Detection of FMDV RNA amplified by the polymerase chain reaction (PCR).

Molecular detection of foot-and-mouth disease virus (FMDV) using the polymerase chain reaction (PCR) is a rapid and accurate method. In this study we present PCR for the detection of FMDV RNA in infected BHK cells. Using PCR and two primers selected from the RNA polymerase gene, a conserved sequence in all types and subtypes of FMDV, we were able to detect FMDV RNA present in RNA extracted from the FMDV-infected cells. RNA from uninfected BHK cells gave negative results. Another set of primers selected from the nucleotide sequence of the variable VP1 gene permitted the demonstration of variations among different FMDV Israeli isolates by PCR. Two 01 type FMDV isolates out of a total of 6 FMDV field isolates (including 01 Geshur) gave a positive PCR while two other 01 isolates and two ASIA isolates were detected with the RNA polymerase gene primers but not with the VP1 primers. Serial dilutions of the RNA used in each reaction showed that a very small amount of RNA may be detected by PCR. The PCR products from the RNA polymerase and the VP1 genes were sequenced and the nucleotide sequences obtained were compared with a known nucleotide sequence of the FMDV 01 genome.     

Detection by PCR of wild-type canine parvovirus which contaminates dog vaccines.

A method for detecting wild-type canine parvovirus (CPV) strains which contaminate vaccines for dogs has been developed by PCR. PCR primers which distinguish vaccine strains from the most common, recent strains of wild-type CPV in many countries, including Japan and the United States, were developed. This PCR is based on the differences in nucleotide sequences which determine the two antigenic types of this virus. CPV vaccine strains derived from antigenically old-type virus prevalent in former times were not detected by PCR with differential primers. Detection sensitivity of PCR was 100- to 10,000-fold higher than that of the culture method in Crandell feline kidney cells.     

Identification of varicella-zoster virus strains by PCR analysis of three repeat elements and a PstI-site-less region.

We established a method of identifying varicella-zoster virus (VZV) strains, especially those of the Oka vaccine, in patients with clinical VZV infections. The DNAs of 30 clinically isolated strains and 4 laboratory strains including the Oka vaccine strain and its parent VZV strain, were analyzed by PCR with four sets of primers for the four variable regions, R2, R4, R5, and a region without a PstI site (PS). R4 was unstable in four laboratory VZV strains and was excluded from the study. The other regions were stable in several passages in cell culture. The number of copies in R2 and R5 were distributed from 2 to 13 and from 1 to 3, respectively, in the strains analyzed. The vaccine strain had seven copies in R2 and two copies in R5, and it was PS negative. Among 30 clinical isolates, 3, 23, and 11 strains had the same characteristics as the vaccine strain in R2, R5, and PS, respectively. Therefore, by this method, 97.2% of the isolates were distinguished from the Oka vaccine strain. This strategy will be useful in diagnosing VZV infections induced by the vaccine strain.     

Development of a non-destructive PCR method for detection of the salivary gland hypertrophy virus (SGHV) in tsetse flies

A PCR based diagnostic method to detect salivary gland hypertrophy virus (SGHV) in tsetse flies is described. Two sets of primers GpSGHV1F/GpSGHV1R and GpSGHV2F/GpSGHV2R were selected from a virus-specific sequence. Both primer sets can detect specifically the virus in individual tsetse flies by generating an amplicon of 401 bp. Attempts were made to develop a simple and reliable non-destructive virus detection method in live flies. PCR reactions were performed on either crude or purified tsetse DNA from saliva and legs. While saliva can be an indicator for the presence of the virus in flies, the method is laborious. Crude extract from an excised middle leg resulted in a positive PCR reaction equivalent to crude extract from whole fly. However, sensitivity could be significantly increased when purified DNA was used as the template. In conclusion, PCR using a purified DNA template from a single tsetse leg represents an efficient, non-destructive method for virus diagnosis in live tsetse flies.     

