scholarly journals Rapid Detection of blaKPC, blaNDM, blaOXA-48-like and blaIMP Carbapenemases in Enterobacterales Using Recombinase Polymerase Amplification Combined With Lateral Flow Strip

2021 ◽  
Vol 11 ◽  
Author(s):  
Fang Wang ◽  
Lei Wang ◽  
Huimin Chen ◽  
Na Li ◽  
Yan Wang ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
High Sensitivity ◽  
Cross Reactivity ◽  
Lateral Flow ◽  
Recombinase Polymerase Amplification ◽  
Global Public Health ◽  
Special Equipment ◽  
Lateral Flow Strip ◽  
Carbapenemase Gene ◽  
Primer Sets

The emergence of carbapenemase-producing Enterobacterales (CPE) infections is a major global public health threat. Rapid and accurate detection of pathogenic bacteria is essential to optimize treatment and timely avoid further transmission of these bacteria. Here, we aimed to develop a rapid on site visualization detection method for CPE using improved recombinase polymerase amplification (RPA) combined with lateral flow strip (LFS) method, based on four most popular carbapenemase genes: blaKPC, blaNDM, blaOXA-48-like, and blaIMP. All available allelic variants of the above carbapenemases were downloaded from the β-lactamase database, and the conserved regions were used as targets for RPA assay. Five primer sets were designed targeting to each carbapenemase gene and the RPA amplification products were analyzed by agarose gel electrophoresis. FITC-labeled specific probes were selected, combined with the best performance primer set (Biotin-labeled on the reverse primer), and detected by RPA-LFS. Mismatches were made to exclude the false positive signals interference. This assay was evaluated in 207 clinically validated carbapenem-resistant Enterobacterales (CRE) isolates and made a comparison with conventional PCR. Results showed that the established RPA-LFS assay for CPE could be realized within 30 min at a constant temperature of 37°C and visually detected amplification products without the need for special equipment. This assay could specifically differentiate the four classes of carbapenemases without cross-reactivity and shared a minimum detection limit of 100 fg/reaction (for blaKPC, blaNDM, and blaOXA-48-like) or 1000 fg/reaction (for blaIMP), which is ten times more sensitive than PCR. Furthermore, the detection of 207 pre-validated clinically CRE strains using the RPA-LFS method resulted in 134 blaKPC, 69 blaNDM, 3 blaOXA-48-like, and 1 blaIMP. The results of the RPA-LFS assay were in consistent with PCR, indicating that this method shared high sensitivity and specificity. Therefore, the RPA-LFS method for CPE may be a simple, specific, and sensitive method for the rapid diagnosis of carbapenemase Enterobacterales.

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

2021 ◽  
Vol 11 ◽  
Author(s):  
Haitao Yang ◽  
Yan Wang ◽  
Qiankun Yang ◽  
Hui Fan ◽  
Lei Wang ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Pathogenic Bacteria ◽  
Detection Method ◽  
Lateral Flow ◽  
Recombinase Polymerase Amplification ◽  
Coincidence Rate ◽  
Kappa Index ◽  
Biochemical Method ◽  
Lateral Flow Strip ◽  
Index Value

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.

scholarly journals Specific Detection of Influenza A and B Viruses by CRISPR-Cas12a-Based Assay

Biosensors ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 88
Author(s):  
Bum Ju Park ◽  
Man Seong Park ◽  
Jae Myun Lee ◽  
Yoon Jae Song
Keyword(s):  
Diagnostic Test ◽  
Reverse Transcription ◽  
Influenza A ◽  
High Sensitivity ◽  
Cross Reactivity ◽  
Lateral Flow ◽  
Influenza Viruses ◽  
Nucleic Acid Amplification ◽  
Lateral Flow Strip ◽  
Influenza Pandemics

