Rapid and visual detection of porcine deltacoronavirus by recombinase polymerase amplification combined with a lateral flow dipstick.

BACKGROUND: Porcine Deltacoronavirus (PDCoV) is a newly emerging Coronavirus that was first identified in 2012 in Hong Kong, China. Since then, PDCoV has subsequently been reported worldwide, causing a high number of neonatal piglet deaths and significant economic losses to the swine industry. Therefore, it is necessary to establish a highly sensitive and specific method for the rapid diagnosis of PDCoV. RESULTS: In the present study, a highly sensitive and specific diagnostic method using recombinase polymerase amplification combined with a lateral flow dipstick (LFD-RPA) was developed for rapid and visual detection of PDCoV. The system can be performed under a broad range of temperature conditions from 10 to 37 °C, and the detection of PDCoV can be completed in 10 min at 37 °C. The sensitivity of this assay was 10 times higher than that of conventional PCR with a lower detection limit of 1 × 102 copies/µl of PDCoV. Meanwhile, the LFD-RPA assay specifically amplified PDCoV, while there was no cross-amplification with other swine-associated viruses, including Porcine epidemic diarrhea virus (PEDV), Transmissible gastroenteritis virus (TGEV), Porcine kobuvirus (PKoV), Foot and mouth disease virus (FMDV), Porcine reproductive and respiratory syndrome virus (PRRSV), Porcine circovirus type 2 (PCV2), Classical swine fever virus (CSFV) and Seneca valley virus (SVV). The repeatability of the test results indicated that this assay had good repeatability. In addition, 68 clinical samples (48 fecal swab specimens and 20 intestinal specimens) were further tested by LFD-RPA and RT-PCR assay. The positive rate of LFD-RPA clinical samples was 26.47% higher than that of conventional PCR (23.53%). CONCLUSIONS: The LFD-RPA assay successfully detected PDCoV in less than 20 min in this study, providing a potentially valuable tool to improve molecular detection for PDCoV and to monitor the outbreak of PDCoV, especially in low-resource areas and laboratories.

Rapid and specific detection of porcine parvovirus by isothermal recombinase polymerase amplification assays.

Porcine parvovirus (PPV) is a major cause of swine reproductive failure and reported in many countries worldwide. Recombinase polymerase amplification (RPA) assays using a real-time fluorescent detection (PPV real-time RPA assay) and a lateral flow dipstick (PPV RPA LFD assay) were developed targeting PPV NS1 gene. The detection limit of PPV real-time RPA assay was 300 copies per reaction within 9 min at 38 °C, while the RPA LFD assay has a detection limit of 400 copies per reaction in less than 20 min at 38 °C. In both assays, there were no cross-reactions with porcine circovirus type 2, pseudorabies virus, porcine reproductive and respiratory syndrome virus, classical swine fever virus, and foot-and-mouth disease virus. Based on a total of 128 clinical samples examined, the sensitivity and the specificity of the developed RPA assays for identification of PPV was 94.4% and 100%, respectively, when compared to real-time (qPCR) assay. Therefore, the RPA assay provides a rapid, sensitive and specific alternative for PPV detection.

Development of a fluorescent probe-based recombinase polymerase amplification assay for rapid detection of Orf virus.

BACKGROUND: Orf virus (ORFV) is the causative agent of Orf (also known as contagious ecthyma or contagious papular dermatitis), a severe infectious skin disease in goats, sheep and other ruminants. The rapid detection of ORFV is of great importance in disease control and highly needed. A isothermal molecular diagnostic approach, termed recombinase polymerase amplification (RPA), is considered as an novel and rapid alternative techonology to PCR assay. RESULTS: In the present study, a novel fluorescent probe based on RPA assay (ORFV exo RPA assay) was developed. The developed ORFV exo RPA assay was capable of as low as 100 copies of ORFV DNA /reaction and was highly specific, with no cross-reaction with closely related viruses (capripox virus, foot-and-mouth disease virus or peste des petits ruminants virus). Further assessment with clinical samples showed that the developed ORFV exo RPA assay has good correlation with qPCR assays for detection of ORFV. CONCLUSIONS: These results suggest that the developed ORFV exo RPA assay is suitable for rapid detection of ORFV.

