORF-V1所编码的蛋白（Rep蛋白）核苷酸同源性可达80.0%以上可设计一种引物来检测DuCV和GoCV感染，但对可能出现的跨种传播仍无相应储备检测方法。
目前，对DuCV和GoCV感染情况用实时荧光定量PCR方法需分别进行实时荧光定量PCR反应，成本较高。
目前，国内外还没有仅需一组实时荧光定量PCR引物同时对DuCV和GoCV感染情况进行可视化鉴别诊断的相关研究报道，本发明的建立可填补国内外相关领域空白。
发明内容
本发明的目的在于提供一组用于水禽圆环病毒可视化鉴别诊断的荧光定量引物及其检测方法，该方法能有效区分鸭圆环病毒（DuCV）和鹅圆环病毒（GoCV）感染，为水禽健康养殖提供技术保证。
本发明根据鸭圆环病毒（DuCV）和鹅圆环病毒（GoCV）基因组中ORF-V1所编码的蛋白（Rep蛋白）特征，设计一组实时荧光定量PCR引物，该引物针对鸭圆环病毒（DuCV）和鹅圆环病毒（GoCV）进行PCR扩增可得到特异性目的条带，该引物对其扩增的DuCV和GoCV基因在该区域存在有GC含量差异，通过建立基于SYBRⅠ实时荧光定量PCR对DuCV和GoCV反应后的溶解曲线Tm值差异存在不同的特异峰，根据溶解曲线的特异峰的差异可直接DuCV和GoCV感染情况进行可视化观察。
本发明采用以下技术方案：
一组用于水禽圆环病毒可视化鉴别诊断的荧光定量引物，所述PCR引物P1和P2的序列为：上游引物P1：5’- TAATAGGGAGCCTCGCGATTGGTA -3’，
下游引物P2：5’- AACCAGGACTTAGTAGTTTATT -3’。
通过所述引物建立基于SYBR GreenⅠ实时荧光定量PCR方法，根据利用该引物扩增的鸭圆环病毒（DuCV）和鹅圆环病毒（GoCV）特异基因片段区域核苷酸GC含量的差异导致溶解曲线温度的差异，根据溶解曲线Tm值的差异可直接对DuCV和GoCV感染进行定量检测。
具体包括以下步骤：
（1）提取DuCV和GoCV基因组DNA；
（2）用所述实时荧光定量引物P1和P2同时对DuCV和GoCV进行基于SYBR GreenⅠ实时荧光定量PCR检测，反应完成后作出溶解曲线；
（3）实时荧光定量PCR反应结束后，根据溶解曲线特异性峰值的差异直接对DuCV和GoCV感染情况进行可视化检测。
所述PCR引物在检测鸭圆环病毒（DuCV）和鹅圆环病毒（GoCV）感染情况方面的应用。
其中，实时荧光定量PCR引物需满足如下要求：
（1）该实时荧光定量PCR引物是选择鸭圆环病毒（DuCV）和鹅圆环病毒（GoCV）基因组中ORF-V1所编码的蛋白（Rep蛋白）的保守区域进行设计，该引物进行常规PCR时，均可对DuCV和GoCV进行扩增，但扩增的PCR产物用常规琼脂糖凝胶电泳无法区别。
（2））该实时荧光定量PCR引物能一个实时荧光定量PCR反应能对鸭圆环病毒（DuCV）和鹅圆环病毒（GoCV）基因组DNA均能阳性扩增，通过生成的溶解曲线获得特异性峰。
（3）该实时荧光定量PCR引物需选择能对DuCV和GoCV进行实时荧光定量PCR阳性扩增，但是实时荧光定量PCR引物针对的DuCV和GoCV在该扩增区域的基因片段存在有GC含量差异。由于DuCV和GoCV在该扩增区域的基因片段存在有GC含量差异导致进行实时荧光定量PCR进行扩增后，生成的溶解曲线Tm值不同而存在有各自特异峰，可通过和实时荧光定量PCR仪器连接的电脑根据观察溶解曲线的Tmz值的差异直接对DuCV和GoCV感染情况进行可视化判断。
其中，所述的步骤（3）的峰值如下：
若在Tm=（82.80±0.08）℃出现单一的特异性峰判为DuCV阳性；若在Tm=（85.98±0.10）℃出现单一的特异性峰判为GoCV阳性。
本发明的有益效果：鉴定方法简单，效率和准确率较高。使用本研究提供的一组实时荧光定量PCR引物对本临床送检疑似鸭圆环病毒（DuCV）和鹅圆环病毒（GoCV）的生长不良鸭鹅各5份感染情况进行可视化鉴别诊断，其中疑似鸭圆环病毒（DuCV）5份样品检测到DuCV感染阳性3份；其中疑似鹅圆环病毒（GoCV）5份样品检测到GoCV感染阳性2份。
附图说明
