scholarly journals Performance Characteristics of Real-time PCRs for African Swine Fever Virus Genome Detection – comparison of Twelve Kits to an OIE Recommended Method

2021 ◽  
Author(s):  
Jutta Pikalo ◽  
Tessa Carrau ◽  
Paul Deutschmann ◽  
Melina Fischer ◽  
Kore Schlottau ◽  
...  
Keyword(s):  
Real Time ◽  
Internal Control ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Laboratory Diagnosis ◽  
Virus Genome ◽  
Test System ◽  
User Friendliness ◽  
Commercial Kits ◽  
Time Required

African swine fever (ASF) is one of the major threats to pig production, and real-time PCR (qPCR) protocols are integral part of ASF laboratory diagnosis. With the pandemic spread of ASF, commercial kits have risen on the market. In Germany, the kits have to go through an approval process and thus, general validation can be assumed. However, they were never compared to each other. In this study, 12 commercial PCR kits were compared to an OIE recommended method. Samples representing different matrices, genome loads, and genotypes were included in a panel that was tested under diagnostic conditions. The comparison included user-friendliness, internal controls, and the time required. All qPCRs were able to detect ASFV genome in different matrices across all genotypes and disease courses. With one exception, there were no significant differences when comparing the overall mean. The overall specificity was 100 % [95 % CI 87.66 - 100], and the sensitivity was between 95 % and 100 % [95 % CI 91.11 - 100]. As can be expected, variability concerned samples with low genome load. Concluding, all tests were fit for purpose. The test system can therefore be chosen based on compatibility and prioritization of the internal control system.

Development of a real‐time PCR assay for detection of African swine fever virus with an endogenous internal control

2020 ◽  
Vol 67 (6) ◽  
pp. 2446-2454 ◽  
Author(s):  
Yin Wang ◽  
Lizhe Xu ◽  
Lance Noll ◽  
Colin Stoy ◽  
Elizabeth Porter ◽  
...  
Keyword(s):  
Real Time ◽  
Internal Control ◽  
Real Time Pcr ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Pcr Assay

scholarly journals African Swine Fever Laboratory Diagnosis—Lessons Learned from Recent Animal Trials

Pathogens ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 177
Author(s):  
Jutta Pikalo ◽  
Paul Deutschmann ◽  
Melina Fischer ◽  
Hanna Roszyk ◽  
Martin Beer ◽  
...  
Keyword(s):  
African Swine Fever Virus ◽  
Clinical Signs ◽  
African Swine Fever ◽  
Laboratory Diagnosis ◽  
Lessons Learned ◽  
Hemorrhagic Disease ◽  
Passive Surveillance ◽  
Animal Trials ◽  
Fit For Purpose ◽  
The Impact

African swine fever virus (ASFV) causes a hemorrhagic disease in pigs with high socio-economic consequences. To lower the impact of disease incursions, early detection is crucial. In the context of experimental animal trials, we evaluated diagnostic workflows for a high sample throughput in active surveillance, alternative sample matrices for passive surveillance, and lateral flow devices (LFD) for rapid testing. We could demonstrate that EDTA blood is significantly better suited for early ASFV detection than serum. Tissues recommended by the respective diagnostic manuals were in general comparable in their performance, with spleen samples giving best results. Superficial lymph nodes, ear punches, and different blood swabs were also evaluated as potential alternatives. In summary, all matrices yielded positive results at the peak of clinical signs and could be fit for purpose in passive surveillance. However, weaknesses were discovered for some matrices when it comes to the early phase of infection or recovery. The antigen LFD showed variable results with best performance in the clinical phase. The antibody LFD was quite comparable with ELISA systems. Concluding, alternative approaches are feasible but have to be embedded in control strategies selecting test methods and sample materials following a “fit-for-purpose” approach.

scholarly journals LAMP assay coupled with CRISPR/Cas12a system for portable detection of African swine fever virus

2021 ◽  
Author(s):  
Bo YANG ◽  
zhengwang shi ◽  
Yuan Ma ◽  
Lijuan Wang ◽  
Liyan Cao ◽  
...  
Keyword(s):  
Real Time ◽  
Detection System ◽  
African Swine Fever Virus ◽  
Low Cost ◽  
African Swine Fever ◽  
Lamp Assay ◽  
Cross Reactivity ◽  
Clinical Samples ◽  
Portable Detection ◽  
Real Time Qpcr

African swine fever (ASF) is one of the most severe infectious diseases of pigs. In this study, a LAMP assay coupled with the CRISPR Cas12a system was established in one tube for the detection of the ASFV p72 gene. The single-strand DNA-fluorophore-quencher (ssDNA-FQ) reporters and CRISPR-derived RNA (crRNAs) were screened and selected for the CRISPR detection system. In combination with LAMP amplification assay, the detection limit for the LAMP-CRISPR assay can reach 7 copies/μl of p72 gene per reaction. Furthermore, this method displays no cross-reactivity with other porcine DNA or RNA viruses. The performance of the LAMP-CRISPR assay was compared with real-time qPCR tests for clinical samples, a good consistency between the LAMP-CRISPR assay and real-time qPCR was observed. In the current study, a LAMP coupled with the CRISPR detection method was developed. The method shed a light on the convenient, portable, low cost, highly sensitive and specific detection of ASFV, demonstrating a great application potential for monitoring on-site ASFV in the field.

