Development of a TaqMan PCR assay with internal amplification control for the detection of African swine fever virus

A closed-tube polymerase chain reaction (PCR) was developed to allow the rapid detection of African swine fever virus (ASFV) DNA. This assay targets the VP72 gene of ASFV and uses the 5'-nuclease assay (TaqMan) system to detect PCR amplicons, avoiding tube opening and potential cross-contamination of post-PCR products. An artificial mimic was engineered with the TaqMan probe site replaced by a larger irrelevant DNA fragment allowing discrimination from ASFV by using two-colour TaqMan probe reporters. When added to the samples, successful amplification of this mimic demonstrated the absence of substances inhibitory to PCR, thereby validating negative results. Assay sensitivity was confirmed by obtaining positive signals with a representative selection of ASFV isolates. Many of the clinical and post-mortem features of ASF resemble those of classical swine fever (CSF) and porcine dermatitis and nephropathy syndrome (PDNS). Therefore, fast and reliable detection of ASFV is essential not only for the implementation of control measures to prevent the spread of ASF, but also in the differential diagnosis from CSF and PDNS. This assay should prove to be a valuable tool in the laboratory diagnosis of ASF and will complement existing molecular methods to provide rapid differential diagnosis in cases of suspected swine fever.     

Development of a primer-probe energy transfer real-time PCR assay for improved detection of classical swine fever virus

Classical swine fever (CSF) is a contagious and devastating disease, causing serious losses in the pig industry worldwide. Vaccination of pigs with the conventional C-strain vaccine has been practised in different regions of the world in order to prevent the disease. In the control programmes of CSF, rapid detection and identification of the causing agent, classical swine fever virus (CSFV) is a crucial step. This study describes a novel real-time PCR assay based on primer-probe energy transfer (PriProET) technology for improved detection of CSFV. The assay is able to detect 20 copies of viral cDNA per reaction, showing a high sensitivity. The specificity has been evaluated by testing 57 pestiviruses, representing all species and unclassified pestiviruses. The assay has been found to be highly reproducible. Following PCR amplification, melting curve analysis allows confirmation of specific amplicons, and differentiation between wild-type CSFV and certain C-strain vaccines. This study provides a new tool for the diagnosis of CSF.     

Preclinical diagnosis of African swine fever in contact-exposed swine by a real-time PCR assay

A fluorogenic probe hydrolysis (TaqMan) PCR assay for African swine fever virus (ASFV) was developed and evaluated in experimentally infected swine. This sensitive and specific one-step single-tube assay, which can be performed in 2 h or less, detected viral DNA in tonsil scraping samples 2 to 4 days prior to onset of clinical disease. Thus, the assay would have application for preclinical diagnosis of African swine fever and surveillance and/or emergency management of a disease outbreak.     

Highly sensitive PCR assay for routine diagnosis of African swine fever virus in clinical samples

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.     

Development of a nested PCR and its internal control for the detection of African swine fever virus (ASFV) in Ornithodoros erraticus

Summary. A nested PCR assay, with an internal control, was developed to detect African swine fever virus (ASFV) DNA in Ornithodoros erraticus. The assay revealed a better analytical sensitivity than virus isolation and the OIE PCR protocol. All ticks collected from the field, which were positive by virus isolation, were also positive by PCR. Viral DNA was detected in a further 19 out of 60 ticks from which no virus was isolated. Our results show that this assay is reliable and can easily be used to screen large tick populations collected in the field for the presence of ASFV.     

Adaptation of an Invader assay for the detection of African swine fever virus DNA

A closed tube isothermal Invader assay (Third Wave Technologies Inc., Madison, Wisconsin, USA) was adapted for the detection of African swine fever virus (ASFV) DNA. Several ASFV Invader assays were designed successfully and tested on a real-time PCR instrument (iCycler, BioRad). The assay exhibiting the lowest signal/noise ratio (VP73 ASFV Invader Assay) was analysed further using serial 10-fold dilutions of Lisbon 60 ASFV viral genome. The assay sensitivity was determined to be in the order of 2500 copies of ASFV DNA and showed a dynamic range of 4 logs, from 2.5x10(6) to 2500 copies. The high specificity of the test was demonstrated by the lack of cross-reactivity to the clinically similar but heterologous virus, classical swine fever virus. The sensitivity of the Invader assay is sufficient for the testing of acutely infected viremic animals in which the viral load will be high. The robustness and ease of use of the ASFV Invader assay, combined with the possibility to run and read the assay using simple and relatively inexpensive equipment, makes it suitable for laboratories lacking containment facilities and/or real-time PCR instrumentation or on a regional basis for on-site diagnosis close to putative sites of ASFV outbreaks.     

