Development of a One-Step Real-Time TaqMan Reverse Transcription Polymerase Chain Reaction (RT-PCR) Assay for the Detection of the Novel Variant Infectious Bursal Disease Virus (nVarIBDV) Circulating in China.

The novel variant IBDV (nVarIBDV, especially genotype A2dB1) mainly affects broilers in China. It causes an infection characterized by the atrophy of the bursa, a decrease in the level of lymphocytes, proliferation of fibrous tissue around the follicle, and severe atrophy of the follicle in the bursa. Poultry vaccinated with live IBDV vaccines do not have the challenge present with bursa atrophy, which is misdiagnosed for nVarIBDV because of the lack of other gross clinical symptoms. The present study sought to explore the potential and reliability of the real-time TaqMan analysis method for the detection and discrimination of the nVarIBDV genotype from that of the non-nVarIBDV, especially in live vaccine strains. This method will help monitor vaccinated poultry to control and manage infection with the nVarIBDV IBDVs. The nucleotide polymorphism in the 5'-UTR region and the vp5/vp2 overlapping region of the segment A sequences of IBDV were used to establish a one-step real-time TaqMan reverse transcription polymerase chain reaction (RT-PCR) method in this study. The results showed that the method accurately distinguished the nVarIBDV and non-nVarIBDV strains (especially live vaccine strains), and there were no cross-reactions with the infectious bronchitis virus (IBV), Newcastle disease virus (NDV), avian influenza virus (AIV), infectious laryngotracheitis virus (ILTV), fowlpox virus (FPV), Mycoplasma gallisepticum (M. gallisepticum), Mycoplasma synoviae (M. synoviae), and IBDV-negative field samples. The method showed a linear dynamic range between 102 and 107 DNA copies/reaction, with an average R2 of 0.99 and an efficiency of 93% for nVarIBDV and an average R2 of 1.00 and an efficiency of 94% for non-nVarIBDV. The method was also used for the detection of 84 clinical bursae of chickens vaccinated with the live vaccine. The results showed that this method accurately distinguished the nVarIBDV and non-nVarIBDV strains (vaccine strains), compared with a strategy based on the sequence analysis of HVRs at the vp2 gene or the reverse transcription PCR (RT-PCR) for the vp5 gene. These findings showed that this one-step real-time TaqMan RT-PCR method provides a rapid, sensitive, specific, and simple approach for detection of infections caused by nVarIBDV and is a useful clinical diagnostic tool for identifying and distinguishing nVarIBDV from non-nVarIBDV, especially live vaccine strains.

Epidemiology and laboratory diagnosis of very virulent infectious bursal disease virus in vaccinated chickens in Khartoum, Sudan.

Background: Infectious Bursal Disease (IBD, Gumboro disease) has become more severe than in early outbreaks in the 1980s. The present research aims to study the epidemiology of IBD in Khartoum state and compare some commonly used laboratory techniques for diagnosis. Method: We collected epidemiological data from 30 farms that showed signs suggestive of IBD, estimated the morbidity and mortality rates, and interviewed the owners about the type and the doses of the used vaccines. We collected bursas of Fabricius for virus assays and histopathology. Samples positive in the agar gel immunodiffusion (AGID) test were inoculated onto chicken embryo fibroblast cell culture and embryonated chicken eggs. Twenty-two-day-old chicks were infected experimentally with three selected isolates, and morbidity and mortality rates were compared. Results: The results showed that 70% of outbreaks occurred between 6 and 8 weeks of age, and the mean mortality rate was 51%. Epidemiologic, clinical, gross, and histopathological findings were characteristic of the severe disease caused by the very virulent IBDvirus (vvIBDV). The farms that used intermediate or the intermediate plus vaccines had lowered mortality compared with the farms that used intermediate vaccines. The AGID was found more sensitive than the counter-immuno-electrophoresis (CIEP) since it detected 83.4% of the IBDV antigen in the samples while the CIEP detected 66.7% of the samples. The reverse transcriptase polymerase chain reaction (RT-PCR) was found to be rapid, specific, and was more sensitive detecting 100% of the tested samples. Virus isolation in embryonated eggs and cell culture was not successful. Conclusion: A vvIBDV is responsible for the recent outbreaks of the disease in Sudan, resulting in a mean high mortality rate of 51%, even in vaccinated flocks. The RT-PCR and AGID are the best methods for laboratory confirmation.

