CORDIS provides links to public deliverables and publications of HORIZON projects.
Links to deliverables and publications from FP7 projects, as well as links to some specific result types such as dataset and software, are dynamically retrieved from OpenAIRE .
Deliverables
A novel hot start PCR (polymerase chain reaction) assay for rapid and specific detection of African swine fever virus (ASFV) has been developed. The method is based on the use of specific primers for ASFV selected in the VP73 coding region of the genome. Viral DNA from Spain 70 ASFV isolate grown in pig leukocytes cultures was used to optimise PCR with the selected primers. The method showed to be highly sensitive, and results can be obtained in less than 5 hours.
Specificity of the assay was assessed by analysis of twenty-two ASFV isolates collected from different outbreaks occurred in Africa, Europe, and America since 1960 to 2001, different experimental and field clinical samples recovered from ASFV infected or healthy pigs, as well as viral genome from other related porcine viruses. A confirmatory method of the specificity of amplified fragments based on restriction endonuclease analysis is also described.
This work provides a novel, highly sensitive, specific and rapid PCR method, for routine laboratory diagnosis of ASF, as a reliable tool in the surveillance, control and eradication programmes worldwide.
A closed tube Invader Squared Assay (Third Wave Technologies, Inc., Madison, Wisconsin, USA) was developed for the detection of African Swine Fever DNA using the real time PCR (polymerase chain reaction) machine iCycler (Biorad). This assay is a linear, isothermal (63degrees C) signal amplification system, which targets the VP73 gene of ASFV. The assay is based on the hybridisation of target specific invader and hybridisation probes to the target of interest. The resulting structure is recognised by a specific enzyme (Cleavase XI), which cleaves the hybridisation probe releasing a flap, which in turn hybridises to a reporter probe. The flap induces a second, cleavase enzyme driven, release of a fluorophore from the proximity of a quencher on the reporter probe resulting in fluorescence. Amplification is achieved by designing the hybridisation probe in such a way that it has a Tm around the temperature optimum of the assay, thus enabling isothermal probe turnover.
Assay sensitivity was determined to be in the order of 2500 copies of DNA using serial 10-fold dilutions of Lisbon 60 ASFV viral genome. The assay showed a linear relationship over the 4 orders of magnitude tested, from 2.5x106 to 2500 copies. The specificity of the test was demonstrated by the inability of the kit to detect a clinically similar but heterologous virus, Classical swine fever virus (Spain 2001).
This assay offers an alternative to PCR based technologies for ASFV diagnosis. This assay is sensitive enough for testing acutely infected animals in which the viral load will be high. Furthermore, additional optimisation of the components in the kit and the use of a detection platform more compatible with the Invader assay could increase the sensitivity at least 10 fold. Manufacturers of real time PCR machines are now getting aware of Invader technology and Corbett research are even providing software to analyse Invader data to their new real time PCR machine Rotorgene 3000.
Invader squared assays are very robust. They can be manufactured in a dried down format. Contamination is less of a problem as compared to PCR technologies. The extraordinary specificity minimises cross detection of other viruses as well as unspecific signals from clinical material. The assay can be performed and read using just a 63 degrees Celsius water bath and a simple fluorescent plate reader.
For the reasons mentioned above Invader squared assays are suitable for under-equipped laboratories close to putative sites of emergence of ASFV (on site diagnosis).
Primer-Probe Energy Transfer system technology transfer from ABI PRISM 7700 to iCycler real-time PCR (polymerase chain reaction) machine was performed using ASFV (African swine fever) as template. The system was quantitative over a range of six orders of magnitude. Melting profiles were obtained as expected. To implement the system for the iCycler the probe was modified for increased sensitivity. A manuscript for the performance of the modified PriProET is in preparation.
The MULTIPLEX PCR project has studied the detection of SVDV (swine vesicular disease virus) and of VSV (vesicular stomatitis virus) Indiana and VSV New Jersey using molecular beacon probe and single-step real-time PCR (polymerase chain reaction) assay.
A single-step real-time PCR assay based on the molecular beacon probe for the detection of all five SVDV serotypes was developed and optimised. Sensitivity of the probe is 1.26 TCID50/ml (UK72 serotype). Cross reactivity with human Coxsackie B5, FMDV and VSV were not observed.
Due to high variability between VSV Indiana (IND) and New Jersey (NJ) serotypes it was not possible to design single molecular beacon probe which could detect both serotypes at the same time. Thus, two real-time PCR (polymerase chain reaction) assays based on the molecular beacon probes against IND and NJ serotypes were developed and optimised. Reverse transcription (RT) step was included in the single PCR run, thus results can be obtained in less than three hours (from RT step to the end). Sensitivity of the probes was 200 TCID50/ml.
A rapid multiplex PCR method for simultaneous detection of Aujeszky's disease virus (ADV), porcine parvovirus (PPV) and porcine reproductive and respiratory syndrome virus (PRRSV) has been developed.
The planned multiplex combination of the five target viruses associated with reproductive disorders (ASF (African swine fever), CSF (classical swine fever), ADV, PPV and PRRSV) gave no satisfactory results. Therefore, the consortium agreed that P5 should divide the group and develop conventional, gel-based multiplex PCR assays for ASF/CSF/ADV and PRRSV/PPV/ADV clusters, respectively. Several new multiplex panels and primer combinations were set up and tested.
