Montserrat Agüero

Highly Sensitive PCR Assay for Routine Diagnosis of African Swine Fever Virus in Clinical Samples

ABSTRACT This work provides a novel, highly sensitive, hot start PCR method for rapid and specific detection of African swine fever virus (ASFV) that can be used as a routine diagnostic test for ASFV in surveillance, control, and eradication programs. A confirmatory test of the specificity of this method based on restriction endonuclease analysis was also developed.

High Throughput Detection of Bluetongue Virus by a New Real-Time Fluorogenic Reverse Transcription—Polymerase Chain Reaction: Application on Clinical Samples from Current Mediterranean Outbreaks

A real-time reverse transcription-polymerase chain reaction (RT-PCR) assay was developed for the detection of bluetongue virus (BTV) in blood samples. A combination of primers specific for a highly conserved region in RNA segment 5 (based on Mediterranean BTV sequences) and a DNA probe bound to 5′-Taq nuclease-3′ minor groove binder (TaqMan© MGB) was used to detect a range of isolates. This real-time RT-PCR assay could detect 5.4 × 10−3 tissue culture infectious doses (TCID50) of virus per milliliter of sample, which was comparable to our current BTV diagnostic nested RT-PCR assay. The assay detected all recent Mediterranean isolates (including serotypes 2, 4, and 16), BTV vaccine strains for serotypes 2 and 4, and 15 out of the 24 BTV reference strains available (all serotypes), but did not detect the related orbiviruses epizootic hemorrhagic disease and African horse sickness viruses. Following assay evaluation, the ability of this assay to identify BTV in recent isolates (2003, 2004) from ovine and bovine samples from an epizootic outbreak in Spain was also tested. Minor nucleotide changes (detected by sequencing viral genomes) within the probe-binding region were found to have a profound effect on virus detection. This assay has the benefits of being fast and simple, and the 96-well format enables large-scale epidemiological screening for BTV, especially when combined with a high-throughput nucleic acid extraction method.

Bagaza Virus in Partridges and Pheasants, Spain, 2010

In September 2010, an unusually high number of wild birds (partridges and pheasants) died in Cádiz in southwestern Spain. Reverse transcription PCR and virus isolation detected flavivirus infections. Complete nucleotide sequence analysis identified Bagaza virus, a flavivirus with a known distribution that includes sub-Saharan Africa and India, as the causative agent.

A New Fluorogenic Real-Time RT-PCR Assay for Detection of Lineage 1 and Lineage 2 West Nile Viruses

West Nile virus represents an emerging threat for animal and human health worldwide. This virus exhibits a marked genetic variation, with at least 2 distinct evolutionary lineages. Lineage 1 has been recognized in Africa, Asia, Europe, Oceania, and more recently in the Americas, whereas lineage 2 is restricted to Africa. Perhaps for this reason, the available real-time RT-PCR methods for detecting West Nile virus genome have mainly focused on lineage 1. However, both viruses may potentially be spread beyond their endemic areas by migratory birds. This report describes a new real-time reverse transcription-PCR (RT-PCR) method based on a 59-Taq nuclease-39 minor groove binder DNA probe (TaqMan MGBE) that allows the detection of a wide range of West Nile virus isolates, including both lineages 1 and 2. This method was able to detect West Nile viruses from different origins (North and Central Africa, Middle East, Europe, and North America), whereas other flaviviruses (Usutu, Dengue, Yellow fever) analyzed in parallel remained negative. The sensitivity achieved by this assay was 1022-1023 pfu/tube. This method, which can be performed in 96-well format, could be suitable for the large-scale surveillance of West Nile virus in areas where both lineages can potentially spread.

