CN112941209A - 一种检测鸡感染肠炎沙门氏菌的分子标记及方法和应用 - Google Patents

一种检测鸡感染肠炎沙门氏菌的分子标记及方法和应用 Download PDF

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CN112941209A
CN112941209A CN202110166622.1A CN202110166622A CN112941209A CN 112941209 A CN112941209 A CN 112941209A CN 202110166622 A CN202110166622 A CN 202110166622A CN 112941209 A CN112941209 A CN 112941209A
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salmonella enteritidis
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李显耀
胡耿
董亚宁
刘丽英
樊新忠
唐辉
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Shandong Agricultural University
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Abstract

本发明公开了一种检测鸡感染肠炎沙门氏菌的分子标记及方法和应用，该分子标记是鸡gga‑miR‑146b‑5p的异构体isomir‑1，核苷酸序列如SEQ ID NO.1所示。通过相应引物和反应条件，检测到鸡盲肠组织isomir‑1的表达量与肠炎沙门氏菌感染显著相关。本发明通过检测鸡盲肠组织gga‑miR‑146b‑5p异构体isomir‑1的表达水平，以确定其是否具有潜在抗肠炎沙门氏菌感染的能力，是一种新型的抗病育种分子标记方法。

Description

一种检测鸡感染肠炎沙门氏菌的分子标记及方法和应用
技术领域
本发明属于生物基因工程技术领域，涉及一种分子标记及检测方法和在鸡抗肠炎沙门氏菌感染中的应用，具体为利用本发明方法可以检测鸡gga-miR-146b-5p异构体的表达水平，进而用于沙门氏菌感染抗性鸡只的筛选。
背景技术
肠炎沙门氏菌(Salmonella enteritidis)为革兰氏阴性菌，在家禽体内普遍存在，一般不表现感染症状，但会降低家禽生产性能，严重时大量致死。肠炎沙门氏菌是一种主要的食源性致病菌，给人类健康带来巨大安全隐患。
microRNA(miRNA)是一类短小、单链、非编码RNA，长度大约20～24nt，广泛存在于动物、植物、病毒体内，能够调控基因的转录后表达水平。miRNA异构体(isomiR)是指在注释序列的3’端、5’端或中间位置发生微小变异的序列，在生长发育和疾病发生过程中具有潜在调控作用。由于isomiR与其注释体通常只有1～2个碱基的差异，目前常规检测miRNA的加尾法和茎环法都不能很好地分辨这种差异，为isomiR的检测造成很大困难。
gga-miR-146b-5p存在异构体，通过本研究构建了其异构体表达的检测方法，并在鸡感染肠炎沙门氏菌后，其中一个异构体isomir-1的表达量与鸡感染肠炎沙门氏菌显著相关，可用于鸡沙门氏菌感染抗性的分子标记。
发明内容
本发明的目的在于提供一种用于检测鸡gga-miR-146b-5p异构体isomir-1表达的方法，并将该方法应用于鸡肠炎沙门氏菌感染抗性的个体筛选，在家禽抗病育种领域具有潜在应用价值。
为了达到上述技术目的，本发明具体通过以下技术方案实现：
一种检测鸡感染肠炎沙门氏菌的分子标记，该分子标记为鸡盲肠组织gga-miR-146b-5p的异构体isomir-1，其核苷酸序列如SEQ ID NO.1所示。
所述gga-miR-146b-5p的核苷酸序列如SEQ ID NO.2所示。
在本发明的另一方面，提供了一种检测鸡感染肠炎沙门氏菌的方法，包括以下步骤：
1)提取鸡盲肠组织总RNA，进行反转录；
2)以反转录的DNA为模板，分别利用检测引物和内参引物进行PCR扩增；
3)经内参校正后用2-△△Ct方法计算待检鸡和阴性鸡isomir-1的差异倍数，待检鸡isomir-1的表达量超过阴性鸡2倍以上判定为肠炎沙门氏菌阳性鸡。
进一步的，反转录引物核苷酸序列如SEQ ID NO.3所示。
进一步的，检测引物如SEQ ID NO.4～5所示，内参引物如SEQ ID NO.6～7所示。
进一步的，实时荧光定量PCR反应体系为：2×ChamQ Universal SYBR qPCRMaster Mix 10.0μl，上下游引物各0.4μl，Template DNA 2.0μl，ddH2O 7.2μl。
进一步的，实时荧光定量PCR反应程序为：95℃预变性30sec；95℃10sec，60℃30sec，40个循环；95℃10sec，65℃60sec，97℃1sec。
在本发明的另一方面，提供一种检测鸡盲肠组织gga-miR-146b-5p异构体isomir-1的引物，包括引物SEQ ID NO.4～5。
在本发明的另一方面，提供了上述分子标记或引物组在检测鸡抗肠炎沙门氏菌感染中的应用。通过实时荧光定量PCR，待检鸡isomir-1的表达量超过阴性鸡2倍以上判定为肠炎沙门氏菌阳性鸡。
本发明的有益效果为：
本发明开发出的gga-miR-146b-5p异构体isomir-1表达的检测方法能够有效检测出异构体isomir-1的表达，其发表量与鸡对肠炎沙门氏菌感染显著相关相关，是肠炎沙门氏菌感染抗性的分子标记。通过检测鸡盲肠组织gga-miR-146b-5p异构体isomir-1的表达水平，可以筛选肠炎沙门氏菌感染抗性的个体。
附图说明
图1是本发明isomir-1和U6特异性PCR产物。
具体实施方式
下面将结合本发明具体的实施例，对本发明技术方案进行清楚、完整地描述，显然，所描述的实施例仅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。
