CN110373478A - 数字PCR检测长牡蛎多巴胺-β-羟化酶表达水平的引物及探针 - Google Patents
数字PCR检测长牡蛎多巴胺-β-羟化酶表达水平的引物及探针 Download PDFInfo
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Abstract
本发明公开一种数字PCR检测长牡蛎多巴胺‑β‑羟化酶表达水平的引物及探针，所述引物及探针核苷酸序列如下：正向引物：5’‑tggcggaccactctctacatc‑3’；反向引物：5’‑tctccgcgacatcatctgaat‑3’；探针：5’‑cttcttccagctagcgaacgcaacaca‑3’；所述探针添加的荧光基团为6‑羧基荧光素。引物及探针的准确性和特异性高，故可利用数字PCR技术检测长牡蛎血淋巴细胞中NE合成酶CgDBH绝对量的时序变化，以此作为评价养殖长牡蛎健康状况的指标，所建立的评价体系在养殖贝类病害预警预报中具有潜在应用价值。不需要设置对照组，也无需内参基因（Internal control），突破了传统荧光实时定量PCR检测方法的应用局限。
Description
技术领域
本发明属于分子生物学和生物化学技术领域，涉及一种数字PCR检测长牡蛎多巴胺-β-羟化酶表达水平的引物及探针。
背景技术
去甲肾上腺素(Norepinephrine, NE)属于儿茶酚胺类(Catecholamines, CAs)神经递质，是神经内分泌免疫系统中的重要调节分子。NE通过结合免疫细胞胞膜上的肾上腺素能受体（Adrenoceptor，ADR）来调节其免疫应答能力，同时，NE在不同的免疫内环境中结合不同类型的ADRs，产生不同的免疫调节效应。例如，在某些免疫细胞中α2 型肾上腺素能受体能通过 PKC 的活化、IκB 的磷酸化和 NF-κB 的激活，促进 TNF-α、IL-1、IL-6 和 IL-10 的产生和释放；β 型肾上腺素能受体（主要是 β2）通过 cAMP-PKA 通路，来抑制 NF-κB对免疫相关基因的转录。当α2 型肾上腺素能受体和β型肾上腺素能受体都被激活时，β型肾上腺素能受体的作用占优势。
近期的研究发现，贝类是具有完善神经内分泌免疫调节系统的最低等动物，NE在贝类免疫应答中发挥了至关重要的作用。鳗弧菌刺激后，栉孔扇贝血淋巴中NE的浓度显著上升，同时血淋巴细胞中NE合成酶（多巴胺-β-羟化酶，CgDBH）、CfMAO和 CfADR的 mRNA表达水平也显著升高。CfMAO被干扰后，血淋巴细胞中超氧化物歧化酶(CfSOD)的 mRNA 表达水平显著低于对照组。此外，研究发现LPS刺激能够诱导长牡蛎血淋巴细胞合成NE，进而调节血细胞的吞噬活性和凋亡指数。
荧光实时定量PCR（Quantitative real-time PCR，简称qPCR）是用于检测基因mRNA表达水平的经典方法。但此方法只能用于检测基因的相对表达水平，需要设置对照组并选取合适的管家基因作为内参，因此qPCR技术的应用具有很大局限性。相对地，数字PCR是一种核酸分子绝对定量技术，能够直接检出DNA分子的个数，是对起始样品的绝对定量，符合野外调查的需要。但是，迄今为止，没有关于数字PCR检测长牡蛎多巴胺-β-羟化酶表达水平的引物及探针的相关报道，以至于并没有基于数字PCR检测长牡蛎多巴胺-β-羟化酶表达水平的时序变化并以此作为评价养殖长牡蛎健康状况的指标。
发明内容
本发明是为了解决现有技术所存在的上述技术问题，提供一种数字PCR检测长牡蛎多巴胺-β-羟化酶表达水平的引物及探针。
本发明的技术解决方案是：一种数字PCR检测长牡蛎多巴胺-β-羟化酶表达水平的引物及探针，其特征在于所述引物及探针核苷酸序列如下：
正向引物：5’- TGGCGGACCACTCTCTACATC-3’；
反向引物：5’- TCTCCGCGACATCATCTGAAT-3’；
探针：5’- CTTCTTCCAGCTAGCGAACGCAACACA-3’；
所述探针添加的荧光基团为6-羧基荧光素。
