Rapid Identification of Salmo salar Using a Combined Isothermal Recombinase Polymerase Amplification–Lateral Flow Strip Approach

2021 ◽  
Author(s):  
Lei Wang ◽  
Danyan Zheng ◽  
Michael Ackah ◽  
Huahua Wu ◽  
Shishi Qi ◽  
...  
Keyword(s):  
Salmo Salar ◽  
Rapid Identification ◽  
Lateral Flow ◽  
Recombinase Polymerase Amplification ◽  
Lateral Flow Strip

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 Rapid and simple detection of Phytophthora cactorum in strawberry using a coupled recombinase polymerase amplification–lateral flow strip assay

2021 ◽  
Vol 3 (1) ◽  
Author(s):  
Xinyu Lu ◽  
Heng Xu ◽  
Wen Song ◽  
Zitong Yang ◽  
Jia Yu ◽  
...  
Keyword(s):  
Crop Production ◽  
Accurate Method ◽  
Fungal Species ◽  
Lateral Flow ◽  
Recombinase Polymerase Amplification ◽  
Economic Losses ◽  
Phytophthora Cactorum ◽  
Lateral Flow Strip ◽  
Alkaline Lysis ◽  
Wide Range

AbstractPhytophthora cactorum is a devastating pathogen that infects a wide range of plants and causes Phytophthora rot disease, which has resulted in great economic losses in crop production. Therefore, the rapid and practicable detection of P. cactorum is important for disease monitoring and forecasting. In this study, we developed a lateral flow recombinase polymerase amplification (LF-RPA) assay for the sensitive visual detection of P. cactorum. Specific primers for P. cactorum were designed based on the ras-related protein gene Ypt1; all 10 P. cactorum isolates yielded positive detection results, whereas no cross-reaction occurred in related oomycete or fungal species. The detection limit for the LF-RPA assay was 100 fg of genomic DNA under optimized conditions. Combined with a simplified alkaline lysis method for plant DNA extraction, the LF-RPA assay successfully detected P. cactorum in naturally diseased strawberry samples without specialized equipment within 40 min. Thus, the LF-RPA assay developed in this study is a rapid, simple, and accurate method for the detection of P. cactorum, with the potential for further application in resource-limited laboratories.

scholarly journals Development and validation of the isothermal recombinase polymerase amplification assays for rapid detection of Mycoplasma ovipneumoniae

2020 ◽  
Author(s):  
Jinfeng Wang ◽  
Ruiwen Li ◽  
Xiaoxia Sun ◽  
Libing Liu ◽  
Xuepiao Hao ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Lateral Flow ◽  
Recombinase Polymerase Amplification ◽  
Metal Bath ◽  
Lateral Flow Strip ◽  
The Real ◽  
Mycoplasmal Pneumonia ◽  
Mycoplasma Ovipneumoniae

Abstract Background: Mycoplasmal pneumonia is an important infectious disease that threatens sheep and goat production worldwide, and Mycoplasma ovipneumoniae is one of major etiological agent causing mycoplasmal pneumonia. Recombinase polymerase amplification (RPA) is an isothermal nucleic acid amplification technique, and RPA-based diagnostic assays have been described for the detection of different types of pathogens. Results: The RPA assays using real-time fluorescence detection (real-time RPA) and lateral flow strip detection (LFS RPA) were developed to detect M. ovipneumoniae targeting a conserved region of the 16S rRNA gene. Real-time RPA was performed in a portable florescence scanner at 39 °C for 20 min. LFS RPA was performed in a portable metal bath incubator at 39 °C for 15 min, and the amplicons were visualized with the naked eyes within 5 min on the lateral flow strip. Both assays were highly specific for M. ovipneumoniae , as there were no cross-reactions with other microorganisms tested, especially the pathogens involved in respiratory complex and other mycoplasmas frequently identified in ruminant s . The limit of detection of LFS RPA assay was 1.0×10 1 copies per reaction using a recombinant plasmid containing target gene as template, which is 10 times lower than the limit of detection of the real-time RPA and real-time PCR assays. The RPA assays were further validated on 111 clinical sheep nasal swab and fresh lung samples, and M. ovipneumoniae DNA was detected in 29 samples in the real-time RPA, 31 samples in the LFS RPA and 32 samples in the real-time PCR assay. Compared to real-time PCR, the real-time RPA and LFS RPA showed diagnostic specificity of 100% and 98.73%, diagnostic sensitivity of 90.63% and 93.75%, and a kappa coefficient of 0.932 and 0.934, respectively. Conclusions: The developed real-time RPA and LFS RPA assays provide the attractive and promising tools for rapid, convenient and reliable detection of M. ovipneumoniae , especially in resource-limited settings.

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.

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