实施例1
本实施例提供一种通过检测鸡盲肠组织内特异性gga-miR-146b-5p异构体isomir-1的表达水平以确定鸡是否感染肠炎沙门氏菌的分子标记方法，在家禽抗病育种领域具有潜在应用价值。具体内容如下：
1)RNA提取及反转录
采用Trizol法从鸡盲肠组织提取总RNA，利用表1的茎环引物及表2、表3的试剂配制方法和反应条件进行反转录。该茎环引物能特异性识别isomir-1的3’末端6个碱基(内参选用细胞内稳定表达的U6小RNA)。经验证，采用所述引物，isomir-1和U6能够分别特异性扩增出63nt和57nt的目的PCR产物(图1)，可进行后续定量工作。
表1检测gga-miR-146b-5p异构体isomir-1的相关引物
Figure BDA0002934776400000041
表2反转录反应混合液
Figure BDA0002934776400000051
表3反转录反应条件
Figure BDA0002934776400000052
2)定量PCR
将上述反转录后的DNA作为模板，采用表1中的上下游引物进行定量PCR，反应混合液及反应条件分别见表4、表5，定量PCR仪为Roche
Figure BDA0002934776400000055
96。
表4 qPCR反应混合液
Figure BDA0002934776400000053
表5 qPCR反应条件
Figure BDA0002934776400000054
3)isomir-1表达量的计算及肠炎沙门氏菌阳性鸡的判定
根据qPCR检测到的待检鸡和肠炎沙门氏菌阴性鸡isomir-1的Ct值，用内参校正后用2-△△Ct方法计算两者isomir-1的差异倍数，待检鸡isomir-1的表达量超过阴性鸡2倍以上判定为肠炎沙门氏菌阳性鸡。
实施例2
本实施例通过测序方法对上述gga-miR-146b-5p异构体isomir-1的检测方法进行应用，包括以下步骤：
1)将20只肠炎沙门氏菌阴性鸡平均分成试验组和对照组，试验组每只鸡灌服0.3ml肠炎沙门氏菌菌液(108cfu/ml)，对照组灌服等体积PBS，随后将两组鸡饲养在独立的隔离器中，自由采食和饮水，避免发生交叉感染。
2)接种后第3天，从试验组和对照组各随机取10只鸡，安乐致死后取盲肠组织和盲肠内容物。将盲肠组织置液氮保存，盲肠内容物置0～4℃低温保存。
3)通过测定盲肠内容物样本中肠炎沙门氏菌的含量，确认人工感染模型构建成功；然后从对照组和试验组分别选取一定数量盲肠组织样本送测序公司进行转录组测序。
4)获得测序数据后，经质控、拼接、比对，筛选出gga-miR-146b-5p的异构体isomir-1(表6)，其基础表达量为2011±794.9(TPM校正值)、肠炎沙门氏菌阳性鸡与阴性鸡的比值为2.39(P＝3.55E-05)，表明gga-miR-146b-5p的异构体isomir-1与肠炎沙门氏菌感染显著相关。
表6 gga-miR-146b-5p异构体isomir-1的测序结果
Figure BDA0002934776400000061
5)采用本发明内容所述的检测方法，经RT-qPCR获得荧光定量结果(表7)，肠炎沙门氏菌阳性鸡与阴性鸡的比值为2.46，与测序结果2.39非常接近，表明本发明的检测方法可以准确检测并定量gga-miR-146b-5p的异构体isomir-1，在家禽抗病育种中具有潜在应用价值。
表7 gga-miR-146b-5p异构体isomir-1的实际定量结果
Figure BDA0002934776400000071
尽管已经示出和描述了本发明的实施例，对于本领域的普通技术人员而言，可以理解在不脱离本发明的原理和精神的情况下可以对这些实例进行多种变化、修改、替换和变型，本发明的范围由所附权利要求及其等同物限定。
序列表
<110> 山东农业大学
<120> 一种检测鸡感染肠炎沙门氏菌的分子标记及方法和应用
<160> 7
<170> SIPOSequenceListing 1.0
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<213> 异构体(isomir-1)
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<210> 2
<211> 22
<212> RNA
<213> 鸡盲肠组织(gga-miR-146b-5p)
<400> 2
ugagaacuga auuccauagg cg 22
<210> 3
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
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gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgacaacgcc 50
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<213> 人工序列(Artificial Sequence)
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cgcgtgagaa ctgaattcca 20
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<213> 人工序列(Artificial Sequence)
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agtgcagggt ccgaggtatt 20
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<213> 人工序列(Artificial Sequence)
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Claims (8)