FLP-FRT-based method to obtain unmarked deletions of CHU_3237 (porU) and large genomic fragments of Cytophaga hutchinsonii.

Cytophaga hutchinsonii is a widely distributed cellulolytic bacterium in the phylum Bacteroidetes. It can digest crystalline cellulose rapidly without free cellulases or cellulosomes. The mechanism of its cellulose utilization remains a mystery. We developed an efficient method based on a linear DNA double-crossover and FLP-FRT recombination system to obtain unmarked deletions of both single genes and large genomic fragments in C. hutchinsonii. Unmarked deletion of CHU_3237 (porU), an ortholog of the C-terminal signal peptidase of a type IX secretion system (T9SS), resulted in defects in colony spreading, cellulose degradation, and protein secretion, indicating that it is a component of the T9SS and that T9SS plays an important role in cellulose degradation by C. hutchinsonii. Furthermore, deletions of four large genomic fragments were obtained using our method, and the sizes of the excised fragments varied from 9 to 19 kb, spanning from 6 to 22 genes. The customized FLP-FRT method provides an efficient tool for more rapid progress in the cellulose degradation mechanism and other physiological aspects of C. hutchinsonii.

An enhanced isothermal amplification assay for viral detection.

Rapid, inexpensive, robust diagnostics are essential to control the spread of infectious diseases. Current state of the art diagnostics are highly sensitive and specific, but slow, and require expensive equipment. Here we report the development of a molecular diagnostic test for SARS-CoV-2 based on an enhanced recombinase polymerase amplification (eRPA) reaction. eRPA has a detection limit on patient samples down to 5 viral copies, requires minimal instrumentation, and is highly scalable and inexpensive. eRPA does not cross-react with other common coronaviruses, does not require RNA purification, and takes ~45 min from sample collection to results. eRPA represents a first step toward at-home SARS-CoV-2 detection and can be adapted to future viruses within days of genomic sequence availability.

Development of real-time reverse transcription recombinase polymerase amplification (RPA) for rapid detection of peste des petits ruminants virus in clinical samples and its comparison with real-time PCR test.

Peste des petits ruminants (PPR), caused by small ruminant morbillivirus (SRMV), formerly called peste des petits ruminants virus (PPRV), is one of the most important pathogens in small ruminants, and has tremendous negative economic impact on the sheep industry worldwide. Current detection of PPRV in clinical samples mainly relies on real-time RT-PCR. Particularly, samples collected from rural area require highly equipped laboratories for screening. A rapid, real-time reverse-transcription recombinase polymerase amplification assay (RT-RPA), employing primers and exo probe, was thus developed to perform at 42 °C for 20 min, and the detection limit at 95% probability was 14.98 copies per reaction and 0.326 TCID50/mL based on plasmid copy number and tissue culture infectivity titre. All the four lineages of PPRV could be detected with no cross-reaction to other pathogens including measles virus (MeV), goatpox virus (GTPV), canine distemper virus (CDV), foot-and-mouth disease virus (FMDV) and Mycoplasma capricolum subsp. capripneumoniae (Mccp). The performance of real-time RT-RPA assay was validated by testing 138 field samples and compared to real-time RT-PCR. The results indicated an excellent diagnostic agreement between real-time RT-RPA and a reference real-time RT-PCR method with the kappa value of 0.968. Compared to real-time RT-PCR, the sensitivity of real-time RT-RPA was 100%, while the specificity was 97.80%. The developed RT-RPA assay offers a promising platform for simple, rapid, and reliable detection of PPRV, especially in the resource-limited settings.

Development of Isothermal Recombinase Polymerase Amplification Assay for Rapid Detection of Porcine Circovirus Type 2.

