CN116287457A - 一种鉴别诊断犬瘟热病毒、犬腺病毒、犬副流感病毒、波氏杆菌的引物组合物及其应用 - Google Patents
一种鉴别诊断犬瘟热病毒、犬腺病毒、犬副流感病毒、波氏杆菌的引物组合物及其应用 Download PDFInfo
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Abstract
本发明公开了一种鉴别诊断犬瘟热病毒、犬腺病毒、犬副流感病毒、波氏杆菌的引物组合物及其应用,属于生物技术领域。包括以下引物:外引物F3和B3,内引物FIP和BIP,环引物LF和LB。本发明可用于鉴别诊断引起犬呼吸道疾病的犬瘟热病毒病原基因、犬腺病毒病原基因、犬副流感病毒病原基因、支气管败血波氏杆菌病原基因。本发明提供的引物组合物检测引起犬呼吸道疾病的病原基因。具有灵敏度好、特异性高、用时短、稳定性高等特点。
Description
技术领域
本发明属于病毒检测技术领域,具体涉及一种鉴别诊断犬瘟热病毒、犬腺病毒、犬副流感病毒、波氏杆菌的引物组合物及其应用。
背景技术
犬呼吸道病原感染严重危害宠物健康与生活质量,是宠物疾病中发病率最高的症状之一,且常因多种呼吸道病原病毒混合感染而使病情加重。引起犬呼吸道疾病的病原主要包括犬瘟热病毒、犬腺病毒、犬副流感病毒、支气管败血波氏杆菌。
犬瘟热病毒(canine distempervirus,CDV)属于副粘病毒科,麻疹病毒属。病毒粒子多呈球形,多在核酸为RNA型。感染犬科的高度接触性、致死性传染病。其特征:双相热型、急性卡他性呼吸道炎、胃肠炎和脑炎。是感染犬的一种最古老的、临床意义重大的病毒。主要通过空气和飞沫水平传播。传染源:病犬的鼻汁、唾液、泪液,也见于血液、脑脊髓液、淋巴液、肝、脾、心包液、胸和腹水中,并能通过尿液长期排毒。传播途径:直接接触;空气飞沫;胎盘CDV感染病型复杂多样,临床上诊断很困难,易与上呼吸道感染、犬传染性肝炎、犬冠状病毒感染等相混淆,必须根据病毒分离鉴定确诊。
犬腺病毒(Canine adeno virus,CAV)属于腺病毒科,哺乳动物腺病毒属,双股DNA病毒。是哺乳动物腺病毒属中致病性最强的一种病毒。有两个血清型。Ⅰ型可引起犬传染性肝炎,Ⅱ型可引起犬传染性喉气管炎和肠炎。犬腺病毒Ⅱ型可引起犬的传染性喉气管炎及肺炎症状。临床特征表现持续性高热、咳嗽、浆液性至粘液性鼻漏、扁桃体炎、喉气管炎和肺炎。从临床发病情况统计,该病多见于4月龄以下的幼犬。在幼犬可以造成全窝或全群咳嗽,因此该病依临床特征常称为“犬窝咳”。犬腺病毒的感染潜伏期为5-6天。持续性发热(体温在39.5℃左右。鼻部流浆液性鼻液,随呼吸向外喷水样鼻液。表现6-7天阵发性干咳,后表现湿咳并有痰液,呼吸喘促,病状继续发展可引起坏死性肺炎。病犬可表现精神沉郁、不食。并有呕吐和腹泻症状出现。该病往往易和犬瘟热、犬副流感病毒及支气管败血波氏杆菌混合感染。混合感染的犬预后大多不良。
犬副流感病毒(Canine ParainfluenzaVirus,CPIV)CPIV在分类上属副粘病毒科,副粘病毒属。核酸型为单股RNA。是犬的主要的呼吸道传染病。临床表现发热、流涕和咳嗽。病理变化以卡他性鼻炎和支气管炎为特征。近年来研究认为,CPIV也可引起急性脑髓炎和脑内积水,临床表现后躯麻痹和运动失调等症状。CPIV可感染玩赏犬、试验犬和军、警犬,在军犬中常发生呼吸道病,在试验犬产生犬瘟热样症状。急性期病犬最是主要的传染来源。自然感染途径主要是呼吸道。