1.一种检测鸡感染肠炎沙门氏菌的分子标记，其特征在于，其核苷酸序列如SEQ IDNO.1所示。
2.一种检测鸡感染肠炎沙门氏菌的方法，其特征在于，包括以下步骤：
1)提取鸡盲肠组织总RNA，进行反转录；
2)以反转录的DNA为模板，分别利用检测引物和内参引物进行PCR扩增；
3)经内参校正后用
Figure FDA0002934776390000011
方法计算待检鸡和阴性鸡isomir-1的差异倍数，待检鸡isomir-1的表达量超过阴性鸡2倍以上判定为肠炎沙门氏菌阳性鸡。
3.根据权利要求2所述的一种检测鸡感染肠炎沙门氏菌的方法，其特征在于，反转录引物核苷酸序列如SEQ ID NO.3所示。
4.根据权利要求2所述的一种检测鸡感染肠炎沙门氏菌的方法，其特征在于，检测引物如SEQ ID NO.4～5所示，内参引物如SEQ ID NO.6～7所示。
5.根据权利要求2所述的一种检测鸡感染肠炎沙门氏菌的方法，其特征在于，实时荧光定量PCR反应体系为：2×ChamQ Universal SYBR qPCR Master Mix 10.0μl，上下游引物各0.4μl，Template DNA 2.0μl，ddH2O 7.2μl。
6.根据权利要求2所述的一种检测鸡感染肠炎沙门氏菌的方法，其特征在于，实时荧光定量PCR反应程序为：95℃预变性30sec；95℃10sec，60℃30sec，40个循环；95℃10sec，65℃60sec，97℃1sec。
7.一种检测鸡盲肠组织gga-miR-146b-5p异构体isomir-1的引物，其特征在于，如SEQID NO.4～5所示的引物对。
8.权利要求1所述分子标记或权利要求7所述引物和方法在鸡肠炎沙门氏菌感染抗性育种中的应用。
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