Quantitative plasmid mixture analysis using the fluorogenic 5'-nuclease polymerase chain reaction assay.

The fluorogenic 5'-nuclease polymerase chain reaction (PCR) assay has been shown to be useful for quantifying a given DNA target in a sample. Here we show how an existing PCR protocol can be amended for quantification by incorporating distinctive dual-labeled, sequence-specific oligonucleotide probes and resulting in a two- to threefold broader and more reliable dynamic range than that of conventional end-point analysis of PCR products. Moreover, we show a multiplex situation in which two targets, one normal and one mutated, can be amplified and quantified simultaneously and in the same reaction tube. Use of this novel approach for quantitative PCR applications eliminates the need for post-PCR processing and has clinical- and research-based diagnostic applications, particularly for measuring levels of mutations in a mixture.

Real-time microchip PCR for detecting single-base differences in viral and human DNA.

This report describes real-time 5' nuclease PCR assays to rapidly distinguish single-base polymorphism using a battery-powered miniature analytical thermal cycling instrument (MATCI). Orthopoxviruses and the human complement component C6 gene served as targets to demonstrate the feasibility of using the MATCI for diagnosis of infectious diseases and genetic disorders. In the Orthopoxvirus assay, consensus Orthopoxvirus PCR primers were designed to amplify 266-281 base-pair (bp) segments of the hemagglutinin (HA) gene in camelpox, cowpox, monkeypox, and vaccinia viruses. A vaccinia virus-specific fluorogenic (TaqMan) probe was designed to detect a single-base (A/G) substitution within the HA gene. In the C6 gene assay, a 73-bp segment of the C6 gene was PCR-amplified from human genomic DNA, and TaqMan probes were used to detect a single-base (A/C) polymorphism in the second position of codon 98. The MATCI correctly identified the nucleotide differences in both viral DNA and human genomic DNA. In addition, using a rapid DNA preparation method, it was possible to achieve sample, preparation of human genomic DNA, DNA amplification, and real-time detection in less than 1 h.

Molecular investigation of a multisource outbreak of Crimean-Congo hemorrhagic fever in the United Arab Emirates.

During the investigation of an outbreak of Crimean-Congo hemorrhagic fever (CCHF) in the United Arab Emirates (UAE) between 1994 and 1995, blood samples from suspected CCHF cases and ticks collected from livestock were tested for CCHF virus by antigen-capture ELISA and by a reverse transcription-polymerase chain reaction. Phylogenetic analysis of partial small (S) segment nucleotide sequences from four ticks and five human samples showed that with one exception, all the human and tick viruses clustered along with samples from Pakistan and Madagascar in one distinct lineage. Within this lineage, sequences from the UAE patients were identical or closely related to those from three Hyalomma spp. ticks obtained from livestock recently imported from Somalia. Another sequence from a UAE patient was more closely related to a CCHF virus from Nigeria. These data indicate that the 1994-1995 CCHF epidemic in the UAE was a multisource outbreak possibly associated with importation of CCHF virus-infected livestock and ticks.

The potential of 5' nuclease PCR for detecting a single-base polymorphism in Orthopoxvirus.

A fluorogenic 5' nuclease PCR assay was evaluated for its ability to specifically detect and differentiate DNA of two Orthopoxvirus species. A pair of consensus primers that target a DNA segment of the Orthopoxvirus haemagglutinin gene, and two oligonucleotide probes; each labelled with a different fluorescent reporter dye and the same quencher dye, were used in a single-tube assay. The assay is based on the 5'-->3' nuclease activity of AmpliTaq DNA polymerase that cleaves a fluorescein-labelled hybridized probe. Probe cleavage generates specific fluorescent signals whose intensity can be quantified by fluorometry. After evaluating the effects of various annealing temperatures and probe concentrations and normalizing the emission intensities of the reporter dyes, it was possible to detect and differentiate monkeypox and vaccinia virus DNAs on the basis of a single-base polymorphism. The sensitivity of the 5' nuclease PCR assay is comparable to the sensitivity of ethidium bromide-stained gels, but the assay provides higher specificity and virtually eliminates the need for laborious post-PCR processing.

Detection of Rift Valley fever virus in mosquitoes by RT-PCR.

A reverse transcriptase-polymerase chain reaction (RT-PCR) assay to detect Rift Valley fever (RVF) virus RNA in experimentally infected mosquitoes was developed. The specificity of the assay was evaluated with three other phleboviruses; sandfly fever Sicilian (Sabin), sandfly fever Naples (Sabin) and Punta Toro (MSP 3) viruses. The relative sensitivity of the assay, determined by using RVF virus RNA extracted from serial dilutions of virus culture, was approximately 50 plaque forming units. This sensitivity level was 100-fold higher when a nested PCR procedure was used. When the RT-PCR assay was used with coded samples of intrathoracically-infected and uninfected mosquito, the assay detected the virus in all infected mosquitoes. With this assay, it was possible to detect RVF virus RNA in a single infected mosquito in the background of 10, 25 or 50 uninfected mosquitoes.

Grouping of hantaviruses by small (S) genome segment polymerase chain reaction and amplification of viral RNA from wild-caught rats.

A single pair of consensus primers in the polymerase chain reaction (PCR) amplified a conserved region of the small genome segment of twenty hantavirus isolates. Isolates tested included representatives of the four recognized hantaviruses, Hantaan, Seoul, Puumala and Prospect Hill, as well as isolates from Mus musculus (Leakey), Bandicota indica (Thailand 749), and Suncus murinus (Thottapalayam). Viruses from the Nairovirus and Phlebovirus genera yielded negative results. The amplification products were 281-nucleotide pairs (np) in length, with the exception of Thottapalayam, which had an amplification product of approximately 320 np. Products of all isolates were detected by Southern hybridization with a 32P-labeled Hantaan 76-118 amplicon, while an oligonucleotide probe to a conserved region of the amplified fragment failed to detect some isolates of Seoul and Puumala viruses. Restriction endonuclease analysis allowed three groupings: Hantaan-like viruses, Seoul-like viruses, and a diverse group of patterns for the other viruses. Differences were found within the Seoul-like virus group by this method, whereas the Hantaan-like viruses were shown to be similar. RNA extracted from tissues of seropositive and seronegative rats trapped in Baltimore showed the practical application of the test. Hantavirus-specific RNA was detected in 12 (92%) of 13 seropositive rats, but not in seronegative rats. This simple method for detecting and characterizing hantaviruses has potential for epidemiologic studies and for diagnosing human hantavirus infections.

Intracranial hemorrhage in congenital deficiency of factor XIII.

We describe a male infant with congenital deficiency of coagulation Factor XIII who presented in the immediate postnatal period with umbilical stump bleeding and suffered a severe intracranial hemorrhage at 2 months of age. Factor XIII, also known as "fibrin-stabilizing factor," is a transpeptidase that produces strong covalent bonds between soluble fibrin monomers formed during coagulation. Presumptive diagnosis of Factor XIII deficiency was made with a clot solubility screening test, and confirmation was accomplished by demonstrating the absence of cross-linked fibrin chains by electrophoresis. This patient had received replacement therapy for 2 years, initially with intravenous fresh frozen plasma, and recently with Fibrogammin (Hoechst-Roussel Pharmaceuticals), a European Factor XIII concentrate soon to be available in the United States. Factor XIII deficiency is associated with a high incidence of life-threatening complications, notably intracranial hemorrhage. In light of the long half-life of this factor and the relatively low risk associated with new Factor XIII concentrates, such as Fibrogammin, prophylactic life-long replacement therapy should be considered for patients with severe Factor XIII deficiency.