支气管败血波氏杆菌(Bordetellabronchiseptica,Bb)为革兰阴性细小球菌,具鞭毛而能运动,极好氧,病犬病初精神、食欲无明显变化,仅出现短而粗的干咳,随后咳嗽症状加剧,出现痉挛性干咳,呈气管炎症状,接着是干呕或作呕以图清除喉中的少量黏液,运动时咳嗽加重,并且晚上特别明显。咳嗽随着运动增加和气管、喉部诱咳或食物的刺激而加剧。有些病犬表现为阵发性吸气性呼吸困难,触压喉头或气管时容易诱发咳嗽,体温升高至39~40℃之间,打开口腔检查扁桃腺和咽部有炎症反应;视诊呼吸频数增多;听诊呼吸音增强,肺区出现哕音,有的犬鼻腔有黏液性、脓性分泌物。
犬呼吸道传染病的临床表现非常相似,不易区别。目前细胞培养时分离和鉴定是目前的最好方法。但细胞培养耗时较长,对实验环境要求高,人工成本高。高通量LAMP微流控芯片检测病毒性腹泻病原效率更高,更便捷。
发明内容
本发明提供一种鉴别诊断犬瘟热病毒、犬腺病毒、犬副流感病毒、波氏杆菌的引物组合物及其应用。具体通过以下技术方案实现:
所述用于检测犬瘟热病毒F基因的引物组合物,包括以下引物:
F3引物为SEQ ID No.1所示的核苷酸序列;
B3引物为SEQ ID No.2所示的核苷酸序列;
FIP引物为SEQ ID No.3所示的核苷酸序列;
BIP引物为SEQ ID No.4所示的核苷酸序列;
F3(SEQ ID No.1):CAAGCTTTGACTCTAATGACC;
B3(SEQ ID No.2):TCAAGGCTAGTTCTCAGAGA;
FIP(SEQ ID No.3):
TGCAAGTACCACTCCTGCAACCTTTACAGTCAATAGGGTCG;
BIP(SEQ ID No.4):
CAGCTTTAGGAGTGGCCACAGATTGCTTGAGCATTGAGG。
所述的引物还包括将上述各F3、B3、FIP、BIP引物序列经过一个或几个核苷酸的取代和/或缺失和/或添加后具有与上述序列具有相同功能的DNA分子。
所述用于检测犬腺病毒p-VIII基因的引物组合物,包括以下引物:
F3引物为SEQ ID No.5所示的核苷酸序列;
B3引物为SEQ ID No.6所示的核苷酸序列;
FIP引物为SEQ ID No.7所示的核苷酸序列;
BIP引物为SEQ ID No.8所示的核苷酸序列;
LF引物为SEQ ID No.9所示的核苷酸序列;
LB引物为SEQ ID No.10所示的核苷酸序列;
F3(SEQ ID No.5):CCAACTTGCGCAAATGCTTA;
B3(SEQ ID No.6):GGAGGCGAGATATTCACAGC;
FIP(SEQ ID No.7):
TCGTGTCCGCTTCATGGCTTAATGTGGTCTCTCCCGACA;
BIP(SEQ ID No.8):
CCATATGACGCCCCAAAACCCTTCCGGGGGATGATTCAGTG;
LF(SEQ ID No.9):GGTGTTGGTGAACGGGAAG;
LB(SEQ ID No.10):CCACTCAACCCCCTTTTTTTAATG。
所述的引物还包括将上述各F3、B3、FIP、BIP、LF、LB引物序列经过一个或几个核苷酸的取代和/或缺失和/或添加后具有与上述序列具有相同功能的DNA分子。
所述用于检测犬副流感病毒NP基因的引物组合物,包括以下引物:
F3引物为SEQ ID No.11所示的核苷酸序列;
B3引物为SEQ ID No.12所示的核苷酸序列;
FIP引物为SEQ ID No.13所示的核苷酸序列;
BIP引物为SEQ ID No.14所示的核苷酸序列;
F3(SEQ ID No.11):AGTTTGGGCAATTTTTCGT;
B3(SEQ ID No.12):AGAAGCCGAGATCTTAGCT;
FIP(SEQ ID No.13):