RPA-Cas12aDS: A visual and fast molecular diagnostics platform based on RPA-CRISPR-Cas12a method for infectious bursal disease virus detection.

Infectious bursal disease (IBD), a major disease of birds, is caused by infectious bursal disease virus (IBDV). The disease can lead to immunosuppression, resulting in huge economic losses in the poultry industry. A specific, rapid, and simple detection method is important for the early diagnosis and prevention and control of IBDV. In this study, we established a naked-eye visual IBDV detection method, named "RPA-Cas12aDS", by combining recombinase polymerase amplification (RPA) with CRISPR-Cas12a-based nucleic acid detection. The detection process can be accomplished in 50 min, and uncapping contamination can be avoided. The detection results can be observed under blue or UV light. We used the RPA-Cas12aDS method to detect IBDV in bursa of Fabricius tissue samples of chickens, and the results were consistent with those obtained using commercial RT-PCR kits. This method presents great potential for visual, rapid, and point-of-care molecular diagnostics of IBDV in poultry.

Retrospective and prospective studies of transmissible viral proventriculitis in broiler chickens in Brazil.

We investigated the occurrence and pathologic findings of transmissible viral proventriculitis (TVP) associated with the chicken proventricular necrosis virus (CPNV) in commercial broiler chickens in southeastern Brazil. Seventy-three broilers, 25-36 d old, with a history of reduced growth, were referred to our veterinary pathology services from 2013 to 2017. Broilers were clinically examined, weighed, and euthanized for postmortem examination. Broilers of different ages with proventricular histologic lesions were positive for CPNV by RT-PCR; however, the intensity of histologic lesions was higher among 33-d-old animals, and viral RNA detection was more frequent among those that were 28 d old. In the proventriculi of 35 of 73 (48%) broilers, lesions were characterized by glandular epithelial necrosis, lymphoplasmacytic and histiocytic infiltrates, and metaplasia of glandular epithelium to ductal epithelium. In 24 of 73 (36%) broilers with histologic TVP-compatible lesions, CPNV was detected by RT-PCR for the viral protein 1 (VP1) gene. Broilers with histologic lesions were lighter than expected compared to the Cobb 500 standard weight. TVP has not been reported previously in broiler chickens in Brazil, to our knowledge.

Characterization of the highly immunogenic VP2 protrusion domain as a diagnostic antigen for members of Birnaviridae family.

Birnaviridae is a family of viruses (birnaviruses) which consists of four genera, members of which cause diseases in fish, birds, mollusks, and insects. The genome of birnaviruses encodes the highly immunogenic VP2 capsid protein. In order to demonstrate that the VP2 protein can be exploited as a diagnostic antigen for birnaviruses, we developed a lateral flow assay based on the surface-exposed VP2 protrusion domain of a representative birnavirus, infectious bursal disease virus (IBDV) of serotype 1 which causes the highly devastating infectious bursal disease in chickens. The biophysical characterization of the purified domain reveals that the domain predominantly consists of ß-sheets, exists in a trimeric form, and remains folded at high temperatures, making it suitable for diagnostic purposes. Owing to its highly immunogenic nature and excellent biophysical properties, we employed the VP2 protrusion domain in a gold nanoparticle-based lateral flow assay for rapid detection of anti-IBDV antibodies in serum samples of infected chickens. Our results indicate that the domain binds anti-IBDV antibodies with high specificity during laboratory testing and on-site testing. The lateral flow assay reported here yields comparable results in a qualitative manner as obtained through a commercial enzyme-linked immunosorbent assay (ELISA). As VP2 is a common capsid protein of birnaviruses, the lateral flow assay can be generalized for other birnaviruses, and members of Tetraviridae and Nodaviridae families which contain homologous VP2 capsid proteins.

