Recombinase polymerase amplification assay for rapid detection of lumpy skin disease virus

Background  Lumpy skin disease virus (LSDV) is aCapripoxvirusinfecting cattle and Buffalos. Lumpy skin disease (LSD) leads to significant economic losses due to hide damage, reduction of milk production, mastitis, infertility and mortalities (10 %). Early detection of the virus is crucial to start appropriate outbreak control measures. Veterinarians rely on the presence of the characteristic clinical signs of LSD. Laboratory diagnostics including virus isolation, sequencing and real-time polymerase chain reaction (PCR) are performed at well-equipped laboratories. In this study, a portable, simple, and rapid recombinase polymerase amplification (RPA) assay for the detection of LSDV-genome for the use on farms was developed.  Results  The LSDV RPA assay was performed at 42 °C and detected down to 179 DNA copies/reaction in a maximum of 15 min. Unspecific amplification was observed with neither LSDV-negative samples (n= 12) nor nucleic acid preparations from orf virus, bovine papular stomatitis virus, cowpoxvirus, Peste des petits ruminants and Blue tongue virus (serotypes 1, 6 and 8). The clinical sensitivity of the LSDV RPA assay matched 100 % (n= 22) to real-time PCR results. In addition, the LSDV RPA assay detected sheep and goat poxviruses.  Conclusion  The LSDV RPA assay is a rapid and sensitive test that could be implemented in field or at quarantine stations for the identification of LSDV infected case

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

Molecular characterization of Marek's Disease virus reveals reticuloendotheliosis virus-long terminal repeat integration in the genome of the field isolates in Egypt

ABSTRACT: The highly contagious, immunosuppressive, and cancer-causing Marek's disease virus (MDV) infects chickens. The financial costs of Marek's disease (MD) are significant for the chicken industry. In this study, a total of 180 samples from chicken farms suspected to be MDV-infected were collected. The chickens were sampled during the period between the months of October 2016 and February 2018 at Dakahlia and Damietta Governorates, Egypt. A total of 36 pooled samples were created. The prepared samples were inoculated into embryonated chicken eggs (ECEs). Indirect fluorescent antibody technique (IFAT) and ICP4 gene-based polymerase chain reaction (PCR) were used for MDV identification. For the genetic characterization of the identified virus, The ICP4 gene sequence was identified and compared with the sequences available from various regions of the world. Furthermore, the genomes of all detected MDVs were screened for the long terminal repeat (LTR) region of reticuloendotheliosis (REV) in their genomes. The results showed that 31 out of 36 pooled samples (86.1%) inoculated into ECEs displayed the characteristic pock lesions. By using IFAT and PCR to identify MDV in ECEs, positive results were found in 27 samples (75%). The Egyptian virus is thought to be genetically closely related to MDVs circulating in Ethiopia, China, and India. REV-LTR was amplified from 6 out of 27 field isolates genomes (22.2 %) while MDV vaccine strains were free from REV-LTR insertion. The integrated REV-LTRs depicted a close genetic relationship with those integrated in fowl poxvirus (FWPV) circulating in Egypt as well as those integrated in FWPVs and MDVs from China, USA, South Africa, and Australia. To the best of our knowledge, this investigation represents the first identification and characterization of REV-LTR insertions in Egyptian MDV field isolates. Given the findings above, additional research in the future seems crucial to determine how the REV-LTR insertions affect MDV pathogenesis, virulence, and insufficient vaccination protection

A Single and Two-Stage, Closed-Tube, Molecular Test for the 2019 Novel Coronavirus (COVID-19) at Home, Clinic, and Points of Entry

The 2019 novel coronavirus (COVID-19) is a newly emerged strain that has never been found in humans before. At present, the laboratory-based reverse transcription-polymerase chain reaction (RT-PCR) is the main method to confirm COVID-19 infection. The intensification of the COVID-19 epidemic overwhelms limited clinical resources in particular, but not only, in developing countries, resulting in many patients not being tested for the infection and in large queues of potentially infected individuals waiting to be tested while providing a breeding ground for the disease. We describe here a rapid, highly sensitive, point-of-care, molecular test amenable for use at home, in the clinic, and at points of entry by minimally trained individuals and with minimal instrumentation. Our test is based on loop mediated isothermal amplification (COVID-19 LAMP) and for higher sensitivity on nested nucleic acid, two stage isothermal amplification (COVID-19 Penn-RAMP). Both tests can be carried out in closed tubes with either fluorescence or colorimetric (e.g., leuco crystal violet LCV) detection. COVID-19 LAMP performs on par with COVID-19 RT-PCR. COVID-19 RAMP has 10 fold better sensitivity than COVID-19 LAMP and COVID-19 RT-PCR when testing purified targets and 100 times better sensitivity than COVID-19 LAMP and COVID-19 RT-PCR when testing rapidly prepared sample mimics. Due to fortunate scarcity of COVID-19 infections in the USA, we were not able to test our assays and methods with patient samples. We hope that such tests will be carried out by colleagues in impacted countries. Our Closed-Tube Penn-RAMP has the potential to significantly reduce false negatives while being amenable to use with minimal instrumentation and training. </p

