CN105463136B - 用于鸡传染性支气管炎病毒rt-pcr分型检测的试剂盒 - Google Patents
用于鸡传染性支气管炎病毒rt-pcr分型检测的试剂盒 Download PDFInfo
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Abstract
本发明涉及生物检测技术领域,旨在提供一种用于鸡传染性支气管炎病毒RT‑PCR分型检测的试剂盒。该试剂盒包括下述三对引物:根据传染性支气管炎病毒的S1基因片段设计的通用引物和4/91类型毒株特异性引物,以及根据nsp12基因片段设计的肾型毒株特异性引物,所述三对引物的序列如SEQ ID NO:6~11所示。本发明提供的试剂盒仅需通过一次RT‑PCR反应就能达到对IBV进行分型检测目的,是业内首次用一次性RT‑PCR的方法实现对肾型、4/91类型、呼吸型三种类型IBV毒株的鉴别检测。本发明具有使用方便快捷、敏感性高、特异性强等优点,为临床上IBV的快速鉴别诊断、流行病学调查以及IB的防控提供了一种新的有效的检测手段。
Description
技术领域
本发明属于生物检测技术领域,具体涉及鸡传染性支气管炎病毒RT-PCR分型检测试剂盒。
背景技术
鸡的传染性支气管炎(Infectious bronchitis,IB)是由传染性支气管炎病毒(Infectious bronchitis Virus,IBV)引起的鸡急性,高度接触性传染病。以呼吸困难,肾炎和蛋品质下降为主要特征,该病在全世界广泛流行,是养鸡业中危害最严重的呼吸道传染病之一。
IBV隶属于套式病毒目(Nidovirilaes)冠状病毒科(Coronaviridae)冠状病毒属(Coronavirus)γ冠状病毒亚属,是该亚群的代表病毒,也是最早分离获得的冠状病毒。鸡传染性支气管炎病毒在世界范围内广泛流行,而且不同的病毒对组织的亲嗜性不同,主要有呼吸型、肾型、肠型、生殖型和腺胃型等。由于IBV RNA特有的转录机制,加上RNA酶缺乏校正系统导致IBV很容易发生变异,出现了复杂的组织嗜性、血清型和基因型。IBV变异株的出现,使现有疫苗不能很好保护鸡群抵抗野毒的攻击,养鸡场IB的暴发时有发生,给IB的防控带来了很大的困难。因此,有必要对流行的IBV毒株类型进行准确的判断,给疫苗的使用提供及时的指导。
IBV为单股正链RNA病毒,基因组长约为27.4kb。在基因组RNA转录过程中形成6种mRNA,即mRNA 1~6,编码6种基因。其中基因1又称多聚酶基因(Ploymerase Gene),编码两个大的融合蛋白,即多聚蛋白1a和1b,多聚蛋白被蛋白酶水解形成15个非结构蛋白(nonstructural protein,nsp),参与病毒的复制和转录。基因2编码纤突蛋白(Spike,S),基因4编码膜蛋白(Membrane,M),基因6编码核衣壳蛋白(Nucleocapsid,N)。另还有基因3编码3a、3b和小膜蛋白(Envelope,E),基因5编码5a和5b蛋白。S、M、N和E是结构蛋白,参与病毒粒子的组成;3a、3b、5a和5b分子量小,功能未知。S蛋白具有免疫原性,被水解为两个亚基,S1和S2,其中S1蛋白决定IBV的血清型,并影响着病毒的组织嗜性。
根据IBV的基因组及其基因片段的遗传进化分析,组织嗜性类似的毒株归入同一遗传进化分支里,这些毒株的基因具有较高的同源性。因此,根据基因的信息可以将毒株进行分型。目前国内流行的IBV毒株从表现型上主要以呼吸型和肾型为主。4/91类型毒株最先在国外报道,此后在世界上许多国家流行起来,该型毒株的S1基因与其它许多类型毒株的异源性超过25%,且没有血清学交叉反应性,是当前世界上用于制备商品化疫苗的主要毒株之一。因此,对4/91类型毒株分型鉴定具有重要意义。
发明内容
本发明要解决的技术问题是,克服现有技术中的不足,提供一种用于鸡传染性支气管炎病毒RT-PCR分型检测的试剂盒。
为解决上述技术问题,本发明的解决方案是:
