RPA Assay Research Articles

Development and evaluation of serotype-specific recombinase polymerase amplification combined with lateral flow dipstick assays for the diagnosis of foot-and-mouth disease virus serotype A, O and Asia1

BackgroundFoot-and-mouth disease (FMD) caused by foot-and-mouth disease virus (FMDV) is one of the most highly infectious diseases in livestock, and leads to huge economic losses. Early diagnosis and rapid differentiation of FMDV serotype is therefore integral to the prevention and control of FMD. In this study, a series of serotype-specific reverse transcription recombinase polymerase amplification assays combined with lateral flow dipstick (RPA-LFD) were establish to differentiate FMDV serotypes A, O or Asia 1, respectively.ResultsThe serotype-specific primers and probes of RPA-LFD were designed to target conserved regions of the FMDV VP1 gene sequence, and three primer and probe sets of serotype-specific RPA-LFD were selected for amplification of FMDV serotypes A, O or Asia 1, respectively. Following incubation at 38 °C for 20 min, the RPA amplification products could be visualized by LFD. Analytical sensitivity of the RPA assay was then determined with ten-fold serial dilutions of RNA of VP1 gene and the recombinant vector respectively containing VP1 gene from FMDV serotypes A, O or Asia1, the detection limits of these assays were 3 copies of plasmid DNA or 50 copies of viral RNA per reaction. Moreover, the specificity of the assay was assessed, and there was no cross reactions with other viruses leading to bovine vesicular lesions. Furthermore, 126 clinical samples were respectively detected with RPA-LFD and real-time PCR (rPCR), there was 98.41% concordance between the two assays, and two samples were positive by RPA-LFD but negative in rPCR, these were confirmed as FMDV-positive through viral isolation in BHK-21 cells. It showed that RPA-LFD assay was more sensitive than the rPCR method in this study.ConclusionThe development of serotype-specific RPA-LFD assay provides a rapid, sensitive, and specific method for differentiation of FMDV serotype A, O or Asia1, respectively. It is possible that the serotype-specific RPA-LFD assay may be used as a integral protocol for field detection of FMDV.

Equipment-free recombinase polymerase amplification assay using body heat for visual and rapid point-of-need detection of canine parvovirus 2

A visible and equipment-free recombinase polymerase amplification assay combined with a lateral flow strip (LFS RPA) was developed to detect canine parvovirus type 2 (CPV-2), which is the etiological agent of canine parvovirus disease. The CPV-2 LFS RPA assay was developed based on the VP2 gene and is performed in a closed fist using body heat for 15 min; the products are visible to the naked eye on the LFS within 5 min. The assay could detect CPV-2a, CPV-2b and CPV-2c, and there was no cross-reaction with the other viruses tested. Using the standard CPV-2 DNA as a template, the analytical sensitivity was 1.0 × 102 copies per reaction, which was the same result as that of a real-time PCR. The assay performance was further evaluated by testing 60 canine fecal samples, and CPV-2 DNA was detected in 46 samples (76.7%, 46/60) by LFS RPA, which was the same result as that of the real-time PCR assay and higher than that of the SNAP method (48.3%, 29/60). The novel CPV-2 LFS RPA assay is an attractive and promising tool for rapid and convenient diagnosis of CPV disease, especially cage side and in underequipped laboratories.

Development of real-time and lateral flow dipstick recombinase polymerase amplification assays for rapid detection of goatpox virus and sheeppox virus

BackgroundGoatpox virus (GTPV) and sheeppox virus (SPPV), which belong to the Capripoxvirus (CaPV), are economically important pathogens of small ruminants. Therefore, a sensitive, specific and rapid diagnostic assay for detection of GTPV and SPPV is necessary to accurately and promptly control these diseases.MethodsRecombinase polymerase amplification (RPA) assays combined with a real-time fluorescent detection (real-time RPA assay) and lateral flow dipstick (RPA LFD assay) were developed targeting the CaPV G-protein-coupled chemokine receptor (GPCR) gene, respectively.ResultsThe sensitivity of both CaPV real-time RPA assay and CaPV RPA LFD assay were 3 × 102 copies per reaction within 20 min at 38 °C. Both assays were highly specific for CaPV, with no cross-reactions with peste des petits ruminants virus, foot-and-mouth disease virus and Orf virus. The evaluation of the performance of these two assays with clinical sample (n = 107) showed that the CaPV real-time RPA assay and CaPV RPA LFD assay were able to specially detect SPPV or GTPV present in samples of ovine in liver, lung, kidney, spleen, skin and blood.ConclusionsThis study provided a highly time-efficient and simple alternative for rapid detection of GTPV and SPPV.