Two Novel Multigene Families, 530 and 300, in the Terminal Variable Regions of African Swine Fever Virus Genome

Virology ◽  
1994 ◽  
Vol 202 (2) ◽  
pp. 997-1002 ◽  
Author(s):  
T. Yozawa ◽  
G.F. Kutish ◽  
C.L. Afonso ◽  
Z. Lu ◽  
D.L. Rock
Keyword(s):  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Virus Genome ◽  
Fever Virus ◽  
Multigene Families ◽  
Variable Regions

Characterization of the African swine fever virus protein p49: a new late structural polypeptide

Microbiology ◽  
2000 ◽  
Vol 81 (1) ◽  
pp. 59-65 ◽  
Author(s):  
Inmaculada Galindo ◽  
Eladio Viñuela ◽  
Angel L. Carrascosa
Keyword(s):  
Cell Attachment ◽  
African Swine Fever Virus ◽  
Rabbit Antiserum ◽  
African Swine Fever ◽  
Virus Genome ◽  
Fever Virus ◽  
Virus Protein ◽  
Significant Similarity ◽  
Reading Frame ◽  
Cell Extracts

The open reading frame B438L, located within the EcoRI B fragment of the African swine fever virus genome, is predicted to encode a protein of 438 amino acids with a molecular mass of 49·3 kDa. It presents a cell attachment RGD (Arg–Gly–Asp) motif but no other significant similarity to protein sequences in databases. Northern blot and primer extension analysis showed that B438L is transcribed only at late times during virus infection. The B438L gene product has been expressed in Escherichia coli, purified and used as an antigen for antibody production. The rabbit antiserum specific for pB438L recognized a protein of about 49 kDa in virus-infected cell extracts. This protein was synthesized late in infection by all the virus strains tested, was located in cytoplasmic virus factories and appeared as a structural component of purified virus particles.

scholarly journals Rapid Sequence-Based Characterization of African Swine Fever Virus by Use of the Oxford Nanopore MinION Sequence Sensing Device and a Companion Analysis Software Tool

2019 ◽  
Vol 58 (1) ◽  
Author(s):  
Vivian K. O’Donnell ◽  
Frederic R. Grau ◽  
Gregory A. Mayr ◽  
Tracy L. Sturgill Samayoa ◽  
Kimberly A. Dodd ◽  
...  
Keyword(s):  
Real Time ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Full Genome Sequence ◽  
Effective Vaccine ◽  
Fast Analysis ◽  
Rapid Acquisition ◽  
Oxford Nanopore

ABSTRACT African swine fever virus (ASFV) is the causative agent of a severe and highly contagious viral disease of pigs that poses serious economic consequences to the swine industry due to the high mortality rate and impact on international trade. There is no effective vaccine to control African swine fever (ASF), and therefore, efficient disease control is dependent on early detection and diagnosis of ASFV. The large size of the ASFV genome (∼180 kb) has historically hindered efforts to rapidly obtain a full-genome sequence. Rapid acquisition of data is critical for characterization of the isolate and to support epidemiological efforts. Here, we investigated the capacity of the Oxford Nanopore MinION sequence sensing device to act as a rapid sequencing tool. When coupled with our novel companion software script, the African swine fever fast analysis sequencing tool (ASF-FAST), the analysis of output data was performed in real time. Complete ASFV genome sequences were generated from cell culture isolates and blood samples obtained from experimentally infected pigs. Removal of the host-methylated DNA from the extracted nucleic acid facilitated rapid ASFV sequence identification, with reads specific to ASFV detected within 6 min after the initiation of sequencing. Regardless of the starting material, sufficient sequence was available for complete genome resolution (up to 100%) within 10 min. Overall, this paper highlights the use of Nanopore sequencing technology in combination with the ASF-FAST software for the purpose of rapid and real-time resolution of the full ASFV genome from a diagnostic sample.

scholarly journals Development and Testing of a Method for Detecting Lujo Virus RNA by Reverse Transcription and Real Time Polymerase Chain Reaction

2021 ◽  
pp. 110-115
Author(s):  
E. V. Naidenova ◽  
V. G. Dedkov ◽  
D. A. Agafonov ◽  
A. M. Senichkina ◽  
M. V. Safonova ◽  
...  
Keyword(s):  
Real Time ◽  
Internal Control ◽  
Biological Samples ◽  
Control Sample ◽  
Virus Genome ◽  
Positive Control ◽  
Control Panel ◽  
Rt Pcr ◽  
Rna Detection ◽  
Virus Rna

The aim of the study was to develop and assess the efficacy of a method for Lujo virus RNA detection in clinical and biological samples using one-step real-time RT-PCR.Materials and methods. In order to select the conservative regions of the genome, we utilized the available in GenBank database Lujo virus sequences (https://www.ncbi. nlm.nih.gov/genbank) aligned in BioEdit 7.2.5 software package ( (IbisBiosciences, USA). To conduct one-round RTPCR, reverse transcriptase and TaqF-polimerase were used. Recombinant Escherichia coli strain, XL1-Blue, containing pGEM-T plasmid with inserted synthetically-generated fragment of the virus genome, was produced to make positive control sample (PCS). Constructed recombinant plasmids were used for creating RNA-containing PCS with protective protein shell of MS2-phage. Determination of specificity of the developed method was performed with the help of control panel of RNA and DNA of 23 viral strains related to 10 families; the sensitivity – the panel of biological samples artificially contaminated with PCS. Further testing was carried out at the premises of laboratory of the Russian-Guinean Center for Epidemiology and Prevention of Infectious Diseases (Kindia, Republic of Guinea) on 265 blood sera from practically healthy persons, 110 blood sera of cattle, 83 suspensions of ticks, and 165 suspensions of organs of small mammals collected in the territory of Guinea.Results and discussion. Two conservative polymerase gene fragments have been chosen as targets for Lujo virus RNA detection using RT-PCR. The combination of primers and probes has been experimentally selected, optimum composition of reaction mixture for PCR and mode of RT-PCR set-up established, as well as control samples C+, internal control, positive control sample developed. Sensitivity of the proposed method is 5·103  GE /ml, specificity – 100 %. 

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