提供一种用于鸡传染性支气管炎病毒RT-PCR分型检测的试剂盒,该试剂盒包括下述三对引物:根据传染性支气管炎病毒的S1基因片段设计的通用引物和4/91类型毒株特异性引物,以及根据nsp12基因片段设计的肾型毒株特异性引物;所述三对引物的序列如SEQID NO:6~11所示:
通用-F:5'-CCAAAGTGCCTTCAGACC-3'
通用-R:5'-GCTAGACCAAGCCATACC-3'
4/91-F:5'-ATGTCTTGGTCAGTTTCA-3'
4/91-R:5'-AACAAGATCACCATTTA-3'
肾型-F:5'-GTGGTTTAGGTGTAGATGTA-3'
肾型-R:5'-TAAGAAATCATCAAGCAAAAGG-3'。
本发明中,所述试剂盒包括下述成分:
(1)RNA提取试剂:Trizol试剂;
(2)反转录试剂:0.2μg/μl的Random primer,DEPC水,5×buffer,10mM的dNTP,20u/μl的Rnase抑制剂,200u/μl的M-MuLV;
(3)PCR反应试剂:20u/μl的ExTaq酶,2.5mM的dNTP,10×buffer;
(4)分型引物:所述通用引物、4/91类型毒株特异性引物和肾型毒株特异性引物,引物浓度均为10mM;
(5)阳性对照样品:包括呼吸型、4/91类型和肾型毒株核酸样品,具体分别为呼吸型M41毒株、4/91毒株和肾型毒株SC021202感染的鸡胚尿囊液抽提RNA的反转录产物cDNA;
(6)阴性对照样品:正常鸡胚尿囊液抽提RNA的反转录产物cDNA。
更进一步,本发明还提供了所述试剂盒的使用方法,其特征在于,包括以下步骤:
(1)从待测拭子、组织或感染鸡胚尿囊液样品中提取总RNA:
在1.5ml EP管中加200μl处理好的拭子、组织或感染鸡胚尿囊液样品后,加入800μl Trizol试剂,混匀,室温静置10-30min;加200μl氯仿,振荡混匀15s,室温静置10-15min;4℃,12000g离心15min;取水层,加等体积的异丙醇,混匀,室温静置10min;4℃,12000g离心10min;弃去上清,加1ml 75%乙醇,4℃,7500g离心5min;弃去上清,沉淀干燥5-10min,加20μl DEPC水溶解,得总RNA,紫外分光光度计测定总RNA浓度;
(2)以上一步骤提取的总RNA为模板,进行反转录反应,反转录反应的步骤为:1μg总RNA加1μl Random primer加DEPC水至12μl,65℃冰浴5min;再加4μl 5×buffer、2μl10mM dNTP、1μl RNase抑制剂、1μl M-MuLV、混匀,25℃5min,42℃1h,70℃5min,得反转录产物cDNA;
(3)以所述反转录产物为模板,利用三对分型引物进行PCR扩增,所述PCR体系为:ExTaq酶0.25μl,10mM的引物通用-F和通用-R、4/91-F和4/91-R、肾型-F和肾型-R各2μl,2.5mM的dNTP 4μl,10×buffer 5μl,模板2.5μl,加水至50μl;所述PCR反应体系的反应条件为:94℃预变性5min,94℃变性30s,54℃退火30s,72℃延伸50s,30个循环,72℃延伸10min;
(4)取所述PCR反应液进行琼脂糖凝胶电泳,EB染色后判断结果。
本发明中,对4/91类型毒株RT-PCR扩增获得位于200bp和260bp的两条带,对肾型毒株扩增获得位于200bp和680bp的两条带,对呼吸型毒株扩增仅得到位于200bp的一条带。
本发明中,4/91毒株扩增获得的PCR产物为SEQ ID NO:1和SEQ ID NO:2所示的两段核苷酸序列;肾型毒株SC021202扩增获得的PCR产物为SEQ ID NO:3和SEQ ID NO:4所示的两段核苷酸序列;M41毒株扩增获得的PCR产物为SEQ ID NO:5所示的一段核苷酸序列。
与现有技术相比,本发明的有益效果在于:
本发明提供的试剂盒仅需通过一次RT-PCR反应就能达到对IBV进行分型检测目的。该试剂盒在同一PCR反应条件下,利用设计的三对分型引物,根据扩增到的产物的片段数和大小,来判断样品中是否含有肾型、4/91类型、呼吸型的IBV毒株。这是业内首次用一次性RT-PCR的方法实现对肾型、4/91类型、呼吸型三种类型IBV毒株的鉴别检测。
