Development of a real‐time recombinase polymerase amplification assay for rapid detection of <i>Aeromonas hydrophila</i>

Qu, Yang; Wang, Qing; Li, Yingying; Wang, Yingying; Yin, Jun; Ren, Yan; Liu, Chun; Liu, Xiaofang; Wang, Yahui; Zeng, Weiwei

doi:10.1111/jfd.13291

Cited by 10 publications

(7 citation statements)

References 36 publications

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“…Increasingly, A. hydrophila is posing a serious threat to human health, aquaculture and food safety, and foodborne outbreaks of its infection have occurred in many countries (Morena et al, 1993;Krovacek et al, 1995;Zhang et al, 2012;Tsheten et al, 2016). Many studies have reported that the pathogenicity of A. hydrophila is determined by multiple virulence factors (Janda and Abbott, 2010;Tichoniuk et al, 2010;Li et al, 2011), and based on this reason, there are many methods targeting one or more virulence genes to detect pathogenic A. hydrophila (Tichoniuk et al, 2010;Uma et al, 2010;Li et al, 2011;Elsheshtawy et al, 2019;Qu et al, 2021). However, these methods have some flaws, such as long operation time, low sensitivity, complicated operation, and missed detection.…”

Section: Discussionmentioning

confidence: 99%

“…In recent years, isothermal nucleic acid amplification technologies, such as recombinase-aided amplification (RAA; Piepenburg et al, 2006;Subsoontorn et al, 2020) and loop-mediated isothermal amplification (Nagamine et al, 2002), have promoted the development of nucleic acid amplification without thermal cyclers. RAA assay that only requires one primer set and can be reacted even at body temperature of operator (Wang et al, 2017) has been used for the detection of pathogens such as A. hydrophila (Qu et al, 2021) and Enterocytozoon hepatopenaei (Zhou et al, 2020), but this method has low sensitivity (Xiao et al, 2021) and the result is complicated to obtain (Zhou et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

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A novel detection method for the pathogenic Aeromonas hydrophila expressing aerA gene and/or hlyA gene based on dualplex RAA and CRISPR/Cas12a

Lin

et al. 2022

Front. Microbiol.

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Aeromonas hydrophila is an emerging waterborne and foodborne pathogen with pathogenicity to humans and warm water fishes, which severely threatens human health, food safety and aquaculture. A novel method for the rapid, accurate, and sensitive detection of pathogenic A. hydrophila is still needed to reduce the impact on human health and aquaculture. In this work, we developed a rapid, accurate, sensitive, and visual detection method (dRAA-CRISPR/Cas12a), without elaborate instruments, integrating the dualplex recombinase-assisted amplification (dRAA) assay and CRISPR/Cas12a system to detect pathogenic A. hydrophila expressing aerA and/or hlyA virulence genes. The dRAA-CRISPR/Cas12a method has high sensitivity, which can rapidly detect (about 45 min) A. hydrophila with the limit of detection in 2 copies of genomic DNA per reaction, and has high specificity for three pathogenic A. hydrophila strains (aerA+hlyA−, aerA−hlyA+, and aerA+hlyA+). Moreover, dRAA-CRISPR/Cas12a method shows satisfactory practicability in the analysis of the spiked human blood and stool and fish samples. These results demonstrate that our developed pathogenic A. hydrophila detection method, dRAA-CRISPR/Cas12a, is a promising potential method for the early diagnosis of human A. hydrophila infection and on-site detection of A. hydrophila in food and aquaculture.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A novel detection method for the pathogenic Aeromonas hydrophila expressing aerA gene and/or hlyA gene based on dualplex RAA and CRISPR/Cas12a

Lin

et al. 2022

Front. Microbiol.

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“…orientalis, with an analytical sensitivity of up to 15 DNA copies per reaction. [115][116][117][118]…”

Section: Recombinase Polymerase Amplificationmentioning

confidence: 99%

“…Thus far, RPA methods have been used to detect a number of pathogens affecting tilapia, such as Aeromonas hydrophila , Flavobacterium columnare and Francisella noatunensis subsp. orientalis , with an analytical sensitivity of up to 15 DNA copies per reaction 115–118 …”

Section: Current and Emerging Molecular Diagnostic Technologiesmentioning

confidence: 99%

From the basics to emerging diagnostic technologies: What is on the horizon for tilapia disease diagnostics?

