Transcription Recombinase Polymerase Amplification Assay Research Articles

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.

Reverse transcription recombinase polymerase amplification assay for the rapid detection of type 2 porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogens in pigs, and has tremendous negative economic impact on the swine industry worldwide. PRRSV is classified into the two distinct genotypes: type 1 and type 2, and most of the described PRRSV isolates in China are type 2. Rapid and sensitive detection of PRRSV is of great importance for the disease control and regional eradication programs. Recombinase polymerase amplification (RPA) has emerged as a novel isothermal amplification technology for the molecular diagnosis of infectious diseases. In this study, a fluorescence reverse transcription RPA (RT-RPA) assay was developed to detect the type 2 PRRSV using primers and exo probe specific for the viral nucleocapsid gene. The reaction was performed at 40°C within 20min. The RT-RPA assay could detect both the classical (C-PRRSV) and highly pathogenic PRRSV (HP-PRRSV), but there was no cross-reaction to other pathogens. Using the in vitro transcribed PRRSV RNA as template, the analytical sensitivity of RT-RPA was 690 copies. The assay performance was evaluated by testing 60 field samples and compared to real-time RT-PCR. The detection rate of RT-RPA was 86.6% (52/60), while the detection rate of real-time RT-PCR was 83.3% (50/60). This simple, rapid and reliable method could be potentially applied for rapid detection of PRRSV in point-of-care and rural areas.

A Field-Deployable Reverse Transcription Recombinase Polymerase Amplification Assay for Rapid Detection of the Chikungunya Virus.

BackgroundChikungunya virus (CHIKV) is a mosquito-borne virus currently transmitted in about 60 countries. CHIKV causes acute flu-like symptoms and in many cases prolonged musculoskeletal and joint pain. Detection of the infection is mostly done using RT-RCR or ELISA, which are not suitable for point-of-care diagnosis.Methodology/Principal FindingsIn this study, a reverse transcription recombinase polymerase amplification (RT-RPA) assay for the detection of the CHIKV was developed. The assay sensitivity, specificity, and cross-reactivity were tested. CHIKV RT-RPA assay detected down to 80 genome copies/reaction in a maximum of 15 minutes. It successfully identified 18 isolates representing the three CHIKV genotypes. No cross-reactivity was detected to other alphaviruses and arboviruses except O'nyong'nyong virus, which could be differentiated by a modified RPA primer pair. Seventy-eight samples were screened both by RT-RPA and real-time RT-PCR. The diagnostic sensitivity and specificity of the CHIKV RT-RPA assay were determined at 100%.Conclusions/SignificanceThe developed RT-RPA assay represents a promising method for the molecular detection of CHIKV at point of need.

Rapid detection of highly pathogenic porcine reproductive and respiratory syndrome virus by a fluorescent probe-based isothermal recombinase polymerase amplification assay.

A novel fluorescent probe-based real-time reverse transcription recombinase polymerase amplification (real-time RT-RPA) assay was developed for rapid detection of highly pathogenic type 2 porcine reproductive and respiratory syndrome virus (HP-PRRSV). The sensitivity analysis showed that the detection limit of RPA was 70 copies of HP-PRRSV RNA/reaction. The real-time RT-RPA highly specific amplified HP-PRRSV with no cross-reaction with classic PRRSV, classic swine fever virus, pseudorabies virus, and foot-and-mouth disease virus. Assessment with 125 clinical samples showed that the developed real-time RT-RPA assay was well correlated with real-time RT-qPCR assays for detection of HP-PRRSV. These results suggest that the developed real-time RT-RPA assay is suitable for rapid detection of HP-PRRSV.

