A Label-Based Polymer Nanoparticles Biosensor Combined with Loop-Mediated Isothermal Amplification for Rapid, Sensitive, and Highly Specific Identification of Brucella abortus

Brucella abortus (B. abortus), an important zoonotic pathogen in Brucella spp., is the major causative agent of abortion in cattle (namely, bovine brucellosis). Currently, although the isolation and identification of the Brucella abortus were commonly accepted as the gold standard method, it cannot...

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Autores principales:	Xinggui Yang, Yue Wang, Ying Liu, Junfei Huang, Qinqin Tan, Xia Ying, Yong Hu, Shijun Li
Formato:	article
Lenguaje:	EN
Publicado:	Frontiers Media S.A. 2021
Materias:	Brucella abortus nanoparticles biosensor loop-mediated isothermal amplification BruAb2_0168 gene blood Biotechnology TP248.13-248.65
Acceso en línea:	https://doaj.org/article/1f5d20f6f0554d1dbd54eccdb0ad063f
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spelling	oai:doaj.org-article:1f5d20f6f0554d1dbd54eccdb0ad063f2021-11-18T05:58:27ZA Label-Based Polymer Nanoparticles Biosensor Combined with Loop-Mediated Isothermal Amplification for Rapid, Sensitive, and Highly Specific Identification of Brucella abortus2296-418510.3389/fbioe.2021.758564https://doaj.org/article/1f5d20f6f0554d1dbd54eccdb0ad063f2021-11-01T00:00:00Zhttps://www.frontiersin.org/articles/10.3389/fbioe.2021.758564/fullhttps://doaj.org/toc/2296-4185Brucella abortus (B. abortus), an important zoonotic pathogen in Brucella spp., is the major causative agent of abortion in cattle (namely, bovine brucellosis). Currently, although the isolation and identification of the Brucella abortus were commonly accepted as the gold standard method, it cannot meet the requirements for early diagnostic strategies. Conventional PCR techniques and immunological tests can realize rapid detection of B. abortus, but the demands for PCR thermal cyclers and/or specific antibodies hinder their application in basic laboratories. Thus, rapid, sensitive, and specific diagnostic strategies are essential to prevent and control the spread of the bovine brucellosis. In this work, a novel detection method for the rapid identification of B. abortus, which uses loop-mediated isothermal amplification (LAMP) combined with a label-based polymer nanoparticles lateral flow immunoassay biosensor (LFIA), was established. One set of specific B. abortus-LAMP primers targeting the BruAb2_0168 gene was designed by the online LAMP primer design tool. The B. abortus-LAMP-LFIA assay was optimized and evaluated using various pathogens and whole blood samples. The optimal amplification temperature and time for B. abortus-LAMP-LFIA were determined to be 65°C and 50 min, respectively. The B. abortus-LAMP-LFIA method limit of detection (LoD) was 100 fg per reaction for pure genomic DNA of B. abortus. Meanwhile, the detection specificity was 100%, and there was no cross-reactivity for other Brucella members and non-Brucella strains. Furthermore, the entire procedure, including the DNA preparation for whole blood samples (30 min), isothermal incubation (50 min), and LFIA detection (2–5 min), can be completed in approximately 85 min. Thus, the B. abortus-LAMP-LFIA assay developed was a simple, rapid, sensitive, and reliable detection technique, which can be used as a screening and/or diagnostic tool for B. abortus in the field and basic laboratories.Xinggui YangXinggui YangYue WangYing LiuJunfei HuangQinqin TanQinqin TanXia YingXia YingYong HuShijun LiShijun LiFrontiers Media S.A.articleBrucella abortusnanoparticles biosensorloop-mediated isothermal amplificationBruAb2_0168 genebloodBiotechnologyTP248.13-248.65ENFrontiers in Bioengineering and Biotechnology, Vol 9 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Brucella abortus nanoparticles biosensor loop-mediated isothermal amplification BruAb2_0168 gene blood Biotechnology TP248.13-248.65
