Recombinase Polymerase Amplification assays for detection of the major tropical root-knot nematodes.
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ABSTRACT:
SUBMITTER: Subbotin SA
PROVIDER: S-EPMC8748504 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Infectious bovine rhinotracheitis virus (IBRV) is a major pathogen in cattle and has led to significant economic losses to the dairy industry worldwide, and therefore a more optimal method for the rapid diagnosis of IBRV infection is highly needed. In this study, we described the development of a lateral flow dipstrip (LFD) of isothermal recombinase polymerase amplification (RPA) method for rapid detection of IBRV.Distinct regions were selected as a candidate target for designing the LFD-RPA primers and probes. The analytical sensitivity of the RPA assay was determined using ten-fold serially diluted IBRV DNA. The specificity of the assay was assessed with other viral pathogens of cattle with similar clinic and other herpesviruses. The clinical performance was evaluated by testing 106 acute-phase high fever clinical specimens.RPA primers and probe were designed to target the specific conserved UL52 region fragment of IBRV. The detection could be completed at a constant temperature of 38 °C for 25 min, and the amplification products were easily visualized on a simple LFD. The detection limit of this assay was 5 copies per reaction of IBRV DNA and there was no cross-reactivity with other viruses causing bovine gastrointestinal and respiratory infections or other herpesviruses. The assay performance on acute-phase high fever clinical samples collected from cattle with no vaccine against IBRV, which were suspected to be infected with IBRV, was validated by detecting 24 fecal, 36 blood, 38 nasal swab and 8 tissue specimens, and compared with SYBR Green I based real-time PCR. The coincidence between IBRV LFD-RPA and real-time PCR was 100%.IBRV LFD-RPA was fast and much easier to serve as an alternative to the common measures used for IBRV diagnosis, as there is reduction in the use of instruments for identification of the infected animals. In addition, this assay may be the potential candidate to be used as point-of-care diagnostics in the field.
| S-EPMC5729238 | biostudies-literature
Project description:SummaryRecombinase 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 implementationPrimedRPA was implemented in Python 3 and supported on Linux and MacOS and is freely available from http://pathogenseq.lshtm.ac.uk/PrimedRPA.html.Supplementary informationSupplementary data are available at Bioinformatics online.
| S-EPMC6379019 | biostudies-literature
Project description:Sensitive, specific and rapid diagnostic tests for the detection of Orientia tsutsugamushi (O. tsutsugamushi) and Rickettsia typhi (R. typhi), the causative agents of scrub typhus and murine typhus, respectively, are necessary to accurately and promptly diagnose patients and ensure that they receive proper treatment. Recombinase polymerase amplification (RPA) assays using a lateral flow test (RPA-nfo) and real-time fluorescent detection (RPA-exo) were developed targeting the 47-kDa gene of O. tsutsugamushi or 17 kDa gene of R. typhi. The RPA assay was capable of detecting O. tsutsugamushi or R. typhi at levels comparable to that of the quantitative PCR method. Both the RPA-nfo and RPA-exo methods performed similarly with regards to sensitivity when detecting the 17 kDa gene of R. typhi. On the contrary, RPA-exo performed better than RPA-nfo in detecting the 47 kDa gene of O. tsutsugamushi. The clinical performance of the O. tsutsugamushi RPA assay was evaluated using either human patient samples or infected mouse samples. Eight out of ten PCR confirmed positives were determined positive by RPA, and all PCR confirmed negative samples were negative by RPA. Similar results were obtained for R. typhi spiked patient sera. The assays were able to differentiate O. tsutsugamushi and R. typhi from other phylogenetically related bacteria as well as mouse and human DNA. Furthermore, the RPA-nfo reaction was completed in 20 minutes at 37°C followed by a 10 minute incubation at room temperature for development of an immunochromatographic strip. The RPA-exo reaction was completed in 20 minutes at 39°C. The implementation of a cross contamination proof cassette to detect the RPA-nfo fluorescent amplicons provided an alternative to regular lateral flow detection strips, which are more prone to cross contamination. The RPA assays provide a highly time-efficient, sensitive and specific alternative to other methods for diagnosing scrub typhus or murine typhus.
| S-EPMC4498641 | biostudies-literature
Project description: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.
| S-EPMC3666764 | biostudies-literature
Project description:Improved access to effective tests for diagnosing tuberculosis (TB) has been designated a public health priority by the World Health Organisation. In high burden TB countries nucleic acid based TB tests have been restricted to centralised laboratories and specialised research settings. Requirements such as a constant electrical supply, air conditioning and skilled, computer literate operators prevent implementation of such tests in many settings. Isothermal DNA amplification technologies permit the use of simpler, less energy intensive detection platforms more suited to low resource settings that allow the accurate diagnosis of a disease within a short timeframe. Recombinase Polymerase Amplification (RPA) is a rapid, low temperature isothermal DNA amplification reaction. We report here RPA-based detection of Mycobacterium tuberculosis complex (MTC) DNA in <20 minutes at 39 °C. Assays for two MTC specific targets were investigated, IS6110 and IS1081. When testing purified MTC genomic DNA, limits of detection of 6.25 fg (IS6110) and 20 fg (IS1081)were consistently achieved. When testing a convenience sample of pulmonary specimens from suspected TB patients, RPA demonstrated superior accuracy to indirect fluorescence microscopy. Compared to culture, sensitivities for the IS1081 RPA and microscopy were 91.4% (95%CI: 85, 97.9) and 86.1% (95%CI: 78.1, 94.1) respectively (n = 71). Specificities were 100% and 88.6% (95% CI: 80.8, 96.1) respectively. For the IS6110 RPA and microscopy sensitivities of 87.5% (95%CI: 81.7, 93.2) and 70.8% (95%CI: 62.9, 78.7) were obtained (n = 90). Specificities were 95.4 (95% CI: 92.3,98.1) and 88% (95% CI: 83.6, 92.4) respectively. The superior specificity of RPA for detecting tuberculosis was due to the reduced ability of fluorescence microscopy to distinguish Mtb complex from other acid fast bacteria. The rapid nature of the RPA assay and its low energy requirement compared to other amplification technologies suggest RPA-based TB assays could be of use for integration into a point-of-care test for use in resource constrained settings.
