A Recombinase Polymerase Amplification and Lateral Flow Strip Combined Method That Detects Salmonella enterica Serotype Typhimurium With No Worry of Primer-Dependent Artifacts

Wu, Huahua; Zhao, Panpan; Yang, Xiaohan; Li, Juan; Zhang, Jingyu; Zhang, Xun; Zeng, Zihan; Dong, Jingquan; Gao, Song; Lu, Chunmei

doi:10.3389/fmicb.2020.01015

Cited by 48 publications

(56 citation statements)

References 35 publications

(39 reference statements)

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“…This significantly decreases the formation of labeled by-product amplicons [ 14 ]. Some research proposed a solution for decreasing the false-positive signal of RPA-LFT by the optimization of the probe design [ 15 ], using blocking antiprimers [ 16 ] and the proper combination of probe–primer sets [ 17 , 18 ] to avoid their cross-dimerization. However, there is no research about the direct comparison of RPA-LFT based on the generation of labeled amplicons through forward and reverse primers and through the oligonucleotide probe and reverse primer.…”

Section: Introductionmentioning

confidence: 99%

Recombinase Polymerase Amplification Assay with and without Nuclease-Dependent-Labeled Oligonucleotide Probe

Иванов

Safenkova

Zherdev

et al. 2021

IJMS

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The combination of recombinase polymerase amplification (RPA) and lateral flow test (LFT) is a strong diagnostic tool for rapid pathogen detection in resource-limited conditions. Here, we compared two methods generating labeled RPA amplicons following their detection by LFT: (1) the basic one with primers modified with different tags at the terminals and (2) the nuclease-dependent one with the primers and labeled oligonucleotide probe for nuclease digestion that was recommended for the high specificity of the assay. Using both methods, we developed an RPA-LFT assay for the detection of worldwide distributed phytopathogen—alfalfa mosaic virus (AMV). A forward primer modified with fluorescein and a reverse primer with biotin and fluorescein-labeled oligonucleotide probe were designed and verified by RPA. Both labeling approaches and their related assays were characterized using the in vitro-transcribed mRNA of AMV and reverse transcription reaction. The results demonstrated that the RPA-LFT assay based on primers-labeling detected 103 copies of RNA in reaction during 30 min and had a half-maximal binding concentration 22 times lower than probe-dependent RPA-LFT. The developed RPA-LFT was successfully applied for the detection of AMV-infected plants. The results can be the main reason for choosing simple labeling with primers for RPA-LFT for the detection of other pathogens.

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Section: Introductionmentioning

confidence: 99%

Recombinase Polymerase Amplification Assay with and without Nuclease-Dependent-Labeled Oligonucleotide Probe

Иванов

Safenkova

Zherdev

et al. 2021

IJMS

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“…RPA was shown to possess a high impurity tolerance of the sample [30]. As expected, the direct addition of bacterial cells to the multiplex RPA reaction also induced the amplification process of the targeted sequences.…”

Section: Discussionmentioning

confidence: 91%

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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“…Thus, the NTC signal should be totally inhibited when designing a specific probe for RPA-LFS. Previous studies demonstrated that the RPA reaction could tolerate a few mismatches between the primer/probe and the template ( Daher et al, 2015 ; Wu et al, 2020 ). As shown in Table 3 , probe-reverse primer dimers were analyzed using Primer Premier 3 software and mismatches were added (indicated by red text in Table 3 ) to break sites that had more than three successive bases or more than one base at the 3′ end.…”

Section: Resultsmentioning

confidence: 99%

“…A specific RPA probe was designed using Primer Premier 3 software based on sequences where screened RPA primers performed well. The parameters of primer and probe design were set referring to previously described parameters ( Wu et al, 2020 ). The reverse primer for RPA-LFS was labeled with biotin at the 5′ end.…”

Section: Methodsmentioning

confidence: 99%

A Rapid and Sensitive Detection Method for Pseudomonas aeruginosa Using Visualized Recombinase Polymerase Amplification and Lateral Flow Strip Technology

Yang¹,

Yan

Yang³

et al. 2021

Front. Cell. Infect. Microbiol.

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Pseudomonas aeruginosa is a common opportunistic pathogen that causes acute nosocomial necrotizing pneumonia and is the predominant source of chronic lung infections in patients with the genetic disorder cystic fibrosis. Early diagnosis in infected patients and monitoring P. aeruginosa contamination is therefore of great importance in controlling disease spread and development with timely drugs intervention treatment and cut off infection source. Traditional culture-biochemical methods are time consuming and highly dependent on technicians and expensive instruments. To address these challenges, the present study aimed to develop a rapid, sensitive, and specific, on-site detection method for P. aeruginosa based on recombinase polymerase amplification (RPA) combined with lateral flow strip (LFS) technology. The experimental process included screening and modification of primer and probe sets targeting the unique virulence gene elastase B (lasB); specificity detection in 29 strains of P. aeruginosa and 23 closely-related pathogenic bacteria; sensitivity measurements with gradient-diluted P. aeruginosa genomic DNA and probit regression analysis; and clinical application evaluation using 574 patients samples and calculating coincidence rate and kappa index value in comparison with the culture-biochemical method. The P. aeruginosa RPA-LFS assay could complete the amplification process at 37°C constant temperature within 30 min and results could be visualized by the naked eye within 10 min on LFS. The assay displayed high sensitivity with a limit of detection of 3.05 CFU/reaction. It also demonstrated high specificity by showing no cross reaction with other pathogenic bacteria, and rapidness by being completed in less than an hour. Furthermore, when used with clinical samples, the assay had a coincidence rate of 98.26% with the culture-biochemical method and a kappa index value of 0.9433. These data indicate that the RPA-LFS assay represents a major improvement for P. aeruginosa detection, especially in resource-limited areas.

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A Recombinase Polymerase Amplification and Lateral Flow Strip Combined Method That Detects Salmonella enterica Serotype Typhimurium With No Worry of Primer-Dependent Artifacts

Cited by 48 publications

References 35 publications

Recombinase Polymerase Amplification Assay with and without Nuclease-Dependent-Labeled Oligonucleotide Probe

Recombinase Polymerase Amplification Assay with and without Nuclease-Dependent-Labeled Oligonucleotide Probe

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

A Rapid and Sensitive Detection Method for Pseudomonas aeruginosa Using Visualized Recombinase Polymerase Amplification and Lateral Flow Strip Technology

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