Development of reverse transcription recombinase polymerase amplification assay for avian influenza H5N1 HA gene detection

The 2006 outbreaks of H5N1 avian influenza in Egypt interrupted poultry production and caused staggering economic damage. In addition, H5N1 avian influenza viruses represent a significant threat to public health. Therefore, the rapid detection of H5 viruses is very important in order to control the disease. In this study, a qualitative reverse transcription recombinase polymerase amplification (RT-RPA) assay for the detection of hemagglutinin gene of H5 subtype influenza viruses was developed. The results were compared to the real-time reverse transcription polymerase chain reaction (RT-PCR). An in vitro transcribed RNA standard of 970 nucleotides of the hemagglutinin gene was developed and used to determine the assay sensitivity. The developed H5 RT-RPA assay was able to detect one RNA molecule within 7min, while in real-time RT-PCR, at least 90min was required. H5 RT-RPA assay did not detect nucleic acid extracted from H5 negative samples or from other pathogens producing respiratory manifestation in poultry. The clinical performance of the H5 RT-RPA assay was tested in 30 samples collected between 2014 and 2015; the sensitivity of H5 RT-RPA and real-time RT-PCR was 100%. In conclusion, H5 RT-RPA was faster than real-time RT-PCR and easily operable in a portable device. Moreover, it had an equivalent sensitivity and specificity.

Diagnostics-in-a-Suitcase: Development of a portable and rapid assay for the detection of the emerging avian influenza A (H7N9) virus

BackgroundIn developing countries, equipment necessary for diagnosis is only available in few central laboratories, which are less accessible and of limited capacity to test large numbers of incoming samples. Moreover, the transport conditions of samples are inadequate, therefore leading to unreliable results. ObjectivesThe development of a rapid, inexpensive, and simple test would allow mobile detection of viruses. Study designA suitcase laboratory “Diagnostics-in-a-Suitcase” (56cm×45.5cm×26.5cm) containing all reagents and devices necessary for performing a reverse transcription recombinase polymerase amplification (RT-RPA) assay was developed. As an example, two RT-RPA assays were established for the detection of hemagglutinin (H) and neuraminidase (N) genes of the novel avian influenza (H7N9) virus. ResultsThe sensitivities of the H7 and the N9 RT-RPA assays were 10 and 100 RNA molecules, respectively. The assays were performed at a single temperature (42°C). The results were obtained within 2–7min. The H7N9 RT-RPA assays did not show a cross-detection either of any other respiratory viruses affecting humans and/or birds or of the human or chicken genomes. All reagents were used, stored, and transported at ambient temperature, that is, cold chain independent. In addition, the Diagnostics-in-a-Suitcase was operated by a solar-powered battery. ConclusionsThe developed assay protocol and mobile setup performed well. Moreover, it can be easily implemented to perform diagnoses at airports, quarantine stations, or farms for rapid on-site viral nucleic acid detection.

Detection of dengue virus in ten minutes using reverse transcription recombinase polymerase amplification assay

Background: Over 2.5 billion are at risk fromdengue fever (DF). Early diagnosis of DF helps to diminish its burden on public health. The real-time reverse transcription polymerase chain reaction (RTPCR) is the standard formolecular detectionof denguevirus (DENV) in acute phase of DF. Real-time RT-PCR analysis is not suitable for on-site of outbreak screening. The PCR devices are large, expensive, and complex. In this study, a reverse transcription recombinase polymerase amplification (RT-RPA) assay was developed to detect DENV 1-4. Methods & Materials: RNA molecular standards comprised of the 3′nontranslated region of DENV 1-4 were prepared. The assay sensitivity was determined by analysis of eight assay runs of serial dilutions of the RNA molecular standard. The assay specificity was evaluated against a panel of viruses considered for differential diagnosis with DENV 1-4. The assay performance was validated by testing 31blood samples in comparison to real-timeRT-PCR. To test the possibility of using RT-RPA assays for outbreak investigations, a mobile RT-RPA unit was operated in Kedougou, Senegal. Results: The DENV 1-4 RT-RPA was rapid (3-10minutes). The analytical sensitivity was 100 and 10 RNA molecules for DENV 1-3 and DENV 4, respectively. No cross reactivity with other viruses causing similar clinical picture was observed. Both RT-RPA and RT-PCR assays showed comparable results (96% sensitivity). The RT-RPA assay was significantly quicker than the real-time RT-PCR assay (10minutes for RT-RPAand90minutes for real-timeRT-PCR). In a field study in Kedougou, Senegal, a magnetic-bead based total nucleic acid extraction method was combined with RT-RPA on a portable instrument (tubescanner). The RT-RPA assaywas successfully used to detect DENV RNA extracted from gamma-irradiation inactivated lyophilized virus supernatants. All operations were carried out at an ambient temperature of 38 ◦C with auxiliary electricity tapped from a vehicle. Conclusion: The developed DENV 1-4 RT-RPA assay constitutes a suitable accurate, sensitive, and rapid assay for DF diagnosis during the first 5 days of infection. Moreover, the use of a portable fluorescence-reading device extends its application potential to point-of-care use or at the outbreak site diagnosis.