spellingShingle	Brucella abortus nanoparticles biosensor loop-mediated isothermal amplification BruAb2_0168 gene blood Biotechnology TP248.13-248.65 Xinggui Yang Xinggui Yang Yue Wang Ying Liu Junfei Huang Qinqin Tan Qinqin Tan Xia Ying Xia Ying Yong Hu Shijun Li Shijun Li A Label-Based Polymer Nanoparticles Biosensor Combined with Loop-Mediated Isothermal Amplification for Rapid, Sensitive, and Highly Specific Identification of Brucella abortus
description	Brucella abortus (B. abortus), an important zoonotic pathogen in Brucella spp., is the major causative agent of abortion in cattle (namely, bovine brucellosis). Currently, although the isolation and identification of the Brucella abortus were commonly accepted as the gold standard method, it cannot meet the requirements for early diagnostic strategies. Conventional PCR techniques and immunological tests can realize rapid detection of B. abortus, but the demands for PCR thermal cyclers and/or specific antibodies hinder their application in basic laboratories. Thus, rapid, sensitive, and specific diagnostic strategies are essential to prevent and control the spread of the bovine brucellosis. In this work, a novel detection method for the rapid identification of B. abortus, which uses loop-mediated isothermal amplification (LAMP) combined with a label-based polymer nanoparticles lateral flow immunoassay biosensor (LFIA), was established. One set of specific B. abortus-LAMP primers targeting the BruAb2_0168 gene was designed by the online LAMP primer design tool. The B. abortus-LAMP-LFIA assay was optimized and evaluated using various pathogens and whole blood samples. The optimal amplification temperature and time for B. abortus-LAMP-LFIA were determined to be 65°C and 50 min, respectively. The B. abortus-LAMP-LFIA method limit of detection (LoD) was 100 fg per reaction for pure genomic DNA of B. abortus. Meanwhile, the detection specificity was 100%, and there was no cross-reactivity for other Brucella members and non-Brucella strains. Furthermore, the entire procedure, including the DNA preparation for whole blood samples (30 min), isothermal incubation (50 min), and LFIA detection (2–5 min), can be completed in approximately 85 min. Thus, the B. abortus-LAMP-LFIA assay developed was a simple, rapid, sensitive, and reliable detection technique, which can be used as a screening and/or diagnostic tool for B. abortus in the field and basic laboratories.
format	article
author	Xinggui Yang Xinggui Yang Yue Wang Ying Liu Junfei Huang Qinqin Tan Qinqin Tan Xia Ying Xia Ying Yong Hu Shijun Li Shijun Li
author_facet	Xinggui Yang Xinggui Yang Yue Wang Ying Liu Junfei Huang Qinqin Tan Qinqin Tan Xia Ying Xia Ying Yong Hu Shijun Li Shijun Li
author_sort	Xinggui Yang
title	A Label-Based Polymer Nanoparticles Biosensor Combined with Loop-Mediated Isothermal Amplification for Rapid, Sensitive, and Highly Specific Identification of Brucella abortus
title_short	A Label-Based Polymer Nanoparticles Biosensor Combined with Loop-Mediated Isothermal Amplification for Rapid, Sensitive, and Highly Specific Identification of Brucella abortus
title_full	A Label-Based Polymer Nanoparticles Biosensor Combined with Loop-Mediated Isothermal Amplification for Rapid, Sensitive, and Highly Specific Identification of Brucella abortus
title_fullStr	A Label-Based Polymer Nanoparticles Biosensor Combined with Loop-Mediated Isothermal Amplification for Rapid, Sensitive, and Highly Specific Identification of Brucella abortus
title_full_unstemmed	A Label-Based Polymer Nanoparticles Biosensor Combined with Loop-Mediated Isothermal Amplification for Rapid, Sensitive, and Highly Specific Identification of Brucella abortus
title_sort	label-based polymer nanoparticles biosensor combined with loop-mediated isothermal amplification for rapid, sensitive, and highly specific identification of brucella abortus
publisher	Frontiers Media S.A.
publishDate	2021
url	https://doaj.org/article/1f5d20f6f0554d1dbd54eccdb0ad063f
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A Label-Based Polymer Nanoparticles Biosensor Combined with Loop-Mediated Isothermal Amplification for Rapid, Sensitive, and Highly Specific Identification of Brucella abortus

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