Porcine circovirus virus type II (PCV2) is the etiology of postweaning multisystemic wasting syndrome (PMWS), porcine dermatitis, nephropathy syndrome (PDNS), and necrotizing pneumonia. Rapid diagnosis tool for detection of PCV2 plays an important role in the disease control and eradication program. Recombinase polymerase amplification (RPA) assays using a real-time fluorescent detection (PCV2 real-time RPA assay) and RPA combined with lateral flow dipstick (PCV2 RPA LFD assay) were developed targeting the PCV2 ORF2 gene. The results showed that the sensitivity of the PCV2 real-time RPA assay was 102 copies per reaction within 20 min at 37°C and the PCV2 RPA LFD assay had a detection limit of 102 copies per reaction in less than 20 min at 37°C. Both assays were highly specific for PCV2, with no cross-reactions with porcine circovirus virus type 1, foot-and-mouth disease virus, pseudorabies virus, porcine parvovirus, porcine reproductive and respiratory syndrome virus, and classical swine fever virus. Therefore, the RPA assays provide a novel alternative for simple, sensitive, and specific identification of PCV2.

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.

A paper and plastic device for performing recombinase polymerase amplification of HIV DNA.

Despite the importance of early diagnosis and treatment of HIV, only a small fraction of HIV-exposed infants in low- and middle-income countries are tested for the disease. The gold standard for early infant diagnosis, DNA PCR, requires resources that are unavailable in poor settings, and no point-of-care HIV DNA test is currently available. We have developed a device constructed of layers of paper, glass fiber, and plastic that is capable of performing isothermal, enzymatic amplification of HIV DNA. The device is inexpensive, small, light-weight, and easy to assemble. The device stores lyophilized enzymes, facilitates mixing of reaction components, and supports recombinase polymerase amplification in five steps of operation. Using commercially available lateral flow strips as a detection method, we demonstrate the ability of our device to amplify 10 copies of HIV DNA to detectable levels in 15 min. Our results suggest that our device, which is designed to be used after DNA extraction from dried-blood spots, may serve in conjunction with lateral flow strips as part of a point-of-care HIV DNA test to be used in low resource settings.

Recombinase-mediated cassette exchange reveals the selective use of Gq/G11-dependent and -independent endothelin 1/endothelin type A receptor signaling in pharyngeal arch development.

The endothelin (Edn) system comprises three ligands (Edn1, Edn2 and Edn3) and their G-protein-coupled type A (Ednra) and type B (Ednrb) receptors. During embryogenesis, the Edn1/Ednra signaling is thought to regulate the dorsoventral axis patterning of pharyngeal arches via Dlx5/Dlx6 upregulation. To further clarify the underlying mechanism, we have established mice in which gene cassettes can be efficiently knocked-in into the Ednra locus using recombinase-mediated cassette exchange (RMCE) based on the Cre-lox system. The first homologous recombination introducing mutant lox-flanked Neo resulted in homeotic transformation of the lower jaw to an upper jaw, as expected. Subsequent RMCE-mediated knock-in of lacZ targeted its expression to the cranial/cardiac neural crest derivatives as well as in mesoderm-derived head mesenchyme. Knock-in of Ednra cDNA resulted in a complete rescue of craniofacial defects of Ednra-null mutants. By contrast, Ednrb cDNA could not rescue them except for the most distal pharyngeal structures. At early stages, the expression of Dlx5, Dlx6 and their downstream genes was downregulated and apoptotic cells distributed distally in the mandible of Ednrb-knock-in embryos. These results, together with similarity in craniofacial defects between Ednrb-knock-in mice and neural-crest-specific Galpha(q)/Galpha(11)-deficient mice, indicate that the dorsoventral axis patterning of pharyngeal arches is regulated by the Ednra-selective, G(q)/G(11)-dependent signaling, while the formation of the distal pharyngeal region is under the control of a G(q)/G(11)-independent signaling, which can be substituted by Ednrb. This RMCE-mediated knock-in system can serve as a useful tool for studies on gene functions in craniofacial development.