图1 特异性实时荧光定量PCR引物对DuCV和GoCV进行检测的溶解曲线。1为DuCV阳性，2为GoCV阳性，3为DuCV和GoCV阴性。
具体实施方式
下面实施例对本发明做进一步的描述。
实施例1
1、毒株：
鸭圆环病毒（DuCV）和鹅圆环病毒（GoCV）均由福建省农业科学院畜牧兽医研究所鉴定和保存。
2、引物设计与合成
根据DuCV和GoCV基因组中ORF-V1所编码的蛋白（Rep蛋白）的保守区域进行设计实时荧光定量PCR引物P1和P2，其中引物P1和P2序列为：
上游引物P1：5’- TAATAGGGAGCCTCGCGATTGGTA -3’，
下游引物P2：5’- AACCAGGACTTAGTAGTTTATT -3’。
3、实时荧光定量PCR扩增
以常规方法提取DuCV和GoCV基因组DNA。用所设计的特异性实时荧光定量PCR引物P1和P2进行实时荧光定量PCR扩增。
优化出的20 μL 最佳反应体系为体系：SYBR Premix Ex TaqTM 10 μL、上、下游引物（10 μmol/L） 各0.4μL、模板1 μL、水补足至20 μL。最佳反应条件为：95 ℃ 2 min预变性；95 ℃ 15 s，60 ℃ 10 s，72 ℃ 15s ，共40个循环，循环结束后，做出溶解曲线。
4、实时荧光定量PCR溶解曲线
实时荧光定量PCR反应结束后，根据做出的溶解曲线，直接在和实时荧光定量PCR仪器连接的电脑上直接分析溶解曲线，对DuCV和GoCV感染情况进行判断，判断方法为：
从生成的溶解曲线可见看出，若在Tm=（82.80±0.08）℃出现单一的特异性峰判为DuCV阳性；若在Tm=（85.98±0.10）℃出现单一的特异性峰判为GoCV阳性。
5、临床应用
使用本研究提供的一组实时荧光定量PCR引物对本临床送检疑似鸭圆环病毒（DuCV）和鹅圆环病毒（GoCV）的生长不良鸭鹅各5份感染情况进行可视化鉴别诊断，其中疑似鸭圆环病毒（DuCV）5份样品检测到DuCV感染阳性3份；其中疑似鹅圆环病毒（GoCV）5份样品检测到GoCV感染阳性2份。本发明通过的方法用一组实时荧光定量PCR引物即可确定感染的水禽圆环病毒类型并进行准确定量分析。
以上所述仅为本发明的较佳实施例，凡依本发明申请专利范围所做的均等变化与修饰，皆应属本发明的涵盖范围。
SEQUENCE LISTING
<110> 福建省农业科学院畜牧兽医研究所
<120> 一组用于水禽圆环病毒可视化鉴别诊断的荧光定量引物
<130> 2
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 24
<212> DNA
<213> 人工序列
<400> 1
taatagggag cctcgcgatt ggta 24
<210> 2
<211> 22
<212> DNA
<213> 人工序列
<400> 2
aaccaggact tagtagttta tt 22

Claims (2)

1.一组用于水禽圆环病毒可视化鉴别诊断的荧光定量引物，其特征在于：所述荧光定量引物P1和P2的序列为：上游引物P1：5’- TAATAGGGAGCCTCGCGATTGGTA -3’，下游引物P2：5’- AACCAGGACTTAGTAGTTTATT -3’。
2.一种如权利要求1所述的一组用于水禽圆环病毒可视化鉴别诊断的荧光定量引物在制备检测鸭圆环病毒和鹅圆环病毒试剂盒上的应用。
CN201410134003.4A 2014-04-04 2014-04-04 一组用于水禽圆环病毒可视化鉴别诊断的荧光定量引物 Active CN103882152B (zh)

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CN101942528A (zh) * 2010-10-20 2011-01-12 福建省农业科学院畜牧兽医研究所 一种用于检测鹅圆环病毒的引物和探针

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