Detecting Infectious Bursal Disease Using a VP1 Gene-Based RT-qPCR Assay Compared to Standard Methods of Virus Isolation, ELISA, and Histopathology.

Infectious bursal disease (IBD) is an immunosuppressive viral disease of chickens, associated with severe economic losses and major threats to poultry production worldwide. Disease prevention programs rely on unequivocal identification of the pathogen, as well as vaccination programs. This study developed a sensitive, one-step, real-time, quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay using a hydrolysis probe system for infectious bursal disease virus (IBDV, VP1 gene) detection and quantification, which was compared to other routinely used diagnostic methods. The assay successfully detected IBD reference viruses and field isolates. The absence of cross-reactivity was detected with negative samples or with other avian viruses in the analytical specificity test. The detection limit of this assay was 70 RNA copies. RT-qPCR was more sensitive in the detection of serially diluted IBDV isolates compared to virus isolation. For clinical samples, the sensitivity and specificity values of RT-qPCR compared to enzyme-linked immunosorbent assay (ELISA) were 97.5% and 100%, respectively, and compared to histopathology, these values were 100% and 93.94%, respectively. RT-qPCR can provide a simple and reliable assay for IBDV surveillance programs and for evaluation of control strategies.

New introduction of a very virulent infectious bursal disease virus in New York, USA.

Bursa tissue samples from a pullet flock in New York State that was experiencing immune suppression related disease were sent to our laboratory in 2018. A very virulent infectious bursal disease virus (vvIBDV) was identified in those samples through molecular and pathogenicity studies and designated 1/chicken/USA/1054NY/18. Phylogenetic analyses of the hypervariable VP2 nucleotide sequence region indicated that this strain belonged to genogroup 3 which comprises the vvIBDV. Partial sequence data of the VP1 gene indicated this virus also had a VP1 typical of vvIBDV. While vvIBDV have previously been identified in the United States in California and Washington State, the 1054NY vvIBDV was most closely related to isolates from Ethiopia, suggesting it is a new introduction into the U.S. The 1054NY vvIBDV was used to challenge four-week old specific-pathogen-free (SPF) layer chicks where it caused 100% morbidity and 68.7% mortality within 4 days. Upon necropsy, gross pathological findings in infected SPF birds included small yellowish coloured bursas, some with haemorrhages on the serosal and mucosal surfaces. Microscopic lesions included inflammation, severe lymphocyte necrosis, atrophy of the follicles and follicular depletion of lymphocytes. RESEARCH HIGHLIGHTS A very virulent infectious bursal disease virus (vvIBDV) was detected in a pullet flock in New York state, USA. Nucleotide sequence analysis of the vvIBDV VP2 gene indicates it is not related to previous US vvIBDV isolates and appears to be a new introduction into the US. The New York vvIBDV caused 100% morbidity and 68.7% mortality in four-week-old specific-pathogen-free chicks.

Robust Bioengineered Apoferritin Nanoprobes for Ultrasensitive Detection of Infectious Pancreatic Necrosis Virus.

Infectious pancreatic necrosis virus (IPNV) has been identified as a viral pathogen for many fish diseases that have become a huge hurdle for the growing fishing industry. Thus, in this work, we report a label-free impedance biosensor to quantify IPNV in real fish samples at point-of-care (POC) level. High specificity IPNV sensor with a detection limit of 2.69 TCID50/mL was achieved by conjugating IPNV antibodies to portable Au disk electrode chips using human heavy chain apoferritin (H-AFN) nanoprobes as a binding agent. H-AFN probes were bioengineered through PCR by incorporating pET-28b(+) resulting in 24 subunits of 6 × his-tag and protein-G units on its outer surface to increase the sensitivity of the IPNV detection. The biosensor surface modifications were characterized by differential pulse voltammetry (DPV) and EIS methods for each modification step. The proposed nanoprobe based sensor showed three-fold enhancement in charge transfer resistance toward IPNV detection in comparison with the traditional linker approach when measured in a group of similar virus molecules. The portable sensor exhibited a linear range of 100-10000 TCID50/mL and sensitivity of 5.40 × 10-4 TCID50/mL in real-fish samples. The performance of the proposed IPNV sensor was fully validated using an enzyme-linked immunosorbent assay (ELISA) technique with a sensitivity of 3.02 × 10-4 TCID50/mL. Results from H-AFN nanoprobe based IPNV sensor indicated high selectivity, sensitivity, and stability could be a promising platform for the detection of similar fish viruses and other biological molecules of interest.