本发明设计的数字PCR检测长牡蛎多巴胺-β-羟化酶表达水平的引物及探针的准确性和特异性高，故可利用数字PCR技术检测长牡蛎血淋巴细胞中NE合成酶CgDBH绝对量的时序变化，以此作为评价养殖长牡蛎健康状况的指标，所建立的评价体系在养殖贝类病害预警预报中具有潜在应用价值。不需要设置对照组，也无需内参基因（Internal control），突破了传统荧光实时定量PCR检测方法的应用局限。
附图说明
图1为本发明实施例中长牡蛎CgDBH的qPCR扩增曲线和反应程序图。
图2为本发明实施例中长牡蛎CgDBH的数字PCR反应的质量控制图。
图3本发明实施例不同时期养殖长牡蛎血淋巴细胞中CgDBH基因的绝对表达量图。
具体实施方式
1. CgDBH序列筛查
从长牡蛎基因组中鉴定得到NE合成酶DBH（CgDBH, CGI_10027734）的全长序列，查找其ORF。
2. CgDBH的数字PCR引物和探针设计
利用ThermoFisher公司的Primer Express V3.0软件根据CgDBH的ORF序列设计数字PCR引物和探针。引物及探针核苷酸序列如下：
正向引物：5’- TGGCGGACCACTCTCTACATC-3’；
反向引物：5’- TCTCCGCGACATCATCTGAAT-3’；
探针：5’- CTTCTTCCAGCTAGCGAACGCAACACA-3’；
数字PCR的正向引物长21 bp，GC含量57%，Tm值58.8°C；反向引物长21 bp，GC含量48.0%，Tm值59.2°C；探针长27 bp，GC含量52%，Tm值68.8°C，制备方式为采用HPLC制备，探针添加的荧光基团为6-羧基荧光素（6-FAM）。
实验例1：长牡蛎血淋巴细胞中CgDBH基因绝对表达量的检测
1. 样品收集
用牡蛎刀撬开长牡蛎外壳之后，以10 mL注射器刺破血窦并抽取血淋巴；500目筛绢过滤后加入1.5 mL EP管中，800 g离心10 min，收集血淋巴细胞。
2. 总RNA提取及cDNA文库构建
向每个盛有血淋巴细胞的EP管中加入1 mL Trizol reagent。向每1 mL样品加入200 μL预冷的氯仿（-20℃保存），剧烈震荡3 min，4℃，12,000 g离心15 min。小心吸取上层透明液体400 μL，加入相同体积的预冷异丙醇（-20℃保存），充分混匀后置于-80℃超低温冰箱中自然沉降。10 min后，取出样品，4℃，12,000 g离心15 min。弃去管中液体，加入1 mL 75%无菌乙醇；用手指轻弹起管底沉淀，4℃，12,000 g离心5 min。弃去管中液体，4℃，12,000 g离心30 s，用1 mL注射器小心吸干管底残留液体。开盖静置2~3 min，使乙醇充分挥发干净。每管加入20 μL DEPC水溶解RNA。取微量样品进行核酸电泳以检测RNA质量。 用DNA酶消化混于RNA中的DNA，采用两步法合成cDNA文库第一条链。
3. qPCR法验证数字PCR引物和探针的有效性
利用本发明实施例的数字PCR引物，按照如下反应体系（14.5 μL）进行实验：
qPCR反应程序：50℃ 2 min；95℃ 10 min，95℃ 15，50个循环；60℃ 1 min。
采用7500分析软件，得到Ct值。利用2-ΔΔCt算法得到基因的相对表达水平值。用Excel和SPSS等软件进行统计学分析。
实验结果如图1所示。图1中A为长牡蛎CgDBH的qPCR扩增曲线，B为反应程序图。结果表明：反应进行到约第28轮时检测到FAM基团的荧光信号，且反应进行至约第45轮时达到平台期。该结果说明所设计的用于检测CgDBH绝对表达量的数字PCR引物和探针有效，符合后续实验要求。
实验例2：数字PCR法检测CgDBH基因的绝对表达水平
利用本发明实施例的数字PCR引物及探针，按照如下反应体系（14.5 μL）进行实验：
借助ProFlex™ 2x Flat PCR System (or Dual Flat Block GeneAmp™ PCR System9700)系统，按照如下反应条件：96℃ 10 min（变性），60℃ 2 min（退火并延伸），98℃ 30s，39个循环；60℃ 2 min，10℃ ∞，检测长牡蛎血淋巴细胞中CgDBH基因的绝对拷贝数。