Detection and genotyping of varicella-zoster virus by TaqMan allelic discrimination real-time PCR

A proportion of individuals vaccinated with live attenuated Oka varicella-zoster virus (VZV) vaccine subsequently develop attenuated chicken pox and/or herpes zoster. To determine whether postvaccination varicella infections are caused by vaccine or wild-type virus, a simple method for distinguishing the vaccine strain from wild-type virus is required. We have developed a TaqMan real-time PCR assay to detect and differentiate wild-type virus from Oka vaccine strains of VZV. The assay utilized two fluorogenic, minor groove binding probes targeted to a single nucleotide polymorphism in open reading frame 62 that distinguishes the Oka vaccine from wild-type strains. VZV DNA could be genotyped and quantified within minutes of thermocycling completion due to real-time monitoring of PCR product formation and allelic discrimination analysis. The allelic discrimination assay was performed in parallel with two standard PCR-restriction fragment length polymorphism (RFLP) methods on 136 clinical and laboratory VZV strains from Canada, Australia, and Japan. The TaqMan assay exhibited a genotyping accuracy of 100% and, when compared to both PCR-RFLP methods, was 100 times more sensitive. In addition, the method was technically simpler and more rapid. The TaqMan assay also allows for high-throughput genotyping, making it ideal for epidemiologic study of the live attenuated varicella vaccine.     

Intracellular equine arteritis virus (EAV)-specific RNAs contain common sequences

Equine arteritis virus (EAV) is a nonarthropod-borne togavirus. Six virus-specific RNA species have been found in EAV-infected cells having the following molecular weights: 4.3 X 10(6) (RNA1), 1.3 X 10(6) (RNA2), 0.9 X 10(6) (RNA3), 0.7 X 10(6) (RNA4), 0.3 X 10(6) (RNA5), and 0.2 X 10(6) (RNA6). RNA1 comigrates with the viral genome (M. F. Van Berlo, M. C. Horzinek, and B. A. M. Van der Zeijst, 1982, Virology 118, 345-352). All RNAs hybridized with a radio-labeled cDNA probe representing RNA6, indicating that they contain common sequences. To study this homology in more detail, RNase T1 oligonucleotide fingerprinting of the RNAs was undertaken. This confirmed the presence of common sequences and showed more specifically that the intracellular viral RNAs form a nested set. The number of oligonucleotides in RNA1, however, is only one-third of the expected value. In all aspects studied the replication mechanism of EAV differs from that of other known positive-stranded RNA viruses.     

Multiplex RT-nested PCR differentiation of gill-associated virus (Australia) from yellow head virus (Thailand) of Penaeus monodon

A multiplex RT-nested PCR has been developed to detect and differentiate the closely related prawn viruses, gill-associated virus (GAV) from Australia and yellow head virus (YHV) from Thailand. RT-PCR using primers to conserved sequences in the ORF1b gene amplified a 794 bp region of either GAV or YHV. Nested PCR using a conserved sense primer and either a GAV- or YHV-specific antisense primer to a divergent sequence differentially amplified a 277 bp region of the primary PCR amplicon. Multiplexing the YHV antisense primer with a GAV antisense primer to another divergent sequence allowed the viruses to be distinguished in a single nested PCR. Nested PCR enhanced detection sensitivity between 100- and 1000-fold and GAV or YHV RNA was detectable in approximately 10 fg lymphoid organ total RNA. The multiplex RT-nested PCR was also able to co-detect GAV and YHV RNA mixed over a wide range of concentrations to simulate potential dual-infection states. The robustness of the test was examined using RNA samples from Penaeus monodon prawns infected either chronically or acutely with GAV or YHV and collected at different locations in Eastern Australia and Thailand between 1994 and 1998. GAV- (406 bp) or YHV-specific (277 bp) amplicons were differentially generated in all cases, including five YHV RNA samples in which no primary RT-PCR amplicon was detected. Sequence analysis of GAV and YHV PCR amplicons identified minor variations in the regions targeted by the virus-specific antisense primers. However, none occurred at positions that critically affected the PCR.     