A multiplex xTAG assay for the simultaneous detection of five chicken immunosuppressive viruses.

BACKGROUND: Chicken anemia virus (CAV), avian reovirus (ARV), infectious bursal disease virus (IBDV), Marek's disease virus (MDV) and reticuloendotheliosis virus (REV) all cause immunosuppressive disease in birds through vertical or horizontal transmission. Mixed infections with these immunosuppressive pathogens lead to atypical clinical signs and obstruct accurate diagnoses and epidemiological investigations. Therefore, it is essential to develop a high-throughput assay for the simultaneous detection of these immunosuppressive viruses with high specificity and sensitivity. The aim of this study was to establish a novel method using a RT-PCR assay combined with fluorescence labeled polystyrene bead microarray (multiplex xTAG assay) to detect single or mixed viral infections. RESULTS: The results showed that the established xTAG assay had no nonspecific reactions with avian influenza virus (AIV), infectious bronchitis virus (IBV), newcastle disease virus (NDV), infectious laryngotracheitis virus (ILTV), Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS). The limit of detection was 1.0 × 103 copies/µL for IBDV and 1.0 × 102copies/µL for the other four viruses. Ninety field samples were tested and the results were confirmed using conventional RT-PCR methods. The detection results of these two methods were 100% consistent. The established multiplex xTAG assay allows a high throughput and simultaneous detection of five chicken immunosuppressive viruses. CONCLUSION: The multiplex xTAG assay has been showed to be an additional tool for molecular epidemiology studies of five chicken immunosuppressive viruses in the poultry industry.

Inter-laboratory ring trial to evaluate real-time reverse transcription polymerase chain reaction methods used for detection of infectious pancreatic necrosis virus in Chile

Background: Infectious Pancreatic Necrosis Virus (IPNV) is the etiological agent of a highly contagious disease that affects salmonids. In Chile, the second worldwide salmon producer, IPNV causes great economic loss and is one of the most frequently detected pathogens. Due to its high level of persistence and the lack of information about the efficiency of its diagnostic techniques, the National Reference Laboratory (NRL) for IPNV in Chile performed the first inter-laboratory ring trial, to evaluate the sensitivity, specificity and repeatability of the qRT-PCR detection methods used in the country. Results: Results showed 100% in sensitivity and specificity in most of the laboratories. Only three of the twelve participant laboratories presented problems in sensitivity and one in specificity. Problems in specificity (false positives) were most likely caused by cross contamination of the samples, while errors in sensitivity (false negatives) were due to detection problems of the least concentrated viral sample. Regarding repeatability, many of the laboratories presented great dispersion of the results (Ct values) for replicate samples over the three days of the trial. Moreover, large differences in the Ct values for each sample were detected among all the laboratories. Conclusions: Overall, the ring trial showed high values of sensitivity and specificity, with some problems of repeatability and inter-laboratory variability. This last issue needs to be addressed in order to allow harmonized diagnostic of IPNV within the country. We recommend the use of the NRL methods as validated and reliable qRT-PCR protocols for the detection of IPNV.

Detection of transmissible viral proventriculitis and chicken proventricular necrosis virus in the UK.