A rapid and accurate on-site diagnostic test for pathogens including influenza viruses is critical for preventing the spread of infectious diseases. Two types of influenza virus, A and B cause seasonal flu epidemics, whereas type A can cause influenza pandemics. To specifically detect influenza A (IAV) and B (IBV) viruses, we developed a clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated (Cas) system-based assay. By coupling reverse transcription recombinase polymerase amplification (RT-RPA) and reverse transcription loop-mediated isothermal amplification (RT-LAMP), a CRISPR-Cas12a DNA endonuclease-targeted CRISPR trans-reporter (DETECTR) detected IAV and IBV titers as low as 1 × 100 plaque forming units (PFUs) per reaction without exhibiting cross-reactivity. Only 75 to 85 min were required to detect IAV and IBV, depending on isothermal nucleic acid amplification methods, and results were verified using a lateral flow strip assay that does not require additional analytic equipment. Taken together, our findings establish RT-RPA and RT-LAMP-coupled DETECTR-based diagnostic tests for rapid, specific and high-sensitivity detection of IAV and IBV using fluorescence and lateral flow assays. The diagnostic test developed in this study can be used to distinguish IAV and IBV infections, a capability that is necessary for monitoring and preventing the spread of influenza epidemics and pandemics.

scholarly journals A Rapid and Sensitive Europium Nanoparticle-Based Lateral Flow Immunoassay Combined with Recombinase Polymerase Amplification for Simultaneous Detection of Three Food-Borne Pathogens

2021 ◽  
Vol 18 (9) ◽  
pp. 4574
Author(s):  
Kai Chen ◽  
Biao Ma ◽  
Jiali Li ◽  
Erjing Chen ◽  
Ying Xu ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Vibrio Parahaemolyticus ◽  
Pathogenic Bacteria ◽  
Simultaneous Detection ◽  
Lateral Flow ◽  
Recombinase Polymerase Amplification ◽  
Quantitative Detection ◽  
Bacterial Strains ◽  
Food Borne Pathogens ◽  
Food Borne

Food-borne pathogens have become an important public threat to human health. There are many kinds of pathogenic bacteria in food consumed daily. A rapid and sensitive testing method for multiple food-borne pathogens is essential. Europium nanoparticles (EuNPs) are used as fluorescent probes in lateral flow immunoassays (LFIAs) to improve sensitivity. Here, recombinase polymerase amplification (RPA) combined with fluorescent LFIA was established for the simultaneous and quantitative detection of Listeria monocytogenes, Vibrio parahaemolyticus, and Escherichia coliO157:H7. In this work, the entire experimental process could be completed in 20 min at 37 °C. The limits of detection (LODs) of EuNP-based LFIA–RPA were 9.0 colony-forming units (CFU)/mL for Listeria monocytogenes, 7.0 CFU/mL for Vibrio parahaemolyticus, and 4.0 CFU/mL for Escherichia coliO157:H7. No cross-reaction could be observed in 22 bacterial strains. The fluorescent LFIA–RPA assay exhibits high sensitivity and good specificity. Moreover, the average recovery of the three food-borne pathogens spiked in food samples was 90.9–114.2%. The experiments indicate the accuracy and reliability of the multiple fluorescent test strips. Our developed EuNP-based LFIA–RPA assay is a promising analytical tool for the rapid and simultaneous detection of multiple low concentrations of food-borne pathogens.

Development of recombinase polymerase amplification combined with lateral flow detection assay for rapid and visual detection of Ralstonia solanacearum in tobacco

Plant Disease ◽  
2021 ◽  
Author(s):  
Changfeng Li ◽  
Yuliang Ju ◽  
Xun Wu ◽  
Pengfei Shen ◽  
Le Cao ◽  
...  
Keyword(s):  
Ralstonia Solanacearum ◽  
Visual Detection ◽  
High Sensitivity ◽  
High Specificity ◽  
Lateral Flow ◽  
Recombinase Polymerase Amplification ◽  
Molecular Diagnostic ◽  
Tobacco Stem ◽  
Detection Assay ◽  
Flow Detection