AGTTCTTGAGTGAGTGTGAATCGCCCCGACACAAAAATGTC;
BIP(SEQ ID No.14):
GCAAGATCAGAGTGAGGAAGGTTAGAGGTTAGTATAAATACCCTGA。
所述的引物还包括将上述各F3、B3、FIP、BIP引物序列经过一个或几个核苷酸的取代和/或缺失和/或添加后具有与上述序列具有相同功能的DNA分子。
所述用于检测支气管败血波氏杆菌FimN基因的引物组合物,包括以下引物:
F3引物为SEQ ID No.15所示的核苷酸序列;
B3引物为SEQ ID No.16所示的核苷酸序列;
FIP引物为SEQ ID No.17所示的核苷酸序列;
BIP引物为SEQ ID No.18所示的核苷酸序列;
LF引物为SEQ ID No.19所示的核苷酸序列;
LB引物为SEQ ID No.20所示的核苷酸序列;
F3(SEQ ID No.15):CGACGCCGTTTTCCATCAA;
B3(SEQ ID No.16):TCTGCACTCCTTCCGCG;
FIP(SEQ ID No.17):
CGGACCGGGCTCGAAGTACACTGCAGAACTGCCCCGAAT;
BIP(SEQ ID No.18):
CTACAGCACCAGGGACCTGACCCTGGCCGGCAACAATGT;
LF(SEQ ID No.19):GGAATCTGGGTGCCATCCA;
LB(SEQ ID No.20):GCGCAAACCGGGCAACA。
所述的引物还包括将上述各F3、B3、FIP、BIP、LF、LB引物序列经过一个或几个核苷酸的取代和/或缺失和/或添加后具有与上述序列具有相同功能的DNA分子。
本发明提供了上述引物组合物在环介导等温扩增方法中用以检测犬瘟热病毒、犬腺病毒、犬副流感病毒、支气管败血波氏杆菌基因的应用。
所述的引物组合物在环介导等温扩增(LAMP)方法中,引物组合物F3、B3、FIP、BIP、LF和LB的摩尔浓度比为1:1:4-16:4-16:2-8:2-8范围。
所述的环介导等温扩增反应条件为60-65℃范围。
本发明还提供了上述组合物在用于引起犬呼吸道疾病的病原基因检测的试剂盒或芯片中的应用,具体为:
在制备检测犬瘟热病毒、犬腺病毒、犬副流感病毒、支气管败血波氏杆菌基因试剂盒和微流控芯片中的应用。包括将各条引物单独包装的步骤。
所述的试剂盒或芯片的检测步骤包括:
1)提取待测病毒或其他类型待检样品中基因组cDNA;
2)以步骤1)提取的基因为模版,采用上述引物组合物进行对犬瘟热病毒、犬腺病毒、犬副流感病毒、支气管败血波氏杆菌的环介导等温扩增。
本发明所述的引物组合物可用于检测样本中是否含有犬呼吸道病原基因:犬瘟热病毒、犬腺病毒、犬副流感病毒、支气管败血波氏杆菌基因。
应用上述引物组合物可以对样品中基因cDNA模版特异性扩增,则待测样品中含有或疑似含有犬瘟热病毒、犬腺病毒、犬副流感病毒、支气管败血波氏杆菌的病原基因;如果不可以对样品中基因cDNA模版特异性扩增,则待测样品中不含有犬瘟热病毒、犬腺病毒、犬副流感病毒、支气管败血波氏杆菌的病原基因。
本发明提供的用于检测犬瘟热病毒、犬腺病毒、犬副流感病毒、支气管败血波氏杆菌的病原基因引物组合物,可便捷、快速、准确的检测出结果。对于实际应用而言,能够在1小时内获得检测结果。
附图说明
图1是CDV扩增典型荧光曲线与CDV引物扩增CAV、CPIV、Bb、犬基因组和空白对照荧光曲线;
图2是CAV扩增典型荧光曲线与CAV引物扩增CDV、CPIV、Bb、犬基因组和空白对照荧光曲线;;
图3是CPIV扩增典型荧光曲线与CPIV引物扩增CDV、CAV、Bb、犬基因组和空白对照荧光曲线;
