Recombinase polymerase amplification with polymer flocculation sedimentation for rapid detection of Staphylococcus aureus in food samples

Hu, Jinqiang; Wang, Yi; Ding, Huimin; Chun-peng, Jiang; Geng, Yangyang; Sun, Xiaoyan; Jianzhou, Jing; Gao, Hui; Wang, Zhangcun; Dong, Caiwen

doi:10.1016/j.ijfoodmicro.2020.108691

Cited by 31 publications

(17 citation statements)

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“…This RPA system derived from bacteriophage T4 has been commercialized and widely applied to molecular diagnosis for many infectious diseases (Dong et al, 2020;Hu et al, 2020;Wang et al, 2020). An effort has been made to apply the RPA technology for the detection of EHP infection that targeted the SSU rRNA gene, but the analysis of the amplification signal was performed by gel electrophoresis, which not only hampered the sensitivity (8 × 10 2 gene copies per reaction) but also restrained the detection assay to the laboratory (Zhou et al, 2020).…”

Section: Discussionmentioning

confidence: 99%

“…The isothermal recombinase polymerase amplification (RPA) assay derived from the recombination-dependent DNA replication (RDR) mechanism of bacteriophage T4 is a potentially suitable method (Li et al, 2018;Lobato and O'Sullivan, 2018;Dong et al, 2020;Hu et al, 2020;Wang et al, 2020). The RPA system uses several enzymes from bacteriophage T4, including the strand-exchange protein UvsX, the mediator protein UvsY, and the single-strand binding protein gp32, to mimic the bacteriophage T4 RDR system in vitro.…”

Section: Introductionmentioning

confidence: 99%

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Rapid Detection of Enterocytozoon hepatopenaei Infection in Shrimp With a Real-Time Isothermal Recombinase Polymerase Amplification Assay

Ma¹,

Fan²,

Wang³

et al. 2021

Front. Cell. Infect. Microbiol.

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Enterocytozoon hepatopenaei (EHP) infection has become a significant threat in shrimp farming industry in recent years, causing major economic losses in Asian countries. As there are a lack of effective therapeutics, prevention of the infection with rapid and reliable pathogen detection methods is fundamental. Molecular detection methods based on polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) have been developed, but improvements on detection speed and convenience are still in demand. The isothermal recombinase polymerase amplification (RPA) assay derived from the recombination-dependent DNA replication (RDR) mechanism of bacteriophage T4 is promising, but the previously developed RPA assay for EHP detection read the signal by gel electrophoresis, which restricted this application to laboratory conditions and hampered the sensitivity. The present study combined fluorescence analysis with the RPA system and developed a real-time RPA assay for the detection of EHP. The detection procedure was completed in 3–7 min at 39°C and showed good specificity. The sensitivity of 13 gene copies per reaction was comparable to the current PCR- and LAMP-based methods, and was much improved than the RPA assay analyzed by gel electrophoresis. For real clinical samples, detection results of the real-time RPA assay were 100% consistent with the industrial standard nested PCR assay. Because of the rapid detection speed and the simple procedure, the real-time RPA assay developed in this study can be easily assembled as an efficient and reliable on-site detection tool to help control EHP infection in shrimp farms.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Rapid Detection of Enterocytozoon hepatopenaei Infection in Shrimp With a Real-Time Isothermal Recombinase Polymerase Amplification Assay

Ma¹,

Fan²,

Wang³

et al. 2021

Front. Cell. Infect. Microbiol.

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“…To promptly prevent the serious consequences of L. monocytogenes infection, the development of a rapid and efficient method for L. monocytogenes detection is needed. Currently, RPA is one of the isothermal amplification techniques that have been applied to detect different infectious bacteria precisely and quickly (Liu et al, 2017;Frimpong et al, 2019;Geng et al, 2019;Hu et al, 2020). In this study, the RPA assay was developed to specifically detect L. monocytogenes in direct crude samples.…”

Section: Discussionmentioning

confidence: 99%

“…Therefore, combination with LF strips can minimize and simplize the detection procedure of RPA product in a resource-limited setting. Previously, the RPA assays were successfully established to identify some types of food poisoning bacteria (Gao et al, 2017;Liu et al, 2017;Du et al, 2018;Geng et al, 2019;Hu et al, 2020). Previously, Gao et al (2017) utilized RPA to detect L. monocytogenes successfully with a limit of detection (LOD) of 10 pg of genomic DNA per reaction.…”