A Fully Automated Procedure for the High-Throughput Detection of Avian Influenza Virus by Real-Time Reverse Transcription–Polymerase Chain Reaction

Abstract The recent spread of highly pathogenic H5N1 avian influenza (AI) has made it important to develop highly sensitive diagnostic systems for the rapid detection of AI genome and the differentiation of H5N1 variants in a high number of samples. In the present paper, we describe a high-throughput procedure that combines automated extraction, amplification, and detection of AI RNA, by an already described TaqMan real-time reverse transcription–polymerase chain reaction (RRT-PCR) assay targeted at the matrix (M) protein gene of AI virus (AIV). The method was tested in cloacal and tracheal swabs, the most common type of samples used in AI surveillance, as well as in tissue and fecal samples. A robotic system (QIAGEN Biosprint 96) extracted RNA and set up reactions for RRT-PCR in a 96-well format. The recovery of the extracted RNA was as efficient as that of a manual RNA extraction kit, and the sensitivity of the detection system was as high as with previously described nonautomated methods. A system with a basic configuration (one extraction robot plus two real-time 96-well thermocyclers) operated by two persons could account for about 360 samples in 5 hr. Further characterization of AI RNA–positive samples with a TaqMan RRT-PCR specific for H5 (also described here) and/or N1 was possible within 2 hr more. As this work shows, the system can analyze up to 1400 samples per working day by using two nucleic acid extraction robots and a 384-well-format thermocycler.

A novel quantitative multiplex real-time RT-PCR for the simultaneous detection and differentiation of West Nile virus lineages 1 and 2, and of Usutu virus.

An increase in activity of two mosquito-borne flaviviruses, West Nile virus (WNV) and Usutu virus (USUV), has been reported in Europe in recent years. The current epidemiological situation calls for RT-PCR methods that are able to detect not only the widespread lineage 1 (L1) WNV, but also lineage 2 (L2) WNV. In addition, the presence in Europe of the closely related USUV requires methods that can identify these three flaviviruses and permit an efficient and accurate differential diagnosis. Here we describe a new one-step real-time multiplex RT-PCR that detects and differentiates efficiently WNV-L1, WNV-L2 and USUV in a single reaction. The assay is based on different sets of primers and fluorogenic probes specific to each virus that are labelled with selective, non-overlapping fluorogen-quencher pairs. This enables the fluorescence emitted by each probe, characterized by distinct wavelengths, to be differentiated. This multiplex assay was very sensitive to all of the target viruses; in addition, there were no cross-reactions between the viruses and the assay did not react to any other phylogenetically or symptomatically related viruses. Quantitation was enabled through the use of in vitro-transcribed RNAs developed specifically for each virus as copy number standards. This new assay was validated using different types of experimental and field samples.

A Real-Time TaqMan RT-PCR Method for Neuraminidase Type 1 (N1) Gene Detection of H5N1 Eurasian Strains of Avian Influenza Virus

Abstract This work describes the development of a real-time RT-PCR (RRT-PCR) procedure for detection of the N1 gene from avian influenza virus (AIV), based on the use of specific primers and a TaqMan-MGB (minor groove binder) probe. Nucleotide sequences of the neuraminidase type 1 gene from a collection of H5N1 Eurasian strains of AIV were aligned using ClustalW software. Conserved regions were located and used to design specific primers and a TaqMan-MGB probe using Primer Express software. A one-step RRT-PCR method was optimized using RNA from the Turkey 2005 H5N1 strain of AIV and can be completed in about 2 hr once the RNA is extracted from the sample. The specificity of the assay was assessed with non-N1 AIV strains, another related avian virus, and different avian tissue samples from healthy animals. Sensitivity was determined using 10-fold serial dilutions of the H5N1 Turkey 2005 strain and was compared with the generic RRT-PCR detection method, targeted at the matrix protein gene of AIV, commonly used at the Spanish AIV National Reference Laboratory. The N1 detection method proved to be even more sensitive than the generic (matrix-based) method, allowing a very quick confirmation (or discarding) of any Eurasian N1 strain when a positive result was obtained with the matrix RRT-PCR assay. Combined with RRT-PCR tests for general detection of AIV and H5 typing in use at the NRL, the procedure here described allows characterizing of any H5N1 Eurasian AIV strain in a field sample within a working day.

