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Development of a panel of recombinase polymerase amplification assays for detection of biothreat agents.

ABSTRACT: Syndromic panels for infectious disease have been suggested to be of value in point-of-care diagnostics for developing countries and for biodefense. To test the performance of isothermal recombinase polymerase amplification (RPA) assays, we developed a panel of 10 RPAs for biothreat agents. The panel included RPAs for Francisella tularensis, Yersinia pestis, Bacillus anthracis, variola virus, and reverse transcriptase RPA (RT-RPA) assays for Rift Valley fever virus, Ebola virus, Sudan virus, and Marburg virus. Their analytical sensitivities ranged from 16 to 21 molecules detected (probit analysis) for the majority of RPA and RT-RPA assays. A magnetic bead-based total nucleic acid extraction method was combined with the RPAs and tested using inactivated whole organisms spiked into plasma. The RPA showed comparable sensitivities to real-time RCR assays in these extracts. The run times of the assays at 42°C ranged from 6 to 10 min, and they showed no cross-detection of any of the target genomes of the panel nor of the human genome. The RPAs therefore seem suitable for the implementation of syndromic panels onto microfluidic platforms.

SUBMITTER: Euler M

PROVIDER: S-EPMC3666764 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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Development of a panel of recombinase polymerase amplification assays for detection of biothreat agents.

Euler Milena M Wang Yongjie Y Heidenreich Doris D Patel Pranav P Strohmeier Oliver O Hakenberg Sydney S Niedrig Matthias M Hufert Frank T FT Weidmann Manfred M

Journal of clinical microbiology 20130123 4

Syndromic panels for infectious disease have been suggested to be of value in point-of-care diagnostics for developing countries and for biodefense. To test the performance of isothermal recombinase polymerase amplification (RPA) assays, we developed a panel of 10 RPAs for biothreat agents. The panel included RPAs for Francisella tularensis, Yersinia pestis, Bacillus anthracis, variola virus, and reverse transcriptase RPA (RT-RPA) assays for Rift Valley fever virus, Ebola virus, Sudan virus, and ...[more]

PMID: 23345286

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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.

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:BackgroundAccurate diagnosis followed by timely treatment is an effective strategy for the prevention of complications together with reducing schistosomiasis transmission. Recombinase Polymerase Amplification (RPA) is a simple, rapid, sensitive, and specific isothermal method with low resource needs. This research aimed at the development and optimisation of a real-time (RT) and a lateral flow (LF) RPA assay for the detection of Schistosoma mansoni.MethodologyRecombinase Polymerase Amplification reactions were performed at full- (as recommended) and half-volumes (to reduce costs), with RT or LF detection systems targeting the S. mansoni mitochondrial minisatellite region. The specificity was assessed using gDNA from other Schistosoma species, helminths co-endemic with S. mansoni, human stool, and urine, and Biomphalaria snail hosts. The analytical sensitivity was evaluated using serial dilutions of gDNA, synthetic copies of the target, and single eggs. The ability of both assays to detect the S. mansoni DNA in human urine and stool samples was also tested. The long-term stability of the RT-RPA reagents was evaluated by storing the reaction components in different temperature conditions for up to 3 weeks.ResultsThe RT- and the LF-RPA (SmMIT- and SmMIT-LF-RPA, respectively) presented similar results when used full- and half-volumes, thus the latter was followed in all experiments. The SmMIT-RPA was 100% specific to S. mansoni, able to detect a single egg, with a limit of detection (LOD) of down to 1 fg of gDNA and one synthetic copy of the target. The assay was able to detect S. mansoni DNA from stool containing 1 egg/g and in spiked urine at a concentration of 10 fg/μl. SmMIT-RPA reagents were stable for up to 3 weeks when kept at 19°C, and 2 weeks when stored at 27°C. The SmMIT-LF-RPA cross-reacted with Clinostomidae, presented the LOD of 10 fg and one synthetic copy of the target, being able to detect a single egg and 1 egg/g in a stool sample. The LOD in spiked urine samples was 10 pg/μl.ConclusionThe half-volume SmMIT-RPA is a promising method to be used in the field. It is specific, sensitive, robust, and tolerant to inhibitors, with a long-term stability of the reaction components and the real-time visualisation of results.

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Development of a panel of recombinase polymerase amplification assays for detection of biothreat agents.

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Development of a panel of recombinase polymerase amplification assays for detection of biothreat agents.

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