Plaque assay for African swine fever virus on swine macrophages

Summary.  A plaque assay developed to detect the infection of African Swine Fever Virus on swine macrophages is described. Plaques were generated by all of the virus isolates tested. The method is suitable not only for virus titration but also for the selection of clones in protocols for isolation/purification of recombinant viruses. Received December 28, 2001; accepted February 20, 2002 Published online April 26, 2002     

A new real-time PCR assay for improved detection of the parasite Babesia microti

Babesiosis is an emerging zoonosis with important public health implications, as the incidence of the disease has risen dramatically over the past decade. Because the current gold standard for detection of Babesia is microscopic examination of blood smears, accurate identification requires trained personnel. Species in the genus cannot be distinguished microscopically, and Babesia can also be confused with the early trophozoite stage (ring forms) of Plasmodium parasites. To allow more accurate diagnosis in a format that is accessible to a wider variety of laboratories, we developed a real-time PCR assay targeting the 18S rRNA gene of Babesia microti, the dominant babesiosis pathogen in the United States. The real-time PCR is performed on DNA extracted from whole-blood specimens and detects Babesia microti with a limit of detection of ～100 gene copies in 5 μl of blood. The real-time PCR assay was shown to be 100% specific when tested against a panel of 24 organisms consisting of Babesia microti, other Babesia species, Plasmodium species, tick-borne and other pathogenic bacteria, and other blood-borne parasites. The results using clinical specimens show that the assay can detect infections of lower parasitemia than can be detected by microscopic examination. This method is therefore a rapid, sensitive, and accurate method for detection of Babesia microti in patient specimens.     

Design and verification of a highly reliable Linear-After-The-Exponential PCR (LATE-PCR) assay for the detection of African swine fever virus

African swine fever virus (ASFV) is a highly pathogenic DNA virus that is the causative agent of African swine fever (ASF), an infectious disease of domestic and wild pigs of all breeds and ages, causing a range of syndromes. Acute disease is characterized by high fever, haemorrhages in the reticuloendothelial system, and a high mortality rate. A powerful novel diagnostic assay based on the Linear-After-The-Exponential-PCR (LATE-PCR) principle was developed to detect ASFV. LATE-PCR is an advanced form of asymmetric PCR which results in direct amplification of large amount of single-stranded DNA. Fluorescent readings are acquired using endpoint analysis after PCR amplification. Amplification of the correct product is verified by melting curve analysis. The assay was designed to amplify the VP72 gene of ASFV genome. Nineteen ASFV DNA cell culture virus strains and three tissue samples (spleen, tonsil, and liver) from infected experimental pigs were tested. Virus was detected in all of the cell culture and tissue samples. None of five ASFV-related viruses tested produced a positive signal, demonstrating the high specificity of the assay. The sensitivity of the LATE-PCR assay was determined in two separate real-time monoplex reactions using samples of synthetic ASFV and synthetic control-DNA targets that were diluted serially from 10? to 1 initial copies per reaction. The detection limit was 1 and 10 copies/reaction, respectively. The sensitivity of the assay was also tested in a duplex end-point reactions comprised of a constant level of 150 copies of synthetic control-DNA and a clinical sample of spleen tissue diluted serially from 10?1 to 10??. The detection limit was 10?? dilution which corresponds to approximately 1 copy/reaction. Since the assay is designed to be used in either laboratory settings or in a portable PCR machine (Bio-Seeq Portable Veterinary Diagnostics Laboratory; Smiths Detection, Watford UK), the LATE-PCR provides a robust and novel tool for the diagnosis of ASF both in the laboratory and in the field.     