Real-time fluorogenic reverse transcription polymerase chain reaction assay for the specific detection of Bagaza virus

In September 2010, an outbreak of disease in 2 wild bird species (red-legged partridge, Alectoris rufa; ring-necked pheasant, Phasianus colchicus) occurred in southern Spain. Bagaza virus (BAGV) was identified as the etiological agent of the outbreak. BAGV had only been reported before in Western Africa (Central African Republic, Senegal) and in India. The first occurrence of BAGV in Spain stimulated a demand for rapid, reliable, and efficacious diagnostic methods to facilitate the surveillance of this disease in the field. This report describes a real-time reverse transcription polymerase chain reaction (RT-PCR) method based on a commercial 5’-Taq nuclease-3’ minor groove binder DNA probe and primers targeting the Bagaza NS5 gene. The method allowed the detection of BAGV with a high sensitivity, whereas other closely related flaviviruses (Usutu virus, West Nile virus, and Japanese encephalitis virus) were not detected. The assay was evaluated using field samples of red-legged partridges dead during the outbreak (n = 11), as well as samples collected from partridges during surveillance programs (n = 81). The results were compared to those obtained with a pan-flaviviral hemi-nested RT-PCR followed by nucleotide sequencing, which was employed originally to identify the virus involved in the outbreak. The results obtained with both techniques were 100% matching, indicating that the newly developed real-time RT-PCR is a valid technique for BAGV genome detection, useful in both diagnosis and surveillance studies.

Development of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification Assay for the Rapid Detection of African Horse Sickness Virus

Summary African horse sickness (AHS) is a disease of equids caused by African Horse Sickness Virus (AHSV) and is transmitted by Culicoides midges. AHS is endemic in sub‐Saharan Africa, but during the past century, outbreaks of significant economic importance and elevated mortality have been recorded in Northern African countries, the Iberian and Arabian Peninsula, the Middle East and the Indian subcontinent. Effective control combines the application of early warning systems, accurate laboratory diagnosis and reporting, animal movement restrictions, suitable vaccination and surveillance programs, and the coordination of all these measures by efficient veterinary services. Conventional reverse‐transcriptase (RT) PCR (RT‐PCR) and real‐time RT‐PCR (rRT‐PCR) assays have improved the sensitivity and rapidity of diagnosing AHS, resulting in the adoption of these methods as recommended tests by the World Organisation for Animal Health (OIE). However, currently these assays are only performed within laboratory settings; therefore, the development of field diagnostics for AHS would improve the fast implementation of control policies. Loop‐mediated isothermal amplification (LAMP) is an isothermal, autocycling, strand‐displacement nucleic acid amplification technique which can be performed in the field. LAMP assays are attractive molecular assays because they are simple to use, rapid, portable and have sensitivity and specificity within the range of rRT‐PCR. This study describes the development of a novel RT‐LAMP assay for the detection of AHSV. The AHSV RT‐LAMP assay has an analytical sensitivity of 96.1% when considering an rRT‐PCR cut‐off value of C T > 36, or 91.3% when no rRT‐PCR cut‐off is applied. Diagnostic sensitivity and specificity were 100%. This assay provides for a rapid and low cost AHS diagnostic for use in the field.

A FULLY AUTOMATED PROCEDURE FOR THE HIGH THROUGHPUT DETECTION OF AVIAN INFLUENZA VIRUS BY REAL-TIME RT-PCR

Rapid detection of AIV is essential in disease outbreak situations, when the rapid identification of viremic animals is crucial to preventing the spread of disease. Viremia can be demonstrated in tracheal or cloacal swabs, tissues (mainly from the respiratory and gastrointestinal tracts) and faecal samples of infected animals both by virus isolation or nucleic acid detection methods. Although virus isolation in embryonated chicken eggs is reliable and sensitive, it is cumbersome and takes longer to perform than nucleic acid-based detection assays. Several reverse transcription (RT)-PCR methods have previously been described for this purpose but conventional RT-PCR methods are difficult to apply when very large numbers of samples are to be analyzed. Recently, realtime PCR protocols for the detection of AIV have become available. This system allows rapid diagnosis and is amenable for automation, which allows high throughput sample monitoring.