Increasing evidence suggests that a new birnavirus, named chicken proventricular necrosis virus (CPNV), is the aetiological agent of transmissible viral proventriculitis (TVP). The present work aimed to explore the possible presence of both TVP and CPNV in the UK. Forty-four chickens showing TVP-compatible gross lesions were classified into three groups based on the histological lesions: (i) TVP-affected chickens: lymphocytic infiltration and glandular necrosis (n = 15); (ii) lymphocytic proventriculitis (LP)-affected chickens: lymphocytic infiltration without necrosis (n = 18); and (iii) without proventriculitis (WP): no lymphocytic infiltration or necrosis (n = 11). Nine proventriculi (seven out of 15 corresponding to TVP, and two out of 11 corresponding to LP) were positive for CPNV by reverse transcriptase polymerase chain reaction (RT-PCR). These results support the previously suggested idea of CPNV as causative agent of TVP. Moreover, these data show that CPNV can also be detected in a number of cases with LP, which do not fulfil the histological TVP criteria. Phylogenetic analysis of partial sequences of gene VP1 showed that British CPNV sequences were closer to other European CPNV sequences and might constitute a different lineage from the American CPNV. TVP cases with negative CPNV PCR results may be due to chronic stages of the disease or to the reduced PCR sensitivity on formalin-fixed paraffin-embedded tissues. However, involvement of other agents in some of the cases cannot totally be ruled out. As far as the authors are aware, this is the first peer-reviewed report of TVP as well as of CPNV in the UK, and the first exploratory CPNV phylogenetic study.

Design and validation of a RT-qPCR procedure for diagnosis and quantification of most types of infectious pancreatic necrosis virus using a single pair of degenerated primers.

Infectious pancreatic necrosis virus (IPNV) is an important virus which affects the salmonid aquaculture industry worldwide; therefore, it is important to develop rapid and reliable methods of diagnosis to detect the disease at early stages. Nowadays, RT-qPCR is replacing other methods because it provides additional information on the viral load, which is important to have a better understanding of the virus replication level and of the stage of the infection and its risk level. The main problem stems from the high diversity of this virus, which can compromise the reliability of the diagnosis. In this study, we have designed an RT-qPCR procedure for diagnosis and quantification of IPNV based on a single pair of primers targeted to segment B. The procedure has been validated, in vitro and in vivo, testing two different types of standards against seven reference strains and 23 field isolates from different types. The procedure is reliable for the detection of any type, with a detection limit of 31 TCID50  mL-1 , 50 pfu mL-1 or 66 RNA copies mL-1 , depending on the standard. All the standard curves showed high reliability (R2  > 0.95). The results support the high reliability of this new procedure for the diagnosis and quantification of IPNV.

Development of an RT-qPCR assay for the specific detection of a distinct genetic lineage of the infectious bursal disease virus.

The infectious bursal disease virus (IBDV) is a major health threat to the world's poultry industry despite intensive controls including proper biosafety practices and vaccination. IBDV (Avibirnavirus, Birnaviridae) is a non-enveloped virus with a bisegmented double-stranded RNA genome. The virus is traditionally classified into classic, variant and very virulent strains, each with different epidemiological relevance and clinical implications. Recently, a novel worldwide spread genetic lineage was described and denoted as distinct (d) IBDV. Here, we report the development and validation of a reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay for the specific detection of dIBDVs in the global poultry industry. The assay employs a TaqMan-MGB probe that hybridizes with a unique molecular signature of dIBDV. The assay successfully detected all the assessed strains belonging to the dIBDV genetic lineage, showing high specificity and absence of cross-reactivity with non-dIBDVs, IBDV-negative samples and other common avian viruses. Using serial dilutions of in vitro-transcribed RNA we obtained acceptable PCR efficiencies and determination coefficients, and relatively small intra- and inter-assay variability. The assay demonstrated a wide dynamic range between 103 and 108 RNA copies/reaction. This rapid, specific and quantitative assay is expected to improve IBDV surveillance and control worldwide and to increase our understanding of the molecular epidemiology of this economically detrimental poultry pathogen.

Recombinant scFv antibodies against infectious pancreatic necrosis virus isolated by flow cytometry.

