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1.
Mol Cell Probes ; 24(4): 219-28, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20412852

ABSTRACT

Flaviviruses are a highly diverse group of RNA viruses classified within the genus Flavivirus, family Flaviviridae. Most flaviviruses are arthropod-borne, requiring a mosquito or tick vector. Several flaviviruses are highly pathogenic to humans; however, their high genetic diversity and immunological relatedness makes them extremely challenging to diagnose. In this study, we developed and evaluated a broad-range Flavivirus assay designed to detect both tick- and mosquito-borne flaviviruses by using RT-PCR/electrospray ionization mass spectrometry (RT-PCR/ESI-MS) on the Ibis T5000 platform. The assay was evaluated with a panel of 13 different flaviviruses. All samples were correctly identified to the species level. To determine the limit of detection for the mosquito-borne primer sets, serial dilutions of RNA from West Nile virus (WNV) were assayed and could be detected down to an equivalent viral titer of 0.2 plaque-forming units/mL. Analysis of flaviviruses in their natural biological background included testing Aedes aegypti mosquitoes that were laboratory-infected with dengue-1 virus. The assay accurately identified the virus within infected mosquitoes, and we determined the average viral genome per mosquito to be 2.0 x 10(6). Using human blood, serum, and urine spiked with WNV and mouse blood and brain tissues from Karshi virus-infected mice, we showed that these clinical matrices did not inhibit the detection of these viruses. Finally, we used the assay to test field-collected Ixodes scapularis ticks collected from sites in New York and Connecticut. We found 16/322 (5% infection rate) ticks positive for deer tick virus, a subtype of Powassan virus. In summary, we developed a single high-throughput Flavivirus assay that could detect multiple tick- and mosquito-borne flaviviruses and thus provides a new analytical tool for their medical diagnosis and epidemiological surveillance.


Subject(s)
Disease Vectors , Flavivirus/genetics , Flavivirus/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction/methods , Spectrometry, Mass, Electrospray Ionization/methods , Animals , Base Composition/genetics , Base Sequence , Culicidae/virology , DNA Primers/metabolism , Dengue Virus/genetics , Dengue Virus/isolation & purification , Encephalitis Viruses, Tick-Borne/genetics , Encephalitis Viruses, Tick-Borne/isolation & purification , Flavivirus Infections/diagnosis , Flavivirus Infections/virology , Mice , Molecular Sequence Data , Sensitivity and Specificity , Sequence Alignment , Ticks/virology , Viral Load/genetics , West Nile virus/genetics , West Nile virus/isolation & purification
2.
Am J Trop Med Hyg ; 82(2): 318-23, 2010 Feb.
Article in English | MEDLINE | ID: mdl-20134011

ABSTRACT

Monkeypox virus (MPXV), a member of the family Poxviridae and genus Orthopoxvirus, causes a smallpox-like disease in humans. A previously described pan-Orthopoxvirus assay, based on a broad-range polymerase chain reaction (PCR) coupled with electrospray ionization mass spectrometry (PCR/ESI-MS), was evaluated for its ability to detect MPXV from spiked human and aerosol-infected cynomolgous macaque (Macaca fascicularis) samples. Detection of MPXV DNA from macaque tissue, blood, and spiked human blood by the PCR/ESI-MS pan-Orthopoxvirus assay was comparable, albeit at slightly higher levels, to the current gold standard method of real-time PCR with the pan-Orthopoxvirus assay and had a limit of detection of 200 plaque-forming units. Furthermore, the platform was able to distinguish MPXV and vaccinia viruses that were spiked into macaque blood samples at various concentrations. This platform provides a new tool for the diagnosis and monitoring of orthopoxviral loads during vaccine or antiviral studies, but also could provide rapid identification during natural outbreaks or bioterrorism attacks.


Subject(s)
Macaca fascicularis , Monkeypox virus , Mpox (monkeypox)/veterinary , Polymerase Chain Reaction/veterinary , Aerosols , Animals , Humans , Mpox (monkeypox)/blood , Mpox (monkeypox)/diagnosis , Mpox (monkeypox)/virology , Polymerase Chain Reaction/methods , Sensitivity and Specificity , Viral Load
3.
Diagn Microbiol Infect Dis ; 63(4): 403-8, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19232863

ABSTRACT

Polymerase chain reaction electrospray ionization mass spectrometry (PCR/ESI-MS) was tested for its ability to accurately identify a blinded panel of 156 diverse bacterial isolates, mostly human and/or animal pathogens. Here, 142/156 (91%) isolates were correctly identified to the genus level and 115/156 (74%) were correctly identified to the species level. Only 9% were misidentified. This study shows that multilocus PCR/ESI-MS has the potential to be a useful technique for identifying a broad range of bacteria.


Subject(s)
Bacteria/classification , Bacteria/isolation & purification , Bacterial Infections/diagnosis , Polymerase Chain Reaction/methods , Spectrometry, Mass, Electrospray Ionization/methods , Animals , Bacteria/genetics , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Humans , Sensitivity and Specificity
4.
Clin Chem ; 52(1): 141-5, 2006 Jan.
Article in English | MEDLINE | ID: mdl-16391330

ABSTRACT

BACKGROUND: Rapid detection of biological threat agents is critical for timely therapeutic administration. Fluorogenic PCR provides a rapid, sensitive, and specific tool for molecular identification of these agents. We compared the performance of assays for 7 biological threat agents on the Idaho Technology, Inc. R.A.P.I.D., the Roche LightCycler, and the Cepheid Smart Cycler. METHODS: Real-time PCR primers and dual-labeled fluorogenic probes were designed to detect Bacillus anthracis, Brucella species, Clostridium botulinum, Coxiella burnetii, Francisella tularensis, Staphylococcus aureus, and Yersinia pestis. DNA amplification assays were optimized by use of Idaho Technology buffers and deoxynucleotide triphosphates supplemented with Invitrogen Platinum Taq DNA polymerase, and were subsequently tested for sensitivity and specificity on the R.A.P.I.D., the LightCycler, and the Smart Cycler. RESULTS: Limit of detection experiments indicated that assay performance was comparable among the platforms tested. Exclusivity and inclusivity testing with a general bacterial nucleic acid cross-reactivity panel containing 60 DNAs and agent-specific panels containing nearest neighbors for the organisms of interest indicated that all assays were specific for their intended targets. CONCLUSION: With minor supplementation, such as the addition of Smart Cycler Additive Reagent to the Idaho Technology buffers, assays for DNA templates from biological threat agents demonstrated similar performance, sensitivity, and specificity on all 3 platforms.


Subject(s)
Bacteria/classification , Biological Warfare , Bacteria/genetics , Bacteriological Techniques , DNA, Bacterial/genetics , Fluorometry , Polymerase Chain Reaction/methods , Sensitivity and Specificity
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