使用QuantStudio™ 3D Digital PCR Instrument系统读取数据，采用QuantStudio™ 3D AnalysisSuite™ Software进行数据分析。利用Excel和SPSS等软件对数据进行统计学分析。
通过对原始数据进行质控分析，结果如图2所示。发现只能检测到特异性的FAM信号的单峰，且FAM阳性信号和ROX阴性对照信号能明显分为两群。结果表明本发明用于检测CgDBH绝对表达量的数字PCR引物和探针具有高度特异性。
实验例3：不同时期养殖长牡蛎血淋巴细胞中CgDBH基因的绝对表达量
应用本发明的数字PCR引物和探针对2019年5月至7月间的长牡蛎血淋巴细胞中CgDBH的 mRNA表达水平进行检测，结果如图3所示。其中，5月份长牡蛎血淋巴细胞中CgDBH的绝对量为12.618个拷贝/微升，而7月份时长牡蛎血淋巴细胞中CgDBH的绝对量为9.545个拷贝/微升，显著低于5月份的水平(p < 0.05) ，出现明显差异。该结果说明利用本发明实施例的引物及探针实施数字PCR，能有效检测养殖长牡蛎体内CgDBH基因的绝对表达水平，从而间接反映养殖长牡蛎的免疫防御能力，相关研究结果在养殖长牡蛎病害预警预报中具有潜在应用价值。
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<212> DNA
<213> 人工序列(Artificial)
<220>
<221> misc_feature
<222> (1)..(21)
<400> 2
tctccgcgac atcatctgaa t 21
<210> 3
<211> 27
<212> DNA
<213> 人工序列(Artificial)
<220>
<221> misc_feature
<222> (1)..(27)
<400> 3
cttcttccag ctagcgaacg caacaca 27
Claims (1)
1.一种数字PCR检测长牡蛎多巴胺-β-羟化酶表达水平的引物及探针，其特征在于所述引物及探针核苷酸序列如下：
正向引物：5’- TGGCGGACCACTCTCTACATC-3’；
反向引物：5’- TCTCCGCGACATCATCTGAAT-3’；
探针：5’- CTTCTTCCAGCTAGCGAACGCAACACA-3’；
所述探针添加的荧光基团为6-羧基荧光素。
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104073557A (zh) * | 2014-06-11 | 2014-10-01 | 中国科学院海洋研究所 | 长牡蛎Poly(I:C)应激实验荧光定量的内参基因及其引物和应用 |
| CN107488705A (zh) * | 2016-06-12 | 2017-12-19 | 中国检验检疫科学研究院 | 用于水貂源性成分数字pcr精准定量检测的引物探针及方法和试剂盒 |
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2019
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Patent Citations (2)
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| CN104073557A (zh) * | 2014-06-11 | 2014-10-01 | 中国科学院海洋研究所 | 长牡蛎Poly(I:C)应激实验荧光定量的内参基因及其引物和应用 |
| CN107488705A (zh) * | 2016-06-12 | 2017-12-19 | 中国检验检疫科学研究院 | 用于水貂源性成分数字pcr精准定量检测的引物探针及方法和试剂盒 |
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