| S-EPMC4138011 | biostudies-literature
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.
| S-EPMC7268655 | biostudies-literature
Project description:This study aimed to detetct Mycoplasma bovis (M. bovis) in bovine milk quickly and directly by developing and validating isothermal recombinase polymerase amplification (RPA) assays. Targeting the uvrC gene of M. bovis, an RPA assay based on the fluorescence monitoring (real-time RPA) and an RPA assay combined with a lateral flow strip (LFS RPA) were conducted. It took 20 min for the real-time RPA to finish in a Genie III at 39°C, and 15 min were required to perform the LFS RPA in an incubator block at 39°C, followed by the visualization of the products on the lateral flow strip within 5 min. Both of the two assays showed high specificity for M. bovis without any cross-reaction with the other tested pathogens. With the standard recombinant plasmid pMbovis-uvrC serving as a template, both RPA assays had a limit of detcion of 1.0 × 101 copies per reaction, equivalent to that of a real-time PCR assay. In the 65 milk samples collected from cattle with mastitis, the M. bovis genomic DNA was detected in 24 samples by both the real-time RPA and the LFS RPA assays. The developed RPA assays could detect M. bovis in bovine milk in an efficient, convenient, and credible manner as attractive and promising tools, and the assays would be helpful in the rapid response to M. bovis infection causing bovine mastitis.
| S-EPMC7946833 | biostudies-literature
Project description:BackgroundCanine distemper virus (CDV) is an enveloped negative-strand RNA virus that exhibits a high mutation rate and continuously expands the range of hosts. Notably, CDV has infected giant panda with spill over from viral reservoirs in canines. Giant pandas (Ailuropoda melanoleuca), especially captive pandas, are known to be susceptible to natural infection with CDV. The high fatality rate of CDV poses a serious threat to the safety of the giant panda population. However, vaccines or drugs for canine distemper in giant pandas have not been developed to date. Therefore, a rapid test that can achieve accurate onsite detection of CDV is important to enable the timely implementation of control measures. In this study, we established a nucleic acid visualization assay for targeting the CDV N gene by using combines reverse transcription recombinase polymerase amplification with a closed vertical flow visualization strip (RT-RPA-VF).ResultsThe RT-RPA-VF assay does not require sophisticated equipment, and it was determined to provide rapid detection at 35 °C for 30 min, while the limit of detection was 5 × 101 copies/μl RNA transcripts and 100.5 TCID50 ml- 1 viruses. The results showed that the assay was high specific to CDV and had no cross-reactivity with other viruses infecting the giant panda. Compared with RT-qPCR, RT-RPA-VF assay had a sensitivity of 100% and a specificity of 100% in 29 clinical samples. The coincidence rate between RT-RPA-VF and RT-qPCR was 100% (kappa = 1), indicating that the RT-RPA-VF assay possessed good diagnostic performance on clinical samples.ConclusionsThe RT-RPA-VF provides a novel alternative for the simple, sensitive, and specific identification of CDV and showed great potential for point of care diagnostics for captive and wild giant panda.
| S-EPMC8063184 | biostudies-literature
Project description:AimsTo develop and evaluate the performance of a panel of isothermal real-time recombinase polymerase amplification (RPA) assays for detection of common bacterial urinary tract infection (UTI) pathogens.Methods and resultsThe panel included RPAs for Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa and Enterococcus faecalis. All five RPAs required reaction times of under 12 min to reach their lower limit of detection of 100 genomes per reaction or less, and did not cross-react with high concentrations of nontarget bacterial genomic DNA. In a 50-sample retrospective clinical study, the five-RPA assay panel was found to have a specificity of 100% (95% CI, 78-100%) and a sensitivity of 89% (95% CI, 75-96%) for UTI detection.ConclusionsThe analytical and clinical validity of RPA for the rapid and sensitive detection of common UTI pathogens was established.Significance and impact of the studyRapid identification of the causative pathogens of UTIs can be valuable in preventing serious complications by helping avoid the empirical treatment necessitated by traditional urine culture's 48-72-h turnaround time. The routine and widespread use of RPA to supplement or replace culture-based methods could profoundly impact UTI management and the emergence of multidrug-resistant pathogens.
| S-EPMC5540129 | biostudies-literature
Project description:Several real-time PCR approaches to develop field detection for Francisella tularensis, the infectious agent causing tularemia, have been explored. We report the development of a novel qualitative real-time isothermal recombinase polymerase amplification (RPA) assay for use on a small ESEQuant Tube Scanner device. The analytical sensitivity and specificity were tested using a plasmid standard and DNA extracts from infected rabbit tissues. The assay showed a performance comparable to real-time PCR but reduced the assay time to 10 min. The rapid RPA method has great application potential for field use or point-of-care diagnostics.
| S-EPMC3405570 | biostudies-literature