Yet, as presented in the meeting held in Malaga, the ASF/CSF/ADV triplex failed to work properly on a considerable percent of field samples with any of the above mentioned primer sets; therefore, no further efforts were made in this direction. On the other hand, in the last working phase we managed to find a proper primer-set to detect all three PRRSV/PPV/ADV viruses in one assay both in conventional PCR and in rapid-cycler (using 10 _l capillary tubes). The reaction gave strong, positive results with all (8) PRRSV, (6) ADV and (5) PPV clinical samples tested, without any non-specific byproducts.
A gel-based one-step multiplex RT-PCR assay for simultaneous and differential detection of haemorragic List A viruses has been developed. These viruses include African swine fever virus (ASFV) and classical swine fever virus (CSFV).
The development and standardisation of a novel, highly sensitive and specific one-step hot start multiplex RT-PCR assay has been described for the simultaneous and differential diagnosis of African swine fever (ASF) and Classical swine fever (CSF). The method uses two primer sets, each one specific for the corresponding virus, amplifying DNA fragments different in length, allowing a gel-based differential detection of the PCR (polymerase chain reaction) products.
Universal detection of ASF and CSF virus strains was achieved through selection of primers in conserved viral genome regions. The detection range was confirmed by analysis of a large collection of isolates of the two viruses. The high specificity of the assay was proven by testing related viruses, uninfected cell line cultures and healthy pig tissues. Additional confirmatory tests of the ASF and CSF virus amplicon specificity, based on restriction endonuclease analysis are also described. The analysis of whole blood and serum samples from experimentally infected animals proved the usefulness of the method for an early diagnosis of both diseases, even before the appearance of the first clinical symptoms.
A study of 150 positive field samples from several ASF and CSF outbreaks showed the suitability of this method for a rapid (less than 5 hours), sensitive and specific differential diagnosis in clinical samples. In addition, a highly sensitive and specific uniplex RT-PCR for CSFV was also developed and standardised as a powerful tool for fast and early diagnosis of the disease.
A novel, highly sensitive and specific one-step hot start multiplex RT-PCR assay has been developed and standardised for the simultaneous and differential diagnosis of vesicular List A viruses: Foot-and-Mouth Disease (FMD), Swine Vesicular Disease (SVD), and Vesicular Stomatitis (VS).
The method uses three primer sets, each one specific for the corresponding virus, amplifying DNA fragments different in length, allowing a gel-based differential detection of the PCR (polymerase chain reaction) products. Detection of the seven serotypes of FMDV (A, O, C, Sat 1, Sat 2, Sat 3, and Asia 1), two main serotypes of VSV (Indiana 1 and New Jersey) and SVDV strains was achieved through selection of primers in conserved viral genome regions. The detection range was confirmed by analysis of a large collection of isolates of the three viruses. The high specificity of the assay was proven by testing related viruses, uninfected cell line cultures and healthy pig tissues.
Additional confirmatory tests of the FMD, SVD, and VS virus amplicon specificity, based on restriction endonuclease analysis, have also been described. The analysis of whole blood and serum samples from experimentally infected animals proved the usefulness of the method for an early diagnosis of the diseases, even before the appearance of the first clinical symptoms. The multiplex RT-PCR assays performed in a collection of samples from experimentally infected animals showed the suitability of this method for a rapid (less than 5 hours), sensitive and specific differential diagnosis in clinical samples. In addition, a highly sensitive and specific uniplex RT-PCR for VSV, that amplifies the two main viral serotypes Indiana-1 and New Jersey, was also developed and standardised as a powerful tool for fast and early diagnosis of the disease.
A real-time PCR (polymerase chain reaction) based on the PriProET system (Primer-Probe Energy Transfer) for pan-Foot and Mouth Disease Virus detection was developed. The system was robust and quantitative over a range of at least seven orders of magnitude. A quantitative multiplex RT-PCR for simultaneous detection and identification of Indiana and New Jersey serotypes of Vesicular Stomatitis Virus (VSV) was also developed.
Development of PriProET assays for FMDV (foot and mouth disease virus):
A real-time PCR based on the PriProET (Primer-probe energy transfer) system was developed for pan-FMDV detection. The detection limit of the PriProET RT-PCR was evaluated by comparing the method with traditional virus cultivation based on CPE and standard gel based RT-PCR. The system was reproducible and furthermore quantitative over a range of at least seven orders of magnitude.
The method has been published: "Rasmussen T.B., Uttenthal A., de Stricker K., Belak S. and Storgaard T. (2003). Development of a novel quantitative real-time RT-PCR assay for the simultaneous detection of all serotypes of Foot-and-mouth disease virus. Arch. Virol. 148: 2005-2021.". A detailed description of the new method, "PriProET" and its use for detection of FMDV. The diagnostic use of the method was assayed on recent Probang samples obtained from Uganda. The method is fast, robust and quantitative. It can be performed in a closed tube format, which will reduce the risk of contamination from other samples. The method will add to the presently available diagnostic methods.
A quantitative multiplex assay for real-time detection and identification of Vesicular Stomatitis Virus (VSV) serotypes:
The detection system was based on the primer-probe energy transfer (PriProET) system using pan-VSV primers and two serotype-specific fluorescent probes labelled with Texas Red and Cy5. Generation of amplicons resulted in fluorescent signals specific for either of the two serotypes and the specificity of the reactions were further confirmed by fluorescent probe melting profiles. The limits of detection were found to be below 10 TCID50/ml for both major VSV serotypes. The diagnostic value of the new method was tested on clinical materials from experimentally infected pigs. The multiplex PriProET method allowed relative quantification of viral load in the clinical material and permitted VSV detection in nasal swabs at second or third day post-inoculation for NJ or Ind infected pigs respectively and even before the typical vesicular lesions appeared in the affected animals.
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