Primer design for specific diagnosis by PCR of highly variable RNA viruses: typing of foot-and-mouth disease virus.

A PCR assay for the specific detection and identification of viral sequences that correlate with established serotypes of foot-and-mouth disease virus (FMDV) has been developed. A new analysis based on homology profiles among reported sequences was used for primer design. RNA replicase (3D) gene regions that showed high homology among FMDVs, and low homology to other picornaviruses, were used for PCR amplification. Specific and highly sensitive detection was achieved for RNA of FMDV types C, A, and O, either purified or extracted from vesicular fluids of infected animals, under reaction conditions permissive for the detection of variants present in the virus population. Similarly, serotype-specific primers were designed to amplify the carboxy-terminal end of VP1 gene of FMDV types either C, A, or O. The results of PCR amplification of 15 different FMDV RNAs using type-specific primers are in agreement with the serological typing of the corresponding viruses and show that the primer-selection procedure developed for FMDV constitutes a reliable method of viral diagnosis.     

Detection of smallpox virus DNA by LightCycler PCR

A 300-bp plasmid fragment of the hemagglutinin gene was used as target DNA to develop a rapid real-time LightCycler (Roche Applied Science, Indianapolis, Ind.) PCR assay for laboratory detection of smallpox virus. PCR primers and probes were designed specifically for detection of smallpox virus DNA, but all viruses of the genus Orthopoxvirus tested could be detected by use of the hemagglutinin gene target sequence. Base pair mismatches in the 204-bp amplicon allowed discrimination of cowpox virus (melting temperature [T(m)], 56.40 degrees C), monkeypox virus (T(m), 56.24 degrees C), and vaccinia virus (T(m), 56.72 degrees C), including the Dryvax vaccine strain, from smallpox virus (T(m), 62.45 degrees C) by melting curve analysis. The analytical sensitivity was 5 to 10 copies of target DNA per sample. The assay was specific for members of the genus Orthopoxvirus; the DNAs of herpes simplex virus and varicella-zoster virus were not detected by the smallpox virus LightCycler PCR.     

Differentiation between wild and vaccine-derived strains of poliovirus by stringent microplate hybridization of PCR products.

Procedures for differentiation between wild and vaccine-derived strains of poliovirus are required, particularly in countries where wild and vaccine-related strains coexist. For this differentiation, we tested the method of Inouye and Hondo (S. Inouye and R. Hondo, Arch. Virol. 129:311-316, 1993) for discrimination of closely related viruses by using stringent microplate hybridization of PCR products. We used a pair of primers with enterovirus common sequences (between these primers there is a variable region for capsid proteins) for PCR using templates from wild and vaccine-derived poliovirus strains which were isolated in tissue culture and serotyped by neutralization assay. We also used the same primers for preparation of probes, which were labelled by incorporation of biotin-dUTP in the PCR, with the three original Sabin vaccine virus strains used as templates. The amplified DNAs from the isolates were immobilized on microplate wells and were then hybridized with the labelled probes. We found that, under the usual hybridization conditions, the Sabin vaccine virus strain probes hybridized with both wild and vaccine-derived viruses, but under stringent conditions, they reacted only with vaccine-derived viruses of the same serotype, clearly differentiating these from wild-type viruses.     