图4是Bb扩增典型荧光曲线与Bb引物扩增CDV、CAV、CPIV、犬基因组和空白对照荧光曲线。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述和展示,所描述的实施例仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
下列实施中所用的试验方法,如无特殊说明,均为本领域常用的实验方法;
下列实施中所用的试验材料,如无特殊说明,均为本领域常用的实验材料;实施例1
利用人工合成含犬瘟热病毒F基因的质粒,用以检测试剂盒检测犬瘟热病毒。
将合成的质粒用作模板,反应体系如下表;
结果如图1所示,CDV质粒荧光曲线为特异S型曲线;CAV、CPIV、Bb、犬基因组cDNA和空白对照荧光曲线为基线。引物组合可以特异检测CDV。
CDV荧光定量PCR反应管25ul反应体系:
CDV各引物的核苷酸序列如下:
F3:CAAGCTTTGACTCTAATGACC;
B3:TCAAGGCTAGTTCTCAGAGA;
FIP:TGCAAGTACCACTCCTGCAACCTTTACAGTCAATAGGGTCG;
BIP:CAGCTTTAGGAGTGGCCACAGATTGCTTGAGCATTGAGG。
实施例2
利用人工合成含犬腺病毒p-VIII基因的质粒,用以检测试剂盒检测CAV。
将合成的质粒用作模板,反应体系同实施例1;
结果如图2所示,CAV质粒荧光曲线为特异S型曲线;CDV、CPIV、Bb、犬基因组cDNA和空白对照荧光曲线为基线。引物组合可以特异检测CAV。
CAV各引物的核苷酸序列如下:
F3:CCAACTTGCGCAAATGCTTA;
B3:GGAGGCGAGATATTCACAGC;
FIP:TCGTGTCCGCTTCATGGCTTAATGTGGTCTCTCCCGACA;
BIP:CCATATGACGCCCCAAAACCCTTCCGGGGGATGATTCAGTG;
LF:GGTGTTGGTGAACGGGAAG;
LB:CCACTCAACCCCCTTTTTTTAATG。
实施例3
利用人工合成含犬副流感病毒NP基因的质粒,用以检测试剂盒检测CPIV。
将合成的质粒用作模板,反应体系同实施例1;
结果如图3所示,图3CIPV质粒荧光曲线为特异S型曲线;CAV、CDV、Bb、犬基因组cDNA和空白对照荧光曲线为基线。引物组合可以特异检测CPIV。
CPIV各引物的核苷酸序列如下:
F3:AGTTTGGGCAATTTTTCGT;
B3:AGAAGCCGAGATCTTAGCT;
FIP:AGTTCTTGAGTGAGTGTGAATCGCCCCGACACAAAAATGTC;
BIP:
GCAAGATCAGAGTGAGGAAGGTTAGAGGTTAGTATAAATACCCTGA。
实施例4
利用人工合成含支气管败血波氏杆菌FimN基因的质粒,用以检测试剂盒检测Bb。
将合成的质粒用作模板,反应体系同实施例1;
结果如图4所示,图3Bb质粒荧光曲线为特异S型曲线;CAV、CDV、CPIV、犬基因组cDNA和空白对照荧光曲线为基线。引物组合可以特异检测Bb。
Bb各引物的核苷酸序列如下:
F3:CGACGCCGTTTTCCATCAA;
B3:TCTGCACTCCTTCCGCG;
FIP:CGGACCGGGCTCGAAGTACA-CTGCAGAACTGCCCCGAAT;
BIP:CTACAGCACCAGGGACCTGACCCTGGCCGGCAACAATGT;
LF:GGAATCTGGGTGCCATCCA;
LB:GCGCAAACCGGGCAACA。
Claims (8)
