Dataset Information

PrimedRPA: primer design for recombinase polymerase amplification assays.

ABSTRACT:

Summary

Recombinase polymerase amplification (RPA), an isothermal nucleic acid amplification method, is enhancing our ability to detect a diverse array of pathogens, thereby assisting the diagnosis of infectious diseases and the detection of microorganisms in food and water. However, new bioinformatics tools are needed to automate and improve the design of the primers and probes sets to be used in RPA, particularly to account for the high genetic diversity of circulating pathogens and cross detection of genetically similar organisms. PrimedRPA is a python-based package that automates the creation and filtering of RPA primers and probe sets. It aligns several sequences to identify conserved targets, and filters regions that cross react with possible background organisms.

Availability and implementation

PrimedRPA was implemented in Python 3 and supported on Linux and MacOS and is freely available from http://pathogenseq.lshtm.ac.uk/PrimedRPA.html.

Supplementary information

Supplementary data are available at Bioinformatics online.

SUBMITTER: Higgins M

PROVIDER: S-EPMC6379019 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Publications

PrimedRPA: primer design for recombinase polymerase amplification assays.

Higgins Matthew M Ravenhall Matt M Ward Daniel D Phelan Jody J Ibrahim Amy A Forrest Matthew S MS Clark Taane G TG Campino Susana S

Bioinformatics (Oxford, England) 20190201 4

<h4>Summary</h4>Recombinase polymerase amplification (RPA), an isothermal nucleic acid amplification method, is enhancing our ability to detect a diverse array of pathogens, thereby assisting the diagnosis of infectious diseases and the detection of microorganisms in food and water. However, new bioinformatics tools are needed to automate and improve the design of the primers and probes sets to be used in RPA, particularly to account for the high genetic diversity of circulating pathogens and cr ...[more]

PMID: 30101342

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Project description:BackgroundBovine rotavirus A (BRVA) is considered to be the most common pathogen of severe diarrhea in cattle worldwide, which could lead to the death of newborn calves and cause the significant economic losses to the cattle industry. As a novel isothermal nucleic acid amplification technique, recombinase polymerase amplification (RPA) has been applied widely for the rapid detection of different important pathogens in human and animals.ResultsAn RT-RPA assay based on the real time fluorescence monitoring (real-time RT-RPA) and an RT-RPA assay combined with a lateral flow strip (LFS RT-RPA) were successfully developed by targeting the VP6 gene of BRVA. The RT-RPA assays allowed the exponential amplification of the target fragment in 20 min. After incubation of the LFS RT-RPA on a metal bath at 40 °C, the results were displayed on the lateral flow strip within 5 min, while real-time RT-RPA allowed the real-time observation of the results in Genie III at 42 °C. Both of the two assays showed high specificity for BRVA without any cross-reaction with the other tested pathogens causing diarrhea in cattle. With the standard RNA of BRVA serving as a template, the limit of detection for real-time RT-RPA and LFS RT-RPA were 1.4 × 102 copies per reaction and 1.4 × 101 copies per reaction, respectively. In the 134 fecal samples collected from cattle with diarrhea, the BRVA positive rate were 45.52% (61/134) and 46.27% (62/134) in real-time RT-RPA and LFS RT-RPA, respectively. Compared to a previously published real-time PCR, the real-time RT-RPA and LFS RT-RPA showed a diagnostic specificity of 100%, diagnostic sensitivity of 98.39% and 100%, and a kappa coefficient of 0.985 and 1.0, respectively.ConclusionsIn this study, BRVA was successfully detected in cattle fecal samples by the developed real-time RT-RPA and LFS RT-RPA assays. The developed RT-RPA assays had great potential for the rapid detection of BRVA in under-equipped diagnostic laboratory and the point-of-need diagnosis at quarantine stations and farms, which is of great importance to control BRVA-associated diarrhea in cattle herds.

Project description: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 ruminants. The limit of detection of LFS RPA assay was 1.0?×?101 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 in sheep, especially in resource-limited settings. However, the effectiveness of the developed RPA assays in the detection of M. ovipneumoniae in goats needs to be further validated.

Dataset Information

PrimedRPA: primer design for recombinase polymerase amplification assays.

Summary

Availability and implementation

Supplementary information

Publications

PrimedRPA: primer design for recombinase polymerase amplification assays.

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