Molecular Detection of Infectious Laryngotracheitis Virus in Chickens with a Microfluidic Chip

Infectious laryngotracheitis (ILT) is a viral disease of chickens’ respiratory system that imposes considerable financial burdens on the chicken industry. Rapid, simple, and specific detection of this virus is crucial to enable proper control measures. Polymerase chain reaction (PCR)-based molecular tests require relatively expensive instruments and skilled personnel, confining their application to centralized laboratories. To enable chicken farms to take timely action and contain the spread of infection, we describe a rapid, simple, semi-quantitative benchtop isothermal amplification (LAMP) assay, and a field-deployable microfluidic device for the diagnosis of ILTV infection in chickens. Our assay performance was compared and favorably agreed with quantitative PCR (qPCR). The sensitivity of our real-time LAMP test is 250 genomic copies/reaction. Clinical performance of our microfluidic device using samples from diseased chickens showed 100% specificity and 100% sensitivity in comparison with benchtop LAMP assay and the gold-standard qPCR. Our method facilitates simple, specific, and rapid molecular ILTV detection in low-resource veterinary diagnostic laboratories and can be used for field molecular diagnosis of suspected ILT cases

[27] Safety of pre-intended en bloc renal pedicle control for laparoscopic nephrectomy

Objective: To evaluate preoperatively intended en bloc renal pedicle control for laparoscopic nephrectomy in our hospital, as the safety and efficacy of en bloc renal pedicle control has been approved for laparoscopic nephrectomy but some authors do not advocate the generalised use of it. Methods: We reviewed all laparoscopic nephrectomies that were carried out by two laparoscopic surgeons (from January 2015 until April 2017) who had a preoperative intention of en bloc renal pedicle control. By creating a window at the lower pole and another window at upper pole then using the Covidien Endo-GIA™ (45,60 mm vascular reload) to control the pedicle. We analysed patients’ demographic data, nephrectomy indication, intraoperative findings, and intra- and postoperative complications. Patients were followed-up with blood pressure measurements and for the presence or absence of any signs of hyperdynamic circulation. The analysis of data was done using SPSS® version 20 (SPSS Inc., IBM Corp., Armonk, NY, USA). Results: We found 38 laparoscopic nephrectomies done for patients with mean (range) age 55.7 (18–94) years and mean (range) body mass index of 29.2 (17–41) kg/m2. The indications for nephrectomy were variable: 22 patients (57.9%) for non-functioning kidney, 14 (36.8%) for renal tumours and two (5.2%) for ureteric tumours. There were 23 left nephrectomies and 15 right nephrectomies performed laparoscopically. There were no conversions to open technique and no intraoperative complications. There was an average blood loss of 75 mL and the mean (range) total laparoscopic time was 85 (45–125) min. All cases had no significant postoperative complications, apart from one who developed a postoperative collection and pleural effusion that required a chest tube and drainage. During the follow-up, none of the patients developed signs of an arteriovenous fistula. Conclusion: Pre-intended en bloc renal pedicle control during laparoscopic nephrectomies is safe using the Endo-GIA vascular stapler without significant complications

Additional file 2: Table S2. of Recombinase polymerase amplification assay for rapid detection of lumpy skin disease virus

Reproducibility of LSDV RPA assay using data sets of eight RPA assay runs using the DNA molecular standards. 107–103 DNA molecules were detected 8 out of 8 runs; 102, 7/8 and 101, 2/8. (DOCX 54 kb

Molecular Detection of Reticuloendotheliosis Virus 5′ Long Terminal Repeat Integration in the Genome of Avipoxvirus Field Strains from Different Avian Species in Egypt

Avipoxviruses (APVs) are among the most complex viruses that infect a wide range of birds&rsquo; species. The infection by APVs is often associated with breathing and swallowing difficulties, reduced growth, decreased egg production, and high mortalities in domestic poultry. In the present study, 200 cutaneous nodular samples were collected from different avian species (chicken, pigeon, turkey, and canary) suspected to be infected with APVs from Dakahlia Governorate, Egypt. Pooled samples (n = 40) were prepared and inoculated in embryonated chicken eggs (ECEs). APVs were then identified by polymerase chain reaction (PCR) and sequence analysis of the APV P4b gene. Furthermore, the forty strains of APVs were screened for the presence of reticuloendotheliosis virus (REV)-5&prime;LTR in their genomes. Interestingly, the phylogenic tree of the APV P4b gene was separated into 2 clades: clade 1, in which our fowlpox virus (FWPV), turkeypox virus (TKPV), and canarypox virus (CNPV) isolates were grouped, along with reference FWPVs and TKPVs retrieved from GenBank, whereas, in clade2, the pigeonpox virus (PGPV) isolate was grouped with PGPVs retrieved from GenBank. Likewise, REV-5&prime;LTR was amplified from 30 strains isolated from chicken, turkey, and canary, while PGPV strains were free from REV-5&prime;LTR integration. To the best of our knowledge, this study involved the detection and characterization of REV-5&prime;LTR insertions in the APVs field isolates in Egypt for the first time. Given the above information, further future research seems recommended to understand the impact of the resulting REV-5&prime;LTR insertions on the pathogenesis, virulence, and inadequate vaccine protection against APVs