Infectious pancreatic necrosis is a significant disease of farmed salmonids in China. In this study, a single chain variable fragment (scFv) antibody library derived from rainbow trout (Oncorhynchus mykiss) and viral protein VP2 of a Chinese infectious pancreatic necrosis virus (IPNV) isolate ChRtm213 were co-expressed by a bacterial display technology. The library was subjected to three rounds of screening by flow cytometry (FCM) to select IPNV specific antibodies. Six antibody clones with different mean fluorescence intensities (MFI) were obtained by picking colonies at random. The antibody clones were expressed and purified. The purified IPNV-specific scFv antibodies were used successfully in Western blotting, enzyme linked immunosorbent assay (ELISA) and an immunofluorescence antibody test (IFAT). This method provides a high throughput means to screen an antibody library by flow cytometry, and isolate a panel of antibody that can be used as potential reagents for the detection and study of IPNV that are prevalent in China.

[IDENTIFICATION OF THE INFECTIOUS PANCREATIC NECROSIS VIRUS (IPNV) USING THE ENZYME IMMUNOASSAY].

The infectious pancreatic necrosis (IPN) caused by a non-enveloped virus of the Birnaviridae family is one of the most important loss factors in the salmonid aquaculture. Virus isolation in the sensitive cell cultures has been approved in the Russian Federation as the diagnostic method for determination of IPNV antigen. This work gives the results of the development of the diagnostic test to reveal IPNV using the antigen-bound ELISA (sandwich ELISA). The developed test supplements a new diagnostic method and verifies some disputable results obtained with classical methods.

Tissue distribution of infectious pancreatic necrosis virus serotype Sp in naturally infected cultured rainbow trout, Oncorhynchus mykiss (Walbaum): an immunohistochemical and nested-PCR study.

This study investigates the occurrence and distribution pattern of infectious pancreatic necrosis virus (IPNV) within the pancreas, liver, kidney and spleen of naturally infected cultured rainbow trout, Oncorhynchus mykiss (Walbaum), using immunohistochemistry (IHC). A nested PCR was also employed to confirm the presence of the virus in the pooled tissues of the specimens. All the examined tissues except spleen were immunohistochemically positive for IPNV, but staining intensity and distribution pattern varied. The kidney tubules had the most intense and widespread staining by IHC, indicating a specific tissue tropism at least for this particular serotype. The nucleotide sequence had the greatest identity with the Sp serotype confirming the presence of the nucleic acid of IPNV in the pooled tissues. Based on the present findings, it could be concluded that the absence of lesions consistent with infectious pancreatic necrosis (IPN) disease in the H&E-stained sections cannot rule out the presence of the IPNV, and the use of an alternative rapid confirmatory method such as IHC with formalin-fixed, paraffin-embedded tissue sections is helpful for the final diagnosis of IPN in rainbow trout.

Novel multiplex RT-PCR/RFLP diagnostic test to differentiate low- from high-pathogenic strains and to detect reassortant infectious bursal disease virus.

Three types of infectious bursal disease virus (IBDV) strains are currently circulating worldwide: the low-pathogenic classic and variant strains and the high-pathogenic very virulent strains. There are also natural reassortant viruses that combine genomic segments A and B from different strains and exhibit particular pathogenic characteristics. Detection and characterization of the different IBDVs is extremely critical for improving disease control and performing epidemiologic studies. Here, we present a novel detection and genotyping method based on the simultaneous characterization of both IBDV genomic segments followed by a simple restriction fragment length polymorphism (RFLP) assay. This single restriction enzyme, multiplex reverse transcriptase-PCR/RFLP diagnostic test not only distinguished typical high-pathogenic from low-pathogenic strains but also detected natural reassortant IBDV. The test was based on the detection of single nucleotide polymorphisms (SNP), in both segments, which were strongly linked to the pathogenic phenotype. These SNPs are embedded in highly conserved genomic regions and can be identified with TfiI endonuclease. The application of this methodology in field samples confirmed that the assay is fast, specific, and may be easily adopted by any molecular diagnostic laboratory as an economical and routine method.

