Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Primer probe combination and kit for detecting bovine respiratory diseases and application of primer probe combination and kit

A primer set and probe set technology, applied in the field of bovine respiratory virus detection, can solve the problems of time-consuming virus isolation and identification, low sensitivity, unfavorable diagnosis, etc., and achieve a wide range of sample application, good repeatability, and no potential biological safety hazard. Effects of ingredients

Pending Publication Date: 2022-06-17
GUANGDONG HAID ANIMAL HUSBANDRY & VETERINARY RES INST
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since it usually takes ten days for the body to produce antibodies after animal infection with the virus, antibody detection is not conducive to the diagnosis of acute infection and early infection of bovine infectious rhinotracheitis virus, bovine viral diarrhea virus and mycoplasma bovis; serum neutralization test , Virus isolation and identification ha

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Primer probe combination and kit for detecting bovine respiratory diseases and application of primer probe combination and kit
  • Primer probe combination and kit for detecting bovine respiratory diseases and application of primer probe combination and kit
  • Primer probe combination and kit for detecting bovine respiratory diseases and application of primer probe combination and kit

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0066]实施例1引物与探针组的设计与筛选

[0067]引物的性能决定了试剂盒检测效果的优劣。本实施例针对检测指标牛传染性鼻气管炎病毒、牛病毒性腹泻病毒和牛支原体筛选了多组引物 / 探针组合，最终筛选到一组最优的引物 / 探针组合。本实施例旨在说明引物 / 探针组合的设计及筛选过程。

[0068]引物和探针的设计：下载GenBank上已登录的具有高度特异性的牛传染性鼻气管炎病毒的gB基因(序列号：MF287966.1)、牛病毒性腹泻病毒的5’-UTR基因(序列号：AF091605.1)和牛支原体的P48基因(序列号：KX772800.1)，利用Primer Express5.0以及Oligo6.0设计软件，设计出扩增上述基因片段的特异性引物和荧光探针，并将设计得到的引物和探针进行BLAST验证，以保证引物的高特异性，经过验证，设计出如表1所示的9组引物和3组探针。

[0069]引物筛选：以牛传染性鼻气管炎病毒、牛病毒性腹泻病毒和牛支原体等病原提取的核酸的混合物的10倍梯度稀释的稀释物为模板，对所设计得到的牛传染性鼻气管炎病毒、牛病毒性腹泻病毒和牛支原体的3组引物分别进行单重荧光PCR反应，筛选出最佳的引物探针组合。单重荧光PCR的初始反应体系为：TaqDNA聚合酶(5U / μL)0.4μL，逆转录酶(5U / μL)0.4μL，2×PCR Buffer10μL，上下游引物(10μM)各0.4μL，探针(10μM)0.2μL，无核酶水6.2μL，模板2μL。反应程序：逆转录42℃5min；预变性94℃30s；变性94℃5s，退火60℃30s，40个循环。由表2可以看出，根据敏感性和扩增效率最终筛选出牛传染性鼻气管炎病毒、牛病毒性腹泻病毒和牛支原体的最佳引物组分别为：IBRV-F1(SEQ ID NO.1)、IBRV-R1(SEQ IDNO.2)，BVDV-F1(SEQ ID NO.8)、BVDV-R1(SEQ ID NO.9)，Mb-F2(SEQ ID NO.17)、Mb-R2(SEQID NO.18)，即将上述三组引物作为本发明后续多重PCR反应的引物组。

[0070]表1引物和探针序列

[0071]

[0072]

[0073]表2引物筛选结果

[0074]

[0075]

[0076]注：0代表无Ct值。

Example Embodiment

[0077]实施例2阳性参考品制备

[0078]分别以牛传染性鼻气管炎病毒的cDNA、牛病毒性腹泻病毒和牛支原体的DNA为模板，使用实施例1筛选出的最佳引物组进行普通PCR扩增，电泳后分别获得117bp、105bp和132bp的目的片段。PCR产物使用琼脂糖凝胶DNA回收试剂盒(天根生化科技有限公司)回收并纯化，将纯化后的DNA片段克隆至pMD 19-T Vector(Takara)，继而转化到DH5α(上海唯地生物技术有限公司)，选取阳性克隆进行测序验证，验证合格后进一步扩增培养。分别将阳性菌液使用质粒小量提取试剂盒(天根生化科技有限公司)提取重组质粒。使用核酸蛋白测定仪测定其蛋白浓度，并根据公式[6.02×1023(拷贝 / moL)]×[浓度(g / μL)] / [MW(g / moL)]＝拷贝数(copies / μL)计算出拷贝数，将三种重组质粒的浓度调整至1×106拷贝 / μL，然后将三种重组质粒按1:1:1进行混合即为阳性参考品。