Section: Introductionmentioning

confidence: 99%

DIRECT DETECTION OF LISTERIA MONOCYTOGENES BY RECOMBINASE POLYMERASE AMPLIFICATION

Phung

2021

Preprint

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Listeria monocytogenes is one of the most common types of food poisoning bacteria which can cause serious foodborne diseases or even lethality. Generally, L. monocytogenes can be detected using traditional microbiology or molecular biology techniques, notably PCR. However, the application of these methods at the field is restricted due to the strict requirement of equipment and skilled personnel. In this study, recombinase polymerase amplification (RPA), an isothermal PCR assay was developed to rapidly detect L. monocytogenes in the crude samples. The results showed that the RPA reaction, without requiring complex thermal cycles, was well-performed in the optimal conditions of 39°C within only 25 minutes. The limit of detection was identified as 310 fg of L. monocytogenes genomic DNA, which was 1000-fold more sensitive than the conventional PCR. In addition, RPA also succeeded to directly detect L. monocytogenes cells at a concentration as low as 2.5×101 Colony Forming Unit (CFU)/ml in pure cultures and 2.5×102 CFU/ml in crude samples without sample extraction or processing. Therefore, RPA established in this study could be an alternative standard method to confirm the presence of L. monocytogenes in food. Accordingly, this rapid and sensitive method could be further applied to clinical testing for the diagnosis of L. monocytogenes infection, especially in areas with limited settings.

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“…This technique simplifies the detection process and allows the in situ detection of the result without instruments. RPA–LFS has been successfully utilized to identify methicillin-resistant Staphylococcus aureus , Mycobacterium tuberculosis , Candida albicans , Klebsiella pneumoniae , and other pathogenic microorganisms ( Figure 1 ) ( Hu et al., 2020 ; Wang et al., 2020 ; Wang et al., 2021a ; Wang et al., 2021b ).…”

Section: Introductionmentioning

confidence: 99%

Rapid, Simple, and Highly Specific Detection of Streptococcus pneumoniae With Visualized Recombinase Polymerase Amplification

Wang

Liu

et al. 2022

Front. Cell. Infect. Microbiol.

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Streptococcus pneumoniae is a major pathogen that causes microbiological illness in humans. The introduction of polyvalent vaccines has resulted in a significant decrease in pneumococcal-related mortality. However, pneumococcal infections continue to be a leading cause of death in children under the age of 5 and adults over the age of 65 worldwide. A speedy and highly sensitive diagnostic tool is necessary for routine adoption to adequately manage patients and control the spread of infection. In this study, we investigated a new nucleic acid amplification technique, isothermal recombinase polymerase amplification (RPA), which amplifies DNA at 37°C under isothermal conditions with high specificity, efficiency, and rapidity. Using the autolysin gene lytA as the molecular diagnostic target, an RPA primer-probe combination was designed and optimized for the detection of S. pneumoniae. This RPA reaction produced amplification products labeled with specific chemical markers, to be detected with gold-nanoparticle-based lateral flow strips (LFS), reducing the reliance on equipment and trained personnel. The high specificity of the RPA-LFS technique was demonstrated with the specific detection of 22 strains of S. pneumoniae but not 25 closely related pathogenic bacteria. The assay showed good sensitivity, and detected S. pneumoniae down to 3.32 colony-forming units/μL. When used on clinical samples, the assay provided accurate and consistent results compared with PCR. The compliance with the culture-biochemistry method was 98.18% and the kappa index was 0.977. These results reveal that the RPA–LFS test significantly improved S. pneumoniae identification, particularly in resource-limited areas.

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Recombinase polymerase amplification with polymer flocculation sedimentation for rapid detection of Staphylococcus aureus in food samples

Cited by 31 publications

References 50 publications

Rapid Detection of Enterocytozoon hepatopenaei Infection in Shrimp With a Real-Time Isothermal Recombinase Polymerase Amplification Assay

Rapid Detection of Enterocytozoon hepatopenaei Infection in Shrimp With a Real-Time Isothermal Recombinase Polymerase Amplification Assay

DIRECT DETECTION OF LISTERIA MONOCYTOGENES BY RECOMBINASE POLYMERASE AMPLIFICATION

Rapid, Simple, and Highly Specific Detection of Streptococcus pneumoniae With Visualized Recombinase Polymerase Amplification

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