Development of an isothermal recombinase polymerase amplification assay for rapid detection of pseudorabies virus

Recombinase polymerase amplification assays using real-time fluorescent detection (real-time RPA assay) and lateral flow dipstick (RPA LFD assay) were developed targeting the gD gene of pseudorabies virus (PRV). Both assays were performed at 39 °C within 20 min. The sensitivity of the real-time RPA assay and the RPA LFD assay was 100 copies per reaction and 160 copies per reaction, respectively. Both assays did not detect DNAs from other virus or PRV negative samples. Therefore, the developed RPA assays provide a rapid, simple, sensitive and specific alternative tool for detection of PRV.

Development of real-time and lateral flow strip reverse transcription recombinase polymerase Amplification assays for rapid detection of peste des petits ruminants virus

BackgroundPeste des petits ruminants (PPR) is an economically important, Office International des Epizooties (OIE) notifiable, transboundary viral disease of small ruminants such as sheep and goat. PPR virus (PPRV), a negative-sense single-stranded RNA virus, is the causal agent of PPR. Therefore, sensitive, specific and rapid diagnostic assay for the detection of PPRV are necessary to accurately and promptly diagnose suspected case of PPR.MethodsIn this study, reverse transcription recombinase polymerase amplification assays using real-time fluorescent detection (real-time RT-RPA assay) and lateral flow strip detection (LFS RT-RPA assay) were developed targeting the N gene of PPRV.ResultsThe sensitivity of the developed real-time RT-RPA assay was as low as 100 copies per reaction within 7 min at 40 °C with 95% reliability; while the sensitivity of the developed LFS RT-RPA assay was as low as 150 copies per reaction at 39 °C in less than 25 min. In both assays, there were no cross-reactions with sheep and goat pox viruses, foot-and-mouth disease virus and Orf virus.ConclusionsThese features make RPA assay promising candidates either in field use or as a point of care diagnostic technique.

A Novel Isothermal Assay of Borrelia burgdorferi by Recombinase Polymerase Amplification with Lateral Flow Detection.

A novel isothermal detection for recombinase polymerase amplification with lateral flow (LF-RPA) was established for Borrelia burgdorferi (B. burgdorferi) detection in this study. This assay with high sensitivity and specificity can get a visible result without any additional equipment in 30 min. We designed a pair of primers according to recA gene of B. burgdorferi strains and a methodology evaluation was performed. The results showed that the RPA assay based on the recA gene was successfully applied in B. burgdorferi detection, and its specific amplification was only achieved from the genomic DNA of B. burgdorferi. The detection limit of the new assay was about 25 copies of the B. burgdorferi genomic DNA. Twenty Lyme borreliosis patients’ serum samples were detected by LF-RPA assay, real-time qPCR and nested-PCR. Results showed the LF-RPA assay is more effective than nested-PCR for its shorter reaction time and considerably higher detection rate. This method is of great value in clinical rapid detection for Lyme borreliosis. Using the RPA assay might be a megatrend for DNA detection in clinics and endemic regions.

Rapid Detection of Salmonella Enterica Serovar Enteritidis from Eggs and Chicken Meat by Real‐Time Recombinase Polymerase Amplification in Comparison with the Two‐Step Real‐Time PCR

AbstractSalmonella is the most common cause of foodborne outbreaks throughout the world and Salmonella enterica serovar Enteritidis is one of the major causes of salmonellosis. We describe a rapid, sensitive and inexpensive method for the detection of S. Enteritidis from eggs and chicken meat using the real‐time recombinase polymerase amplification (Real‐time RPA) and compared this assay with the two‐step real‐time polymerase chain reaction (PCR) (Real‐time PCR). The detection sensitivity of the RPA and PCR assays was identical in pure culture, however, the reaction time was as short as 10 min compared with 40 min for the PCR assay. In food application, the detection sensitivity of the RPA assay was 10 cfu/g from eggs and 100 cfu/g from chicken samples without additional DNA purification steps before the amplification – therefore, the sensitivity of the RPA assay was 100 times greater from eggs and 10 times greater from chicken than the PCR assay.Practical ApplicationsNumerous epidemiological investigations attributed the source of sporadic Salmonella outbreaks as poultry and poultry‐by‐products including eggs. This is the first study in which a real‐time RPA was developed to detect the Salmonella Enteritidis specifically. The whole procedure is fairly simple and does not require specific equipment, making it potentially applicable in the monitoring and detection of S. Enteritidis in eggs and poultry products.

Development of a rapid recombinase polymerase amplification assay for the detection of Streptococcus pneumoniae in whole blood.