Induction of nAbs and protection of mice immunized with VLP's expressing CHIKV env

Background: Over 2.5 billion are at risk fromdengue fever (DF). Early diagnosis of DF helps to diminish its burden on public health. The real-time reverse transcription polymerase chain reaction (RTPCR) is the standard formolecular detectionof denguevirus (DENV) in acute phase of DF. Real-time RT-PCR analysis is not suitable for on-site of outbreak screening. The PCR devices are large, expensive, and complex. In this study, a reverse transcription recombinase polymerase amplification (RT-RPA) assay was developed to detect DENV 1-4. Methods & Materials: RNA molecular standards comprised of the 3′nontranslated region of DENV 1-4 were prepared. The assay sensitivity was determined by analysis of eight assay runs of serial dilutions of the RNA molecular standard. The assay specificity was evaluated against a panel of viruses considered for differential diagnosis with DENV 1-4. The assay performance was validated by testing 31blood samples in comparison to real-timeRT-PCR. To test the possibility of using RT-RPA assays for outbreak investigations, a mobile RT-RPA unit was operated in Kedougou, Senegal. Results: The DENV 1-4 RT-RPA was rapid (3-10minutes). The analytical sensitivity was 100 and 10 RNA molecules for DENV 1-3 and DENV 4, respectively. No cross reactivity with other viruses causing similar clinical picture was observed. Both RT-RPA and RT-PCR assays showed comparable results (96% sensitivity). The RT-RPA assay was significantly quicker than the real-time RT-PCR assay (10minutes for RT-RPAand90minutes for real-timeRT-PCR). In a field study in Kedougou, Senegal, a magnetic-bead based total nucleic acid extraction method was combined with RT-RPA on a portable instrument (tubescanner). The RT-RPA assaywas successfully used to detect DENV RNA extracted from gamma-irradiation inactivated lyophilized virus supernatants. All operations were carried out at an ambient temperature of 38 ◦C with auxiliary electricity tapped from a vehicle. Conclusion: The developed DENV 1-4 RT-RPA assay constitutes a suitable accurate, sensitive, and rapid assay for DF diagnosis during the first 5 days of infection. Moreover, the use of a portable fluorescence-reading device extends its application potential to point-of-care use or at the outbreak site diagnosis.

A Portable Reverse Transcription Recombinase Polymerase Amplification Assay for Rapid Detection of Foot-and-Mouth Disease Virus

Foot-and-mouth disease (FMD) is a trans-boundary viral disease of livestock, which causes huge economic losses and constitutes a serious infectious threat for livestock farming worldwide. Early diagnosis of FMD helps to diminish its impact by adequate outbreak management. In this study, we describe the development of a real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay for the detection of FMD virus (FMDV). The FMDV RT-RPA design targeted the 3D gene of FMDV and a 260 nt molecular RNA standard was used for assay validation. The RT-RPA assay was fast (4–10 minutes) and the analytical sensitivity was determined at 1436 RNA molecules detected by probit regression analysis. The FMDV RT-RPA assay detected RNA prepared from all seven FMDV serotypes but did not detect classical swine fever virus or swine vesicular disease virus. The FMDV RT-RPA assay was used in the field during the recent FMD outbreak in Egypt. In clinical samples, reverse transcription polymerase chain reaction (RT-PCR) and RT-RPA showed a diagnostic sensitivity of 100% and 98%, respectively. In conclusion, FMDV RT-RPA was quicker and much easier to handle in the field than real-time RT-PCR. Thus RT-RPA could be easily implemented to perform diagnostics at quarantine stations or farms for rapid spot-of-infection detection.