Dong

Chaijarasphong

Barnes

et al. 2023

Reviews in Aquaculture

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Tilapia is an affordable protein source farmed in over 140 countries with the majority of production in low‐ and middle‐income countries. Intensification of tilapia farming has exacerbated losses caused by emerging and re‐emerging infectious diseases. Disease diagnostics play a crucial role in biosecurity and health management to mitigate disease loss and improve animal welfare in aquaculture. Three continuous levels of diagnostics (I, II and III) for aquatic species have been proposed by Food and Agriculture Organization of the United Nations (FAO) and the Network of Aquaculture Centers in Asia and the Pacific (NACA) to promote the integration of basic and advanced methods to achieve accurate and meaningful interpretation of diagnostic results. However, the recent proliferation of cutting‐edge molecular methods applied in the diagnosis of diseases of aquacultured animals has shifted the focus of researchers and users away from basic approaches and toward molecular diagnostics, despite the fact that many diseases can be rapidly diagnosed using inexpensive, simple microscopic examination and that most emerging diseases in aquaculture were discovered by histopathology. This review, therefore, revisits and highlights the importance of the three levels of diagnostics for diseases of tilapia, particularly the frequently overlooked basic procedures (e.g., case history records, gross pathology, presumptive diagnostic methods and histopathology). The review also covers current and emerging molecular diagnostic technologies for tilapia pathogens including polymerase chain reaction methods (conventional, quantitative, digital), isothermal amplification methods Loop‐mediated Isothermal Amplification (LAMP), recombinase polymerase amplification (RPA), clustered regularly interspaced short palindromic repeats (CRISPR)‐based detection, lateral flow immunoassays, as well as discussing what is on the horizon for tilapia disease diagnostics (next generation sequencing, artificial intelligence, environmental Deoxyribonucleic Acid (DNA) and Ribonucleic Acid (RNA) and point‐of‐care testing) providing a future vision for transferring these technologies to farmers and stakeholders for a sustainable aquatic food system transformation.

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“…At present, antibiotic therapy is the primary treatment for Aeromonas hydrophila infections, and no effective cure exists. Consequently, early detection of this pathogen is crucial for prevention and control of these infections [ 9 ]. Developing a rapid, sensitive, and precise detection method for Aeromonas hydrophila is therefore essential to enhancing treatment and control strategies, mitigating the risk of bacterial infection, and safeguarding the safety of fish, as well as human consumption and health.…”

Section: Introductionmentioning

confidence: 99%

Establishment of a Rapid LAMP Assay for Aeromonas hydrophila and Comparison with the Application of qPCR

et al. 2023

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The development of an exceptionally sensitive diagnostic technique for early identification of aquaculture diseases, specifically Aeromonas hydrophila, is essential for efficient management of disease outbreaks at aquaculture locations. In this research, a swift and sensitive diagnostic assay employing Loop-mediated isothermal amplification (LAMP) of Aeromonas hydrophila was devised and compared to the conventional qPCR method documented by Rong Wang. Validation of the diagnostic assay was carried out using actual samples obtained from aquaculture fish. The findings revealed that based on the rapid detection of crude bacterial genomic DNA, the fluorescent LAMP assay possessed a lower limit of detection (LOD) of 0.559 ng/μL (0.315–1.693, 95% CI), while the LOD for qPCR stood at 4.301 ng/μL (2.084–8.876, 95% CI). Both techniques demonstrated outstanding specificity, exhibiting no cross-reactivity with bacteria from the same or closely related genera. A total of 74 fish samples suspected to be infected with the fish disease were gathered, with 26 and 23 samples testing positive for Aeromonas hydrophila via LAMP and qPCR, respectively. The concordance analysis for LAMP and qPCR methods generated a Kappa value of 0.909 (0.778–1.000, 95% CI), signifying a high degree of diagnostic consensus. This study highlights that the LAMP assay eliminates the thermal cycle temperature change process of qPCR, uses lysate to crudely extract bacterial genomic DNA, and can complete the detection within 40 min, rendering it a practical and efficient alternative for monitoring disease outbreaks at aquaculture sites.

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Development of a real‐time recombinase polymerase amplification assay for rapid detection of Aeromonas hydrophila

Cited by 10 publications

References 36 publications

A novel detection method for the pathogenic Aeromonas hydrophila expressing aerA gene and/or hlyA gene based on dualplex RAA and CRISPR/Cas12a

A novel detection method for the pathogenic Aeromonas hydrophila expressing aerA gene and/or hlyA gene based on dualplex RAA and CRISPR/Cas12a

From the basics to emerging diagnostic technologies: What is on the horizon for tilapia disease diagnostics?

Establishment of a Rapid LAMP Assay for Aeromonas hydrophila and Comparison with the Application of qPCR

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