Rapid molecular detection of Senecavirus A based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) and CRISPR/Cas12a.

IF 4.3 3区工程技术 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Frontiers in Bioengineering and Biotechnology Pub Date : 2025-04-04 eCollection Date: 2025-01-01 DOI:10.3389/fbioe.2025.1451125

Chenghui Jiang, Huibao Wang, Rongxia Guo, Rui Yang, Xiaoming Li, Ping Liu, Jing Wang, Jincai Yang, Yanyan Chang, Qiaoying Zeng

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引用次数: 0

Abstract

Introduction: Senecavirus A (SVA), an emerging vesicular pathogen, is responsible for porcine idiopathic vesicular disease (PIVD). This disease is closely associated with porcine vesicular disease and acute neonatal piglet mortality, presenting a substantial threat to the global swine industry. At present, the absence of effective drugs or vaccines for treating the disease makes accurate diagnosis of SVA of paramount importance for the effective prevention and control of the disease.

Methods: In this study, we combined reverse transcription loop-mediated isothermal amplification (RT-LAMP) and Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein12a (CRISPR/Cas12a) using a dual-labelled fluorescence quencher or fluorescent biotin single-stranded DNA reporter molecule to develop two rapid, reliable, and portable visual SVA assays: RT-LAMP-Cas12a-FQ and RT-LAMP-Cas12a-FB.

Results: The two methods exhibited comparable detection limits, with 9.6 copies/μL achieved in 40 and 45 minutes, respectively. They did not cross-react with non-target nucleic acids extracted from other related viruses and showed high specificity for SVA RNA detection. Furthermore, the methods demonstrated satisfactory performance in detecting 69 porcine adventitious samples, with no significant differences from that of quantitative reverse transcription polymerase chain reaction (RT-qPCR).

Discussion: In summary, the RT-LAMP-Cas12a-FQ and RT-LAMP-Cas12a-FB methods developed are promising for early detection and routine surveillance of porcine SVA in resource-poor areas.

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基于逆转录环介导等温扩增（RT-LAMP）和CRISPR/Cas12a的塞尼卡病毒A快速分子检测

塞内卡病毒A （SVA）是一种新兴的水疱病原体，是猪特发性水疱病（PIVD）的罪魁祸首。这种疾病与猪水疱病和急性新生儿仔猪死亡率密切相关，对全球养猪业构成重大威胁。目前，由于缺乏有效的药物或疫苗治疗该病，因此准确诊断SVA对于有效预防和控制该病至关重要。方法：在本研究中，我们结合逆转录环介导的等温扩增（RT-LAMP）和聚集规则间隔短回文重复序列和CRISPR相关蛋白12a (CRISPR/Cas12a)，使用双标记荧光猝灭剂或荧光生物素单链DNA报告分子，建立了两种快速、可靠、便携式的视觉SVA检测方法：RT-LAMP-Cas12a- fq和RT-LAMP-Cas12a- fb。结果：两种方法的检出限相当，40 min和45 min检出限分别为9.6拷贝/μL。它们不与从其他相关病毒中提取的非靶核酸发生交叉反应，对SVA RNA检测具有高特异性。此外，该方法对69份猪不确定样本的检测结果令人满意，与定量逆转录聚合酶链反应（RT-qPCR）的检测结果无显著差异。综上所述，RT-LAMP-Cas12a-FQ和RT-LAMP-Cas12a-FB方法在资源贫乏地区的猪SVA早期检测和常规监测中具有广阔的应用前景。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Frontiers in Bioengineering and Biotechnology Chemical Engineering-Bioengineering

CiteScore

8.30

自引率

5.30%

发文量

2270

审稿时长

12 weeks

期刊介绍： The translation of new discoveries in medicine to clinical routine has never been easy. During the second half of the last century, thanks to the progress in chemistry, biochemistry and pharmacology, we have seen the development and the application of a large number of drugs and devices aimed at the treatment of symptoms, blocking unwanted pathways and, in the case of infectious diseases, fighting the micro-organisms responsible. However, we are facing, today, a dramatic change in the therapeutic approach to pathologies and diseases. Indeed, the challenge of the present and the next decade is to fully restore the physiological status of the diseased organism and to completely regenerate tissue and organs when they are so seriously affected that treatments cannot be limited to the repression of symptoms or to the repair of damage. This is being made possible thanks to the major developments made in basic cell and molecular biology, including stem cell science, growth factor delivery, gene isolation and transfection, the advances in bioengineering and nanotechnology, including development of new biomaterials, biofabrication technologies and use of bioreactors, and the big improvements in diagnostic tools and imaging of cells, tissues and organs. In today`s world, an enhancement of communication between multidisciplinary experts, together with the promotion of joint projects and close collaborations among scientists, engineers, industry people, regulatory agencies and physicians are absolute requirements for the success of any attempt to develop and clinically apply a new biological therapy or an innovative device involving the collective use of biomaterials, cells and/or bioactive molecules. “Frontiers in Bioengineering and Biotechnology” aspires to be a forum for all people involved in the process by bridging the gap too often existing between a discovery in the basic sciences and its clinical application.