Rapid Detection of Staphylococcus aureus in Food Using a Recombinase Polymerase Amplification-Based Assay

Geng, Yunyun; Liu, Siying; Wang, Jinfeng; Nan, Huizhu; Liu, Libing; Sun, Xiaoxia; Li, Danyu; Liu, Ming; Wang, Jianchang; Tan, Karsoon

doi:10.1007/s12161-018-1267-1

Cited by 18 publications

(16 citation statements)

References 30 publications

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“…Real-time PCR was performed on the ABI 7500 instrument in which premix Ex Taq TM (Takara Co., Ltd., Dalian, China) was employed (Geng et al, 2019). The reaction was performed as follows: 95°C for 30 s, followed by 35 cycles of 95°C for 10 s and then 60°C for 34 s. The sequences of the primers and probes used for real-time PCR were listed in Table 2.…”

Section: Real-time Pcrmentioning

confidence: 99%

“…Recombinase polymerase amplification (RPA) acts as one of isothermal gene amplification techniques. RPA has the merit of amplification at a relatively low temperature (37–42°C) within 10–20 min ( Piepenburg et al, 2006 ; Geng et al, 2018 ). The use of RPA-based methods has been proved to be a success in detecting pathogenic bacteria and viruses in clinical and food samples ( Euler et al, 2012 ; Abd El Wahed et al, 2015 ; Wang et al, 2020 ).…”

Section: Introductionmentioning

confidence: 99%

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Development and Evaluation of the Rapid and Sensitive RPA Assays for Specific Detection of Salmonella spp. in Food Samples

Zhao

Wang²,

Sun³

et al. 2021

Front. Cell. Infect. Microbiol.

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Salmonella spp. is among the main foodborne pathogens which cause serious foodborne diseases. An isothermal real-time recombinase polymerase amplification (RPA) and lateral flow strip detection (LFS RPA) were used to detect Salmonella spp. targeting the conserved sequence of invasion protein A (invA). The Real-time RPA was performed in a portable florescence scanner at 39°C for 20 min. The LFS RPA was performed in an incubator block at 39°C for 15 min, under the same condition that the amplifications could be inspected by the naked eyes on the LFS within 5 min. The detection limit of Salmonella spp. DNA using real-time RPA was 1.1 × 101 fg, which was the same with real-time PCR but 10 times higher than that of LFS RPA assay. Moreover, the practicality of discovering Salmonella spp. was validated with artificially contaminated lamb, chicken, and broccoli samples. The analyzing time dropped from 60 min to proximately 5–12 min on the basis of the real-time and LFS RPA assays compared with the real-time PCR assay. Real-time and LFS RPA assays’ results were equally reliable. There was no cross-reactivity with other pathogens in both assays. In addition, the assays had good stability. All of these helped to show that the developed RPA assays were simple, rapid, sensitive, credible, and could be a potential point-of-need (PON) test required mere resources.

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Section: Real-time Pcrmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Development and Evaluation of the Rapid and Sensitive RPA Assays for Specific Detection of Salmonella spp. in Food Samples

Zhao

Wang²,

Sun³

et al. 2021

Front. Cell. Infect. Microbiol.

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“…Therefore, accurate methods to detect and discriminate Staphylococcus species contamination in food are needed to reduce disease outbreaks and ensure food safety. Traditional methods for detecting pathogenic bacteria require multiple steps, including pre-enrichment, selective growth, and selective plating, which can then be characterized by analyzing additional biochemical tests [7]. The process is labor-intensive and timeconsuming, is not cost-effective, and is also unable to detect viable but nonculturable cells [8,9].…”

Section: Introductionmentioning

confidence: 99%

Real-Time PCR Method for the Rapid Detection and Quantification of Pathogenic Staphylococcus Species Based on Novel Molecular Target Genes

Kim

Yang

Won

et al. 2021

Foods

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Coagulase-positive Staphylococcus aureus is a foodborne pathogen considered one of the causes of food-related disease outbreaks. Like S. aureus, Staphylococcus capitis, Staphylococcus caprae, and S. epidermidis are opportunistic pathogens causing clinical infections and food contamination. The objective of our study was to develop a rapid, accurate, and monitoring technique to detect four Staphylococcus species in food. Four novel molecular targets (GntR family transcriptional regulator for S. aureus, phosphomannomutase for S. epidermidis, FAD-dependent urate hydroxylase for S. capitis, and Gram-positive signal peptide protein for S. caprae) were mined based on pan-genome analysis. Primers targeting molecular target genes showed 100% specificity for 100 non-target reference strains. The detection limit in pure cultures and artificially contaminated food samples was 102 colony-forming unit/mL for S. aureus, S. capitis, S. caprae, and S. epidermidis. Moreover, real-time polymerase chain reaction successfully detected strains isolated from various food matrices. Thus, our method allows an accurate and rapid monitoring of Staphylococcus species and may help control staphylococcal contamination of food.

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“…Previously, RPA was successfully applied to rapidly detect S. aureus and P. aeruginosa [19, 20]. Nevertheless, different bacterial pathogens can usually co-infect food, thus quickly and accurately diagnose their presences at the same time will significantly save time, labor, and cost and bring more useful testing information as well.…”

Section: Introductionmentioning

confidence: 99%

Direct multiplex recombinase polymerase amplification for rapid detection of Staphylococcus aureus and Pseudomonas aeruginosa in food

Tran

Pham

et al. 2021

Preprint

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Foodborne illness undermines human health by causing fever, stomachache and even lethality. Among foodborne bacterial pathogens, Staphylococcus aureus and Pseudomonas aeruginosa are of extraordinary significance which drive reasons of food and beverage poisoning in numerous cases. Today, PCR has been widely used to examine the presence of different foodborne pathogens. However, PCR requires specialized equipment and skillful personnel which limit its application in the field. Recently, there is an emerging of isothermal PCR methods in which the reactions occur at low and constant temperatures, allowing their application in restricted-resource settings. In this work, multiplex Recombinase Polymerase Amplification (RPA) was used to simultaneously detect S. aureus and P. aeruginosa with high sensitivity and specificity. The limit detection of multiplex RPA was 10 and 30 fg/reaction of genomic DNAs of S. aureus and P. aeruginosa, respectively. Besides, the reaction time was reduced to only 25 minutes with a low incubation temperature of 39 °C. Markedly, multiplex RPA reactions succeeded to directly detect as low as 1 and 5 CFU/reaction of S. aureus and P. aeruginosa cells, respectively without the requirement of extracting DNA genome. Moreover, the multiplex RPA reliably detected the two foodborne bacteria in milk, fruit juice and bottled water samples. In general, the direct multiplex RPA described in this study is a rapid, simple, sensitive and efficient alternative tool that could be used to detect the presence of S. aureus and P. aeruginosa without the necessity of costly devices and high-trained staff.

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Rapid Detection of Staphylococcus aureus in Food Using a Recombinase Polymerase Amplification-Based Assay

Cited by 18 publications

References 30 publications

Development and Evaluation of the Rapid and Sensitive RPA Assays for Specific Detection of Salmonella spp. in Food Samples

Development and Evaluation of the Rapid and Sensitive RPA Assays for Specific Detection of Salmonella spp. in Food Samples

Real-Time PCR Method for the Rapid Detection and Quantification of Pathogenic Staphylococcus Species Based on Novel Molecular Target Genes

Direct multiplex recombinase polymerase amplification for rapid detection of Staphylococcus aureus and Pseudomonas aeruginosa in food

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