Development of TaqMan real-time PCR assay for detection and quantitation of reticuloendotheliosis virus

A highly sensitive real-time PCR method was developed in this study for reticuloendotheliosis virus (REV) detection and quantitation. The real-time PCR method, with a minimum detection limit of 10 proviral DNA copies, was 100 times more sensitive than the conventional PCR. It was also shown to be highly specific, as no positive signals were detected for other common avian DNA viruses. The coefficients of variation of intra- and inter-assay reproducibility were both less than 2%. The chicken β-actin gene was co-amplified and used as the internal control to monitor the efficiency of DNA extraction and PCR amplification. Specific pathogen free chickens were infected with REV at different ages and the blood was detected with the real-time PCR method. High levels of proviral DNA were detected in the blood of REV-infected chickens during the experiment and chickens infected early had higher proviral loads from 2 weeks post-infection compared with late infected chickens. This study provides an excellent research and diagnostic tool that can be used for REV detection and quantitation.     

Development of a real-time PCR assay based on primer-probe energy transfer for the detection of swine vesicular disease virus

Summary. A real-time PCR assay based on primer-probe energy transfer (PriProET) was developed to detect swine vesicular disease virus (SVDV). Specificity tests of SVDV and heterologous virus showed specific amplification of SVDV strains only. The amplification plot for the closely related Coxsackievirus B5 remained negative. The sensitivity of assay was five copies of viral genome equivalents. A key point of the assay is tolerance toward mutations in the probe region. Melting curve analysis directly after PCR, with determination of probe melting point, confirmed specific hybridisation of the SVDV strains. Eight of twenty SVDV strains tested, revealed shifted melting points that indicated mutations in the probe region. All predicted mutations were confirmed by nucleotide sequencing. With the PriProET system there is a chance to identify phylogenetically divergent strains of SVDV, which may appear negative in other probe-based real-time PCR assays. At the same time, any difference in melting points may provide an indication of divergence in the probe region. The high sensitivity, specificity, and tolerance toward mutations in the probe region of the SVDV PriProET assay may improve the early and rapid detection of a wide range of SVDV strains, allowing reduced turnaround time and the use of high-throughput, automated technology.     

Development and validation of a duplex real-time PCR assay for the simultaneous detection and quantification of porcine circovirus type 2 and an internal control on porcine semen samples

A duplex real-time quantitative PCR (qPCR) method for the simultaneous detection of porcine circovirus type 2 (PCV2) and an exogenous internal positive control (IPC) in porcine semen samples was developed. The IPC was included to monitor DNA extraction and PCR inhibition and consisted of a mutated PCV2 plasmid clone which differed from the target PCV2 in the probe binding region and thus was detected by the use of a second probe with different end-labeling. The sensitivity, specificity and repeatability of the assay were validated by testing semen samples from 12 boars inoculated experimentally with PCV2, 10 boars infected naturally with PCV2, and 3 PCV2 negative control boars. The duplex qPCR assay was found to be more sensitive, specific, rapid, and repeatable than nested PCR (nPCR) methods for the detection of PCV2 DNA in semen. Analysis of separated semen fractions by the duplex qPCR assay showed PCV2 DNA to be present mainly in the cell fraction as opposed to the seminal plasma fraction which is in contrast to previous reports. The duplex qPCR assay was found to be a valuable tool for accurate and quantitative detection of PCV2 DNA in boar semen.     

Development and validation of a real-time Taqman PCR assay for the detection and quantitation of infectious laryngotracheitis virus in poultry

In this study, the development and validation of a real-time (ReTi) PCR assay is described using a Taqman labeled probe for the detection and quantitation of infectious larygotracheitis virus (ILTV) in chickens. The ReTi ILTV assay was highly specific with a quantitation limit of 100 viral template copies per amplification reaction. In experimentally infected, birds during early acute stages of infection, an average of 6.67 log(10) viral template copies/amplification reaction were detected, while at chronic late stages of infection an average of 2.86-3.27 log(10) viral template copies/amplification reaction were detected. A total of 246 tracheal swab samples collected from natural outbreaks of the disease were tested by virus isolation and the ReTi ILTV assay. Both assays agreed in 37% of the samples tested and the ReTi ILTV assay detected approximately 3.7 times more positives samples than virus isolation. A minimum of 5 log(10) viral template copies/amplification reaction were required from a tracheal swab to render a virus isolation positive result. In conclusion, the ReTi ILTV assay was highly specific, sensitive, reproducible, and capable of reliably quantifying viral nucleic acid directly from clinical samples.