BackgroundStreptococcus pneumoniae is an important cause of microbial disease in humans. The introduction of multivalent vaccines has coincided with a dramatic decrease in the number of pneumococcal-related deaths. In spite of this, at a global level, pneumococcal infection remains an important cause of death among children under 5 years of age and in adults 65 years of age or older. In order to properly manage patients and control the spread of infection, a rapid and highly sensitive diagnostic method is needed for routine implementation, especially in resource-limited regions where pneumococcal disease is most prevalent.MethodsUsing the gene encoding leader peptidase A as a molecular diagnostics target, a real-time RPA assay was designed and optimised for the detection of S. pneumoniae in whole blood. The performance of the assay was compared to real-time PCR in terms of its analytical limit of detection and specificity. The inhibitory effect of human genomic DNA on amplification was investigated. The potential clinical utility of the assay was investigated using a small number of clinical samples.ResultsThe RPA assay has a limit of detection equivalent to PCR (4.0 and 5.1 genome equivalents per reaction, respectively) and was capable of detecting the equivalent of <1 colony forming unit of S. pneumoniae when spiked into human whole blood. The RPA assay was 100 % inclusive (38/38 laboratory reference strains and 19/19 invasive clinical isolates) and 100 % exclusive; differentiating strains of S. pneumoniae species from other viridans group streptococci, including S. pseudopneumoniae. When applied to the analysis of a small number (n = 11) of clinical samples (blood culture positive for S. pneumoniae), the RPA assay was demonstrated to be both rapid and sensitive.ConclusionsThe RPA assay developed in this work is shown to be as sensitive and as specific as PCR. In terms of reaction kinetics, the RPA assay is shown to exceed those of the PCR, with the RPA running to completion in 20 minutes and capable generating a positive signal in as little as 6 minutes. This work represents a potentially suitable assay for application in point-of-care settings.

Evaluation of three alternative methods for diagnosis of equine herpesvirus abortion

use of a labeled probe, to enable detection of amplified products using a LFD. The assay to identify T. equigenitalis successfully amplified a 121bp fragment of the 23S rDNA genes. The assay detected as few as 46 gene copies in 12 minutes. The assay did not cross-hybridise with any of the 38 different bacterial organisms tested. The RPA assay was tested against a panel of 60 clinical equine genital swabs. All of the culture negative DNA extracts were correctly identified. Of the 30 culture positive DNA extracts, 24 were correctly identified. Culture positive, RPA negative DNA extracts had high real-time PCR CT values, indicating low levels of target DNA present. The introduction of a second probe into the assay enabled the simultaneous detection and differentiation of T. equigenitalis and T. asinigenitalis. The T. equigenitalis assaywas successfully adapted to enable detection of amplification products LFDs. We have successfully developed a rapid and sensitive isothermal assay to identify T. equigenitalis. Additionally, the inclusion of a second probe enables the detection of T. asinigenitalis. The amplification products can be detected in real time or via LFD. This test provides a fast (under 15 minutes) and accurate means of identification. The low energy requirements, lack of expensive equipment and LFD readout makes this test a suitable candidate for rapid diagnosis in low technology laboratories or near to the point of sampling.

Selection and validation of a real-time rt-PCR assay for the detection of equine arteritis virus and preliminary investigations into the prevalence of carrier stallions in the Netherlands

use of a labeled probe, to enable detection of amplified products using a LFD. The assay to identify T. equigenitalis successfully amplified a 121bp fragment of the 23S rDNA genes. The assay detected as few as 46 gene copies in 12 minutes. The assay did not cross-hybridise with any of the 38 different bacterial organisms tested. The RPA assay was tested against a panel of 60 clinical equine genital swabs. All of the culture negative DNA extracts were correctly identified. Of the 30 culture positive DNA extracts, 24 were correctly identified. Culture positive, RPA negative DNA extracts had high real-time PCR CT values, indicating low levels of target DNA present. The introduction of a second probe into the assay enabled the simultaneous detection and differentiation of T. equigenitalis and T. asinigenitalis. The T. equigenitalis assaywas successfully adapted to enable detection of amplification products LFDs. We have successfully developed a rapid and sensitive isothermal assay to identify T. equigenitalis. Additionally, the inclusion of a second probe enables the detection of T. asinigenitalis. The amplification products can be detected in real time or via LFD. This test provides a fast (under 15 minutes) and accurate means of identification. The low energy requirements, lack of expensive equipment and LFD readout makes this test a suitable candidate for rapid diagnosis in low technology laboratories or near to the point of sampling.

A homogeneous method to quantify mRNA levels: a hybridization of RNase protection and scintillation proximity assay technologies

A novel method to measure mRNA levels has been developed by combining the detection capabilities of RNase protection (RPA) with the quantification advantages of scintillation proximity assay (SPA) technology. Sample processing is reduced to the addition of a single reagent post RNase digestion. As a model system, the inducible expression of rat apolipoprotein-A1 mRNA has been measured by both traditional gel-based RPAs and the SPA-based RPA assay. Results demonstrate that the ribonuclease protection proximity assay (RiPPA) faithfully reproduces the gel-based results and is at least as sensitive as many existing methods.