Real-time PCR and its application to mumps rapid diagnosis

A real-time polymerase chain reaction assay was initially developed in China to detect mumps genome. The primers and TaqMan-MGB probe were selected from regions of the hemagglutinin gene of mumps virus. The primers and probe for the real-time PCR were evaluated by both laboratories in China and in the UK using three different pieces of equipment, LightCycler (Roche), MJ DNA Engine Option 2 (BIO-RAD) and TaqMan (ABI Prism) on different samples. The reaction was performed with either a one-step (China) or two-step (UK) process. The sensitivity (10 copies) was estimated using a serial dilution of constructed mumps-plasmid DNA and a linear standard curve was obtained between 10 and 10(7) DNA copies/reaction, which can be used to quantify viral loads. The detection limit on cell culture-grown virus was approximately 2 pfu/ml with a two-step assay on TaqMan, which was equivalent to the sensitivity of the nested PCR routinely used in the UK. The specificity was proved by testing a range of respiratory viruses and several genotypes of mumps strains. The concentration of primers and probe is 22 pmol and 6.25 or 7 pmol respectively for a 25 microl reaction. The assay took 3 hr from viral RNA extraction to complete the detection using any of the three pieces of equipment. Three hundred forty-one (35 in China and 306 in the UK) clinical specimens were tested, the results showing that this real-time PCR assay is suitable for rapid and accurate detection of mumps virus RNA in various types of clinical specimens.     

A real‐time PCR assay to identify and discriminate among wild‐type and vaccine strains of varicella‐zoster virus and herpes simplex virus in clinical specimens,and comparison with the clinical diagnoses

A real‐time PCR assay was developed to identify varicella‐zoster virus (VZV) and herpes simplex virus (HSV) DNA in clinical specimens from subjects with suspected herpes zoster (HZ; shingles). Three sets of primers and probes were used in separate PCR reactions to detect and discriminate among wild‐type VZV (VZV‐WT), Oka vaccine strain VZV (VZV‐Oka), and HSV DNA, and the reaction for each virus DNA was multiplexed with primers and probe specific for the human β‐globin gene to assess specimen adequacy. Discrimination of all VZV‐WT strains, including Japanese isolates and the Oka parent strain, from VZV‐Oka was based upon a single nucleotide polymorphism at position 106262 in ORF 62, resulting in preferential amplification by the homologous primer pair. The assay was highly sensitive and specific for the target virus DNA, and no cross‐reactions were detected with any other infectious agent. With the PCR assay as the gold standard, the sensitivity of virus culture was 53% for VZV and 77% for HSV. There was 92% agreement between the clinical diagnosis of HZ by the Clinical Evaluation Committee and the PCR assay results. J. Med. Virol. 81:1310–1322, 2009. Published 2009 Wiley‐Liss, Inc.     

Evaluation of a degenerate primer for the PCR detection of human caliciviruses

Summary Numerous outbreaks of gastroenteritis have been associated with Norwalk virus and Small Round Structured Viruses (SRSVs). These single-stranded RNA viruses, recently classified in the Caliciviridae, have been divided into three genogroups. Antigenic relationships also have been established among the different strains. As both an in vitro culture system and an animal model are lacking for these viruses, virus detection depends primarily on electron microscopy, immunological assays or molecular detection. In this study we first analyzed the genetic homology of the RNA polymerase region for 40 SRSV strains. From a consensus sequence for these strains, we designed a degenerate oligonucleotide to prime cDNA synthesis from viral RNA. We evaluated the degenerate primer in combination with three previously described primers in PCR reactions. A panel of 15 stools containing SRSVs, typed when possible by solid phase immune electron microscopy (SPIEM), were selected to represent all three genogroups and four different SPIEM antigenic types. Serial dilutions of the purified viral nucleic acids were amplified using the three different primer sets. Virus-specific probes were used to characterize the amplicons obtained. Virus-specific amplicons were obtained with at least one primer pair for each strain, but apparent viral RNA titers differed as much as 1 000-fold between primer sets. Amplicons from all but one of the 15 strains were confirmed as virus-specific using a panel of 10 different probes. Correlations between the most sensitive primer pair and SPIEM type were seen. This study showed that a single degenerate primer could be used in cDNA synthesis for a variety of SRSVs but that the sensitivity of the RT-PCR assay depended upon the second primer and virus-specific probes used.