Bacterial wilt caused by Ralstonia solanacearum is a serious soil-borne disease that results in severe losses to tobacco (Nicotiana tabacum) production in China. In this study, a novel RPA-LFD assay for the rapid visual detection of R. solanacearum was established using recombinase polymerase amplification (RPA) and lateral-flow dipstick (LFD). The RPA-LFD assay was performed at 37°C in 30 min without complex equipment. Targeting the sequence of the RipTALI-9 gene, we designed RPA primers (Rs-rpa-F/R) and an LF probe (Rs-LF-probe) that showed high specificity to R. solanacearum. The sensitivity of RPA-LFD assay to R. solanacearum was the same as that in conventional PCR at 1 pg genomic DNA, 102 CFU/g artificially inoculated tobacco stem, and 103 CFU/g artificially inoculated soil. The RPA-LFD assay could also detect R. solanacearum from plant and soil samples collected from naturally infested tobacco fields. These results suggest that the RPA-LFD assay developed in this study is a rapid, accurate molecular diagnostic tool with high sensitivity for the detection of R. solanacearum.

scholarly journals One-Step Reverse-Transcription Recombinase Polymerase Amplification Using Lateral Flow Strips for the Detection of Coxsackievirus A6

2021 ◽  
Vol 12 ◽  
Author(s):  
Jia Xie ◽  
Xiaohan Yang ◽  
Lei Duan ◽  
Keyi Chen ◽  
Pan Liu ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Clinical Performance ◽  
Quantitative Polymerase Chain Reaction ◽  
Lateral Flow ◽  
Recombinase Polymerase Amplification ◽  
Clinical Samples ◽  
Effective Vaccine ◽  
Lateral Flow Strip ◽  
Coxsackievirus A6 ◽  
One Step

Hand, foot, and mouth disease (HFMD) is a common infectious disease affecting mainly children under 5 years of age. Coxsackievirus A6 (CVA-6), a major causative pathogen of HFMD, has caused outbreaks in recent years. Currently, no effective vaccine or antiviral treatments are available. In this study, one-step reverse-transcription recombinase polymerase amplification (RT-RPA), combined with a disposable lateral flow strip (LFS) assay, was developed to detect CVA-6. This assay can be performed in less than 35 min at 37°C without expensive instruments, and the result can be observed directly with the naked eye. The sensitivity of the RT-RPA-LFS was 10 copies per reaction, which was comparable to that of the conventional real-time quantitative polymerase chain reaction (qPCR) assays. Moreover, the assay specificity was 100%. The clinical performance of the RT-RPA-LFS assay was evaluated using 142 clinical samples, and the coincidence rate between RT-RPA-LFS and qPCR was 100%. Therefore, our RT-RPA-LFS assay provides a simple and rapid approach for point-of-care CVA-6 diagnosis.

scholarly journals Detection of Helminth Ova in Wastewater Using Recombinase Polymerase Amplification Coupled to Lateral Flow Strips

Water ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 691
Author(s):  
Vivek B. Ravindran ◽  
Basma Khallaf ◽  
Aravind Surapaneni ◽  
Nicholas D. Crosbie ◽  
Sarvesh K. Soni ◽  
...  
Keyword(s):  
Wastewater Treatment ◽  
Wastewater Treatment Plants ◽  
Cost Effective ◽  
Lateral Flow ◽  
Recombinase Polymerase Amplification ◽  
Detection Methods ◽  
Water Current ◽  
Recycled Water ◽  
Lateral Flow Strip ◽  
Skilled Personnel

Ascaris lumbricoides is a major soil-transmitted helminth that is highly infective to humans. The ova of A. lumbricoides are able to survive wastewater treatment, thus making it an indicator organism for effective water treatment and sanitation. Hence, Ascaris ova must be removed from wastewater matrices for the safe use of recycled water. Current microscopic techniques for identification and enumeration of Ascaris ova are laborious and cumbersome. Polymerase chain reaction (PCR)-based techniques are sensitive and specific, however, major constraints lie in having to transport samples to a centralised laboratory, the requirement for sophisticated instrumentation and skilled personnel. To address this issue, a rapid, highly specific, sensitive, and affordable method for the detection of helminth ova was developed utilising recombinase polymerase amplification (RPA) coupled with lateral flow (LF) strips. In this study, Ascaris suum ova were used to demonstrate the potential use of the RPA-LF assay. The method was faster (< 30 min) with optimal temperature at 37 °C and greater sensitivity than PCR-based approaches with detection as low as 2 femtograms of DNA. Furthermore, ova from two different helminth genera were able to be detected as a multiplex assay using a single lateral flow strip, which could significantly reduce the time and the cost of helminth identification. The RPA-LF system represents an accurate, rapid, and cost-effective technology that could replace the existing detection methods, which are technically challenged and not ideal for on-site detection in wastewater treatment plants.