本方法具有使用方便快捷、敏感性高、特异性强等优点,为临床上IBV的快速鉴别诊断、流行病学调查以及IB的防控提供了一种新的有效的检测手段。
附图说明
图1为试剂盒RT-PCR扩增IBV SC021202(肾型)毒株、4/91、M41(呼吸型)、的结果;
图中,M.100bp marker;1.SC021202;2.4/91;3.M41,4.阴性对照。
图2为试剂盒RT-PCR特异性扩增IBV的结果;
图中,M.100bp marker;1.4/91;2.SC021202;3.M41;4-8.AIV H1N1亚型、AIV H9N2亚型、NDV、IBDV、FPV;9.阴性对照。
图3为试剂盒分型检测IBV毒株的验证结果;
图中,M.100bp marker;1.ZJ971;2.ARK99;3.X;4.H120;5.Y98;6.28/86;7.AH07091;8.SD0611;9.Ark DPI;10.BJ;11.N;12.Ark Vaccine;13.H52;14.Gray;15.HK;16.HN99;17.Cal99;18.R2;19.T;20.W93;21.SD07011;22.阴性对照。
具体实施方式
本发明中所利用的试剂盒组成包括:
(1)RNA提取试剂:Trizol
(2)反转录PCR试剂:包括0.2μg/μl的Random primer、DEPC水、5×buffer、10mM的dNTP、20u/μl的Rnase抑制剂、200u/μl的M-MuLV
(3)PCR反应试剂:包括20u/μl的ExTaq酶、2.5mM的dNTP、10×buffer;
(4)三对分型引物:通用引物、4/91型特异性引物和肾型特异性引物(如SEQ IDNO:6~11所示),浓度均为10μM。
(5)阳性对照样品:包括呼吸型、4/91类型和肾型毒株核酸样品,具体分别为呼吸型毒株M41、4/91毒株和肾型毒株SC021202感染的鸡胚尿囊液抽提RNA的反转录产物cDNA。
(6)阴性对照样品:正常鸡胚尿囊液抽提RNA的反转录产物cDNA。
上述试剂盒的使用方法,依次包括下列步骤:
(1)RNA的提取:从待测拭子、组织或感染鸡胚尿囊液样品中提取总RNA,其提取方法是:1.5ml EP管中加200μl处理好的拭子、组织或感染鸡胚尿囊液样品后,加入800μlTrizol试剂,混匀,室温静置10-30min;加200μl氯仿,振荡混匀15s,室温静置10-15min;4℃,12000g离心15min;取水层,加等体积的异丙醇,混匀,室温静置10min;4℃,12000g离心10min;弃去上清,加1ml75%乙醇,4℃,7500g离心5min;弃去上清,沉淀干燥5-10min,加20μl DEPC水溶解,得总RNA。紫外分光光度计测定总RNA浓度;
(2)反转录反应:上述反应提取的1μg总RNA加1μl Random primer加DEPC水至12μl,65℃冰浴5min;再加4μl 5×buffer、2μl 10mM dNTP、1μl RNase抑制剂、1μl M-MuLV、混匀,25℃5min,42℃1h,70℃5min,得反转录产物;
(3)PCR反应:以上述反转录产物为模板,进行PCR扩增,所述PCR体系为:ExTaq酶0.25μl,10mM的引物通用-F和通用-R、4/91-F和4/91-R、肾型-F和肾型-R各2μl,2.5mM的dNTP 4μl,10×buffer 5μl,模板2.5μl,加水至50μl;所述PCR反应体系的反应条件为:94℃预变性5min,94℃变性30s,54℃退火30s,72℃延伸50s,30个循环,72℃延伸10min;
(4)扩增产物电泳检测:取上述反应产物进行琼脂糖凝胶电泳,取5μl扩增产物与1μl上样缓冲液混匀后点样于1.5%琼脂糖凝胶中,90V电泳30min,凝胶成像系统观察。对4/91毒株RT-PCR扩增获得200bp和260bp左右的两条带,对肾型毒株SC021202扩增获得200bp和680bp左右两条带,对呼吸型毒株M41扩增仅得到200bp左右的一条带。