Evaluation of rapid and sensitive reverse transcription loop-mediated isothermal amplification method for detecting infectious pancreatic necrosis virus in chum salmon (Oncorhynchus keta).

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed for detecting Infectious pancreatic necrosis virus (IPNV) in chum salmon (Oncorhynchus keta) in Korea. The RT-LAMP is a novel approach of nucleic acid gene amplification with high specificity, sensitivity, and rapidity under isothermal conditions. Based on the VP2/NS gene sequence of VR-299 and Jasper strains, a set of 6 IPNV-specific primers was designed to recognize 8 diverse sequences of the IPNV RNA. The assay was successfully optimized to detect IPNV at 65°C in 30 min. The detection limit was 0.075 tissue culture infectious dose infecting 50% of inoculated cultures per milliliter (TCID(50)/ml) from IPNV-infected rainbow trout gonad (RTG)-2 cells, whereas nested reverse transcription polymerase chain reaction (nRT-PCR) had a sensitivity of 7.5 TCID(50)/ml. Using RT-LAMP assay, field samples were analyzed and the results compared with those of nRT-PCR assay. Two hundred and sixty-six out of 659 (40.4%) samples were IPNV-positive by RT-LAMP, whereas 182 of 659 samples (27.6%) were IPNV-positive by nRT-PCR. The results indicate that RT-LAMP can be a useful tool for early field diagnosis of IPNV.

One-step reverse-transcription loop-mediated isothermal amplification for detection of infectious bursal disease virus.

A fast, sensitive, and specific reverse-transcription (RT) loop-mediated isothermal amplification (RT-LAMP) assay was developed that involved a single tube and a 1-step reaction for detecting infectious bursal disease virus (IBDV). Four specific primers were used for amplification of the VP2 gene of IBDV. The amplified LAMP products were detected by DNA electrophoresis and by direct observation with the naked eye in the presence of SYBR Green I. The sensitivity of RT-LAMP was determined to be 0.01 fg of IBDV viral RNA. This assay for IBDV is more sensitive than the conventional RT-polymerase chain reaction assay, which has a detection limit of 1 ng. The LAMP assay was also assessed for specificity and was found to precisely discriminate between positive and negative test samples. This newly established LAMP assay, combined with RT, is a practical diagnostic tool because IBDV-infected and uninfected clinical samples collected from an experimental farm could be discriminated. Full verification of a sample's IBDV status was obtained within 40 min of extraction of the viral RNA, which could then be directly added to the RT-LAMP reaction mixture.

A one-step reverse transcription loop-mediated isothermal amplification for detection and discrimination of infectious bursal disease virus.

BACKGROUND: Infectious bursal disease (IBD) is a highly contagious immunosuppressive disease in young chickens caused by infectious bursal disease virus (IBDV). It causes huge economic losses to the poultry industry. The objective of this study is to develop a loop-mediated isothermal amplification (LAMP) method for the detection and discrimination of IBDV. RESULTS: In this study, we applied reverse transcription loop-mediated isothermal amplification (RT-LAMP) to detect IBDV in one simple step and further identified the very virulent strain from non-vvIBDVs with a simply post-amplification restriction enzyme analysis. Based on sequence analysis, a set of two inner, two outer and two loop primers were designed to target the VP5 gene and they showed great specificity with no cross reaction to the other common avian pathogens. The detection limit determined by both color change inspection and agarose gel electrophoresis was 28 copies viral RNA, which was almost as sensitive as a real-time RT-PCR previous developed in our laboratory. We also identified a unique Tfi I restriction site located exclusively in non-vvIBDVs, so very virulent strain could be distinguished from current vaccine strains. By screening a panel of clinical specimens, results showed that this method is high feasible in clinical settings, and it obtained results 100% correlated with real-time RT-PCR. CONCLUSION: RT-LAMP is a rapid, simple and sensitive assay. In combination with the Tfi I restriction analysis, this method holds great promises not only in laboratory detection and discrimination of IBDV but also in large scale field and clinical studies.