Example Embodiment

[0079]实施例3三重荧光PCR检测方法建立并优化

[0080]以3倍稀释的阳性参考品(由实施例2制得)为模板，分别进行牛传染性鼻气管炎病毒、牛病毒性腹泻病毒和牛支原体单重荧光PCR引物(上下引物浓度为1:1)和探针浓度梯度试验，根据扩增效率和敏感性分别确定牛传染性鼻气管炎病毒、牛病毒性腹泻病毒和牛支原体引物 / 探针浓度范围(得到引物终浓度范围为0.2～0.6μM，探针终浓度范围为0.1～0.3μM)；然后根据确定的引物浓度范围选择牛传染性鼻气管炎病毒、牛病毒性腹泻病毒和牛支原体引物的三个浓度，先后设计8组引物浓度配比组合(表3)，在保证扩增效率的基础上，考虑试剂成本后筛选出引物浓度的最佳配比组合。使用同样的方法筛选出探针浓度的最佳配比组合。引物和探针浓度的最佳配比组合确定后，对退火温度(52～62℃)(表5)进行进一步的优化，最终确定三重荧光PCR的反应体系和反应程序。由表3～表5可以得出，牛传染性鼻气管炎病毒、牛病毒性腹泻病毒和牛支原体的最佳引物组合为IBRV+BVDV+Mb＝0.3μM+0.4μM+0.3μM，最佳探针组合为IBRV+BVDV+Mb＝0.15μM+0.2μM+0.15μM，最佳退火温度为52℃，因此，三重荧光PCR的最终反应体系见表6，反应程序具体为：反转录42℃5min；预变性94℃30s；变性94℃5s，退火55℃30s，40个循环数。使用最佳反应体系和反应程序扩增阳性参考品和阴性参考品(无核酶水)，其扩增曲线如图1所示，阳性参考品有扩增曲线，而阴性参考品无扩增曲线，表明该反应体系和反应程序对牛传染性鼻气管炎病毒、牛病毒性腹泻病毒和牛支原体的混合样品均有良好的扩增效果。

[0081]表3引物浓度组合优化结果

[0082]

[0083]表4探针浓度组合优化结果

[0084]

[0085]表5退火温度优化结果

[0086]

[0087]

[0088]表6三重荧光PCR检测反应体系

[0089]

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention belongs to the technical field of bovine respiratory pathogen detection, discloses a primer probe combination and a kit for detecting bovine respiratory diseases and application of the primer probe combination and the kit, and particularly discloses a reagent which comprises a primer group and/or a probe group. The reagent provided by the invention can be used for simultaneously detecting the infectious bovine rhinotracheitis virus, the bovine viral diarrhea virus and the mycoplasma bovis, and the pathogens of three bovine respiratory diseases can be detected by only carrying out PCR amplification once in one-tube reaction.

Description

technical field [0001] The invention belongs to the technical field of bovine respiratory virus detection, and in particular relates to a primer-probe combination, a kit and an application thereof for detecting bovine respiratory diseases. Background technique [0002] Infectious bovine rhinotracheitis virus (IBRV), also known as bovine herpesvirus type I (BHV-1), is a DNA virus. IBRV-infected cattle often show respiratory symptoms accompanied by clinical symptoms such as abortion, decreased milk production, metritis, infectious vulvaginitis and enteritis. Bovine viral diarrhea virus (Bovine viral diarrhea virus, BVDV) is a single-stranded positive-sense, enveloped RNA virus. Infected cattle will show acute infection such as pneumonia, diarrhea, abortion, persistent infection and mucosal disease. Mycoplasma bovis (Mycoplasma bovis, M.bovis) belongs to Mycoplasma family, Mycoplasma genus, no cell wall, double-stranded DNA and ribosomes exist in the cytoplasm. Mycoplasma bov...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12Q1/70C12N15/11C12Q1/686
CPCC12Q1/705C12Q1/686C12Q2600/16C12Q2537/143C12Q2563/107
Inventor 陶攀贾爱卿王贵平东笑
Owner GUANGDONG HAID ANIMAL HUSBANDRY & VETERINARY RES INST
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com