scholarly journals Nucleic Acid Lateral Flow Immunoassay for the Detection of Pathogenic Bacteria from Food

2009 ◽  
Vol 27 (Special Issue 1) ◽  
pp. S350-S353 ◽  
Author(s):  
M. Blažková ◽  
M. Koets ◽  
J. H Wichers ◽  
A. van Amerongen ◽  
L. Fukal ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Pathogenic Bacteria ◽  
Simultaneous Detection ◽  
Lateral Flow ◽  
Lateral Flow Immunoassay ◽  
Carbon Particles ◽  
Microbiological Methods ◽  
Biological Principle ◽  
Primer Sets ◽  
Food Bacteria

Nucleic acid lateral flow immunoassay (NALFIA) is a method combining molecular biological principle of detection with immunochemical principle of visualisation. Following isolation of DNA from the sample, a duplex PCR with two primer sets, of which one was labelled with biotin and the other with digoxigenin or fluorescein, respectively, was performed. The PCR solution and carbon particles conjugated with avidin are directly added to the nitrocellulose membrane with two test lines of immobilised antibodies specific for digoxigenin and fluorescein. The appearance of a black line indicates the presence of specific amplicon. We would like to present the NALFIA for the simultaneous detection of L. monocytogenes in particular and the genus Listeria in general, in food. Bacteria from the genus Listeria frequently contaminate a large variety of foods. Occurrence of Listeria strains in food may indicate errors in good hygienic and manufacturing practice, only L. monocytogenes is a significant human and animal pathogen responsible for the serious illness listeriosis. Conventional microbiological methods for L. monocytogenes detection are laborious and take several days to achieve a confirmed identification.

scholarly journals Applicability of duplex real time and lateral flow strip reverse-transcription recombinase aided amplification assays for the detection of Enterovirus 71 and Coxsackievirus A16

2019 ◽  
Vol 16 (1) ◽  
Author(s):  
Xin-na Li ◽  
Xin-xin Shen ◽  
Ming-hui Li ◽  
Ju-ju Qi ◽  
Rui-huan Wang ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Limit Of Detection ◽  
Enterovirus 71 ◽  
Cross Reactivity ◽  
Lateral Flow ◽  
Coxsackievirus A16 ◽  
Fluorescence Detector ◽  
Lateral Flow Strip ◽  
Clinical Diagnostic

Abstract Background Enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are the two main etiological agents of Hand, Foot and Mouth Disease (HFMD). Simple and rapid detection of EV71 and CA16 is critical in resource-limited settings. Methods Duplex real time reverse-transcription recombinase aided amplification (RT-RAA) assays incorporating competitive internal amplification controls (IAC) and visible RT-RAA assays combined with lateral flow strip (LFS) for detection of EV71 and CA16 were developed respectively. Duplex real time RT-RAA assays were performed at 42 °C within 30 min using a portable real-time fluorescence detector, while LFS RT-RAA assays were performed at 42 °C within 30 min in an incubator. Recombinant plasmids containing conserved VP1 genes were used to analyze the sensitivities of these two methods. A total of 445 clinical specimens from patients who were suspected of being infected with HFMD were used to evaluate the performance of the assays. Results The limit of detection (LoD) of the duplex real time RT-RAA for EV71 and CA16 was 47 copies and 38 copies per reaction, respectively. The LoD of the LFS RT-RAA for EV71 and CA16 were both 91 copies per reaction. There was no cross reactivity with other enteroviruses. Compared to reverse transcription-quantitative PCR (RT-qPCR), the clinical diagnostic sensitivities of the duplex real time RT-RAA assay were 92.3% for EV71 and 99.0% for CA16, and the clinical diagnostic specificities were 99.7 and 100%, respectively. The clinical diagnostic sensitivities of the LFS RT-RAA assay were 90.1% for EV71 and 94.9% for CA16, and the clinical diagnostic specificities were 99.7 and 100%, respectively. Conclusions The developed duplex real time RT-RAA and LFS RT-RAA assays for detection of EV71 and CA16 are potentially suitable in primary clinical settings.

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