1.一种用于检测引起犬呼吸道疾病的病原基因的引物组合物,其特征在于,所述的引物组合物用于检测犬瘟热病毒F基因、犬腺病毒p-VIII基因、犬副流感病毒NP基因、支气管败血波氏杆菌FimN基因;
所述用于检测犬瘟热病毒F基因的引物组合物,包括以下引物:
F3引物为SEQ ID No.1所示的核苷酸序列;
B3引物为SEQ ID No.2所示的核苷酸序列;
FIP引物为SEQ ID No.3所示的核苷酸序列;
BIP引物为SEQ ID No.4所示的核苷酸序列;
所述用于检测犬腺病毒p-VIII基因的引物组合物,包括以下引物:
F3引物为SEQ ID No.5所示的核苷酸序列;
B3引物为SEQ ID No.6所示的核苷酸序列;
FIP引物为SEQ ID No.7所示的核苷酸序列;
BIP引物为SEQ ID No.8所示的核苷酸序列;
LF引物为SEQ ID No.9所示的核苷酸序列;
LB引物为SEQ ID No.10所示的核苷酸序列;
所述用于检测犬副流感病毒NP基因的引物组合物,包括以下引物:
F3引物为SEQ ID No.11所示的核苷酸序列;
B3引物为SEQ ID No.12所示的核苷酸序列;
FIP引物为SEQ ID No.13所示的核苷酸序列;
BIP引物为SEQ ID No.14所示的核苷酸序列;
所述用于检测犬支气管败血波氏杆菌FimN基因的引物组合物,包括以下引物:
F3引物为SEQ ID No.15所示的核苷酸序列;
B3引物为SEQ ID No.16所示的核苷酸序列;
FIP引物为SEQ ID No.17所示的核苷酸序列;
BIP引物为SEQ ID No.18所示的核苷酸序列;
LF引物为SEQ ID No.19所示的核苷酸序列;
LB引物为SEQ ID No.20所示的核苷酸序列。
2.根据权利要求1所述的一种用于检测引起犬呼吸道疾病的病原基因的引物组合物,其特征在于,所述的组合物包括将F3、B3、FIP、BIP、LF和LB引物序列经过一个或几个核苷酸的取代和/或缺失和/或添加后具有与所描述核苷酸序列具有相同功能的DNA分子。
3.根据权利要求2所述一种用于检测引起犬呼吸道的病原基因的引物组合物,其特征在于,在环介导等温扩增方法中用以检测犬瘟热病毒F基因、犬腺病毒p-VIII基因、犬副流感病毒NP基因、支气管败血波氏杆菌FimN基因。
4.根据权利要求3所述一种用于检测引起犬呼吸道疾病的病原基因的引物组合物,其特征在于,所述的引物组合物在环介导等温扩增方法中,引物组合物F3、B3、FIP、BIP、LF和LB的摩尔浓度比为1:1:4-16:4-16:2-8:2-8。
5.根据权利要求3所述的一种用于检测引起犬呼吸道疾病的病原基因的引物组合物,其特征在于,所述的环介导等温扩增反应条件为60-65℃,恒温60min。
6.根据权利要求1所述的一种用于检测引起犬呼吸道疾病的病原基因的引物组合物,其特征在于,在制备检测犬瘟热病毒F基因、犬腺病毒p-VIII基因、犬副流感病毒NP基因、支气管败血波氏杆菌FimN基因的试剂盒和基因芯片中的应用。
7.一种权利要求1所述的引物组合物的应用,其特征在于,在用于引起犬呼吸道疾病的病原基因检测的试剂盒或芯片中的应用。
8.根据权利要求7所述的应用,其特征在于,所述的试剂盒和芯片的检测步骤包括:
1)提取待测病毒或其他类型待检样品中基因组RNA;
2)以步骤(1)提取的基因为模版,采用上述引物组合物对犬瘟热病毒F基因、犬腺病毒p-VIII基因、犬副流感病毒NP基因、支气管败血波氏杆菌FimN基因RNA反转录和环介导等温扩增。
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