(5)扩增产物测序:分别取上述扩增获得的阳性对照样品特异性条带送测序公司进行测序确定。4/91毒株扩增获得的PCR产物为SEQ ID NO:1所示的两段核苷酸序列,大小分别为193bp和261bp;肾型毒株SC021202扩增获得的PCR产物为SEQ ID NO:2所示的两段核苷酸序列,大小分别为213bp和686bp;M41毒株扩增获得的PCR产物为SEQ ID NO:3所示的核苷酸序列,大小为193bp。
(6)本设计的方法对其它4/91类型、肾型和呼吸型毒株扩增获得类似的结果:扩增4/91类型毒株同样获得大约200bp和260bp左右两条带,扩增肾型毒株时,同样获得200bp和680bp左右两条带,扩增呼吸型毒株时,获得200bp左右条带。
下面结合实施例和附图作进一步说明。应该理解这些实施例仅用于说明目的,而不用于限制本发明的范围。
[实施例1]RNA抽提
1检测样品处理
组织样品的处理:采取病死鸡气管、肾脏等易分离IBV的组织,用剪刀剪碎后按照1:3体积比加入灭菌PBS充分研磨。将匀浆好的组织悬液于-20℃(或更低温度)-室温反复冻融3次后12,000g离心10min,取上清液200μl于新的灭菌离心管中。
拭子样品的处理:新鲜的咽或泄殖腔拭子加入1ml灭菌PBS,振荡搅匀之后,弃掉拭子(尽量将拭子中的液体收集完全)。浸出液12,000g离心10min,取上清液200μl于新的灭菌离心管中。
尿囊液处理:收集病毒感染的鸡胚尿囊液进行12,000g离心10min,取上清液200μl于新的灭菌离心管中。
2病毒RNA提取
上述处理的样品200μl加入800μl Trizol,剧烈震荡混匀,室温静置10-30min;加200μl氯仿,振荡混匀15s,室温静置10-15min;4℃,12000g离心15min;取水层,加等体积的异丙醇,混匀,室温静置10min;4℃,12000g离心10min;弃去上清,加1ml75%乙醇,4℃,7500g离心5min;弃去上清,沉淀干燥5-10min,加20μl DEPC水溶解,得总RNA。紫外分光光度计测定总RNA浓度后用于反转录反应。
[实施例2]反转录反应(RT反应)
取1μg上述总RNA加入1μl Random primer后,加DEPC水至12μl。65℃冰浴5min后,体系中再加入4μl 5×buffer、2μl 10mM dNTP、1μl RNase抑制剂、1μl M-MuLV,混匀,然后经25℃5min,42℃1h,70℃5min进行RT反应,最后获得反转录产物。反转录产物也经紫外分光光度计测定浓度。-20℃保存备用。
[实施例3]PCR反应
1.引物设计
选取GeneBank上收录的400条有代表性的IBV毒株的S1基因序列,进行比对分析。根据比对分析的结果,选择其保守序列,利用primer premier 5软件设计了一对IBV通用引物(通用-F和通用-R)以及4/91类型毒株特异性的鉴别引物(4/91-F和24/91-R);为了进一步鉴别呼吸型和肾型IBV,再选取GENBANK上收录的30株经典IBV毒株全序列(其中包括H120、M41、H52等呼吸型毒株,以及BJ、SC021202、GX-YL5等肾型毒株)进行比对,并根据S1基因序列绘制进化树。根据进化树的远近关系进行分型,选择肾型毒株nsp12基因的保守序列设计了一对肾型毒株的鉴别引物(肾型-F和肾型-R)。引物均由上海生工生物有限公司合成,引物信息如表1所示。
表1试剂盒分型引物信息
2.阳性标准品制备
IBV 4/91、SC021202(肾型)和M41(呼吸型)毒株接种9-11日龄SPF鸡胚,鸡胚感染后36-72h收获感染鸡胚尿囊液。按前述方法处理好鸡胚尿囊液后,从尿囊液中提取总RNA,反转录成cDNA。然后测定cDNA浓度,将每个毒株对应cDNA浓度调整一致,-20℃保存备用。
3.PCR反应体系优化
使用上述制备的IBV 4/91、SC021202(肾型)和M41(呼吸型)毒株的cDNA作为模板,分别对PCR程序中的引物浓度、退火温度、延伸时间、PCR循环次数等条件进行优化。
对反应体系和扩增程序优化的结果如下:
在50μLPCR反应体系中,ExTaq酶0.25μl,10mM的引物通用-F和通用-R、4/91-F和4/91-R、肾型-F和肾型-R各2μl,2.5mM的dNTP 4μl,10×buffer 5μl,模板2.5μl,加水至50μl;所述PCR反应体系的反应条件为:94℃预变性5min,94℃变性30s,54℃退火30s,72℃延伸50s,30个循环,72℃延伸10min;
利用优化后的体系对阳性标准品PCR扩增结果如图1所示,4/91类型毒株(4/91)扩增出200bp和260bp左右的两条带,肾型毒株(SC021202)扩增出200bp和680bp左右的两条带,而呼吸型毒株(M41)只能扩增出200bp左右的一条带(图1)。扩增结果与预期设计的相符。
4.PCR产物测序验证
分别将扩增得到的PCR产物进行回收,送测序公司测序分析验证扩增的序列与预期的理论序列完全一致,经BLAST分析也显示,扩增的序列为用作扩增模板的各IBV毒株的相应片段。具体的4/91毒株扩增片段序列为SEQ ID NO1所示,SC021202毒株扩增片段序列为SEQ ID NO2所示,M41毒株扩增片段序列为SEQ ID NO3所示。
[实施例4]试剂盒的特异性验证
选取常见禽类病毒包括禽流感病毒(AIV)H1N1和H9N2亚型、鸡新城疫病毒(NDV)、传染性法氏囊病毒(IBDV)、鸡痘病毒(FPV)等感染的鸡胚尿囊液或尿囊膜,抽提RNA,反转录成cDNA,用IBV分型检测试剂盒优化好的体系进行PCR检测。特异性验证结果如图2所示,用于阳性对照的4/91毒株、SC021202毒株和M41毒株的cDNA扩增到了特异性的目的条带,而对常见禽类病毒AIV(H1N1和H9N2亚型)、NDV、IBDV、FPV等不能扩出任何条带(图2)。
[实施例5]不同亚型毒株样品检测验证
将本实验室保存有的毒株包括4/91类型毒株(N株和X株)、肾型毒株(AH07091、BJ、Gray、HN99、R2、T、SD07011)和呼吸型毒株(ZJ971、ArK99、Ark Vaccine、H120、H52、Y98、28/86、SD0611、HK、Cal99、W93)用本发明的试剂盒检测,结果4/91类型毒株均扩增出200bp和260bp左右的DNA条带,肾型毒株扩增出200bp和680bp左右的条带,呼吸型毒株扩增出200bp左右的DNA条带(图3)。验证了IBV RT-PCR分型检测试剂盒的特异性和有效性。
Claims (2)
1.一种用于鸡传染性支气管炎病毒RT-PCR分型检测的试剂盒,其特征在于,该试剂盒包括下述三对引物:根据传染性支气管炎病毒的S1基因片段设计的通用引物和4/91类型毒株特异性引物,以及根据nsp12基因片段设计的肾型毒株特异性引物;
所述三对引物的序列如下:
通用-F:5'-CCAAAGTGCCTTCAGACC-3'
通用-R:5'-GCTAGACCAAGCCATACC-3'
4/91-F:5'-ATGTCTTGGTCAGTTTCA-3'
4/91-R:5'-AACAAGATCACCATTTA-3'
肾型-F:5'-GTGGTTTAGGTGTAGATGTA-3'
肾型-R:5'-TAAGAAATCATCAAGCAAAAGG-3'。
2.根据权利要求1所述的试剂盒,其特征在于,所述试剂盒包括下述成分:
(1)RNA提取试剂:Trizol试剂;
(2)反转录试剂:0.2µg/µl的Random primer,DEPC水,5×buffer,10mM的dNTP,20u/µl的Rnase抑制剂,200u/µl 的M-MuLV;
(3)PCR反应试剂:20u/µl的 ExTaq酶,2.5mM的 dNTP,10×buffer;
(4)分型引物:所述通用引物、4/91类型毒株特异性引物和肾型毒株特异性引物,引物浓度均为10mM;
(5)阳性对照样品:包括呼吸型M41毒株、4/91毒株和肾型毒株SC021202感染的鸡胚尿囊液抽提RNA的反转录产物cDNA;
(6)阴性对照样品:正常鸡胚尿囊液抽提RNA的反转录产物cDNA。
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