Quantitative real-time PCR assays for detection of human adenoviruses and identification of serotypes 40 and 41. 2005

Narayanan Jothikumar, and Theresa L Cromeans, and Vincent R Hill, and Xiaoyan Lu, and Mark D Sobsey, and Dean D Erdman
Centers for Disease Control and Prevention, 4770 Buford Highway, Mailstop F-36, Atlanta, Georgia 30341, USA. JIN2@cdc.gov

A quantitative real-time TaqMan PCR assay for detection of human adenoviruses (HAdV) was developed using broadly reactive consensus primers and a TaqMan probe targeting a conserved region of the hexon gene. The TaqMan assay correctly identified 56 representative adenovirus prototype strains and field isolates from all six adenovirus species (A to F). Based on infectious units, the TaqMan assay was able to detect as few as 0.4 and 0.004 infectious units of adenovirus serotype 2 (AdV2) and AdV41, respectively, with results obtained in less than 90 min. Using genomic equivalents, the broadly reactive TaqMan assay was able to detect 5 copies of AdV40 (which had zero mismatches with the PCR primers and probe), 8 copies of AdV41, and 350 copies of AdV3 (which had the most mismatches [seven] of any adenovirus serotype tested). For specific detection and identification of F species serotypes AdV40 and AdV41, a second real-time PCR assay was developed using fluorescence resonance energy transfer (FRET) probes that target the adenovirus fiber gene. The FRET-based assay had a detection limit of 3 to 5 copies of AdV40 and AdV41 standard DNA and was able to distinguish between AdV40 and AdV41 based on melting curve analysis. Both the TaqMan and FRET PCR assays were quantitative over a wide range of virus titers. Application of these assays for detection of adenoviruses and type-specific identification of AdV40 and AdV41 will be useful for identifying these viruses in environmental and clinical samples.

UI MeSH Term Description Entries
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000260 Adenoviruses, Human Species of the genus MASTADENOVIRUS, causing a wide range of diseases in humans. Infections are mostly asymptomatic, but can be associated with diseases of the respiratory, ocular, and gastrointestinal systems. Serotypes (named with Arabic numbers) have been grouped into species designated Human adenovirus A-G. APC Viruses,APC Virus,Adenovirus, Human,Human Adenovirus,Human Adenoviruses
D012680 Sensitivity and Specificity Binary classification measures to assess test results. Sensitivity or recall rate is the proportion of true positives. Specificity is the probability of correctly determining the absence of a condition. (From Last, Dictionary of Epidemiology, 2d ed) Specificity,Sensitivity,Specificity and Sensitivity
D012703 Serotyping Process of determining and distinguishing species of bacteria or viruses based on antigens they share. Serotypings
D013045 Species Specificity The restriction of a characteristic behavior, anatomical structure or physical system, such as immune response; metabolic response, or gene or gene variant to the members of one species. It refers to that property which differentiates one species from another but it is also used for phylogenetic levels higher or lower than the species. Species Specificities,Specificities, Species,Specificity, Species
D016133 Polymerase Chain Reaction In vitro method for producing large amounts of specific DNA or RNA fragments of defined length and sequence from small amounts of short oligonucleotide flanking sequences (primers). The essential steps include thermal denaturation of the double-stranded target molecules, annealing of the primers to their complementary sequences, and extension of the annealed primers by enzymatic synthesis with DNA polymerase. The reaction is efficient, specific, and extremely sensitive. Uses for the reaction include disease diagnosis, detection of difficult-to-isolate pathogens, mutation analysis, genetic testing, DNA sequencing, and analyzing evolutionary relationships. Anchored PCR,Inverse PCR,Nested PCR,PCR,Anchored Polymerase Chain Reaction,Inverse Polymerase Chain Reaction,Nested Polymerase Chain Reaction,PCR, Anchored,PCR, Inverse,PCR, Nested,Polymerase Chain Reactions,Reaction, Polymerase Chain,Reactions, Polymerase Chain
D017931 DNA Primers Short sequences (generally about 10 base pairs) of DNA that are complementary to sequences of messenger RNA and allow reverse transcriptases to start copying the adjacent sequences of mRNA. Primers are used extensively in genetic and molecular biology techniques. DNA Primer,Oligodeoxyribonucleotide Primer,Oligodeoxyribonucleotide Primers,Oligonucleotide Primer,Oligonucleotide Primers,Primer, DNA,Primer, Oligodeoxyribonucleotide,Primer, Oligonucleotide,Primers, DNA,Primers, Oligodeoxyribonucleotide,Primers, Oligonucleotide
D019914 Taq Polymerase A heat stable DNA-DIRECTED DNA POLYMERASE from the bacteria Thermus aquaticus. It is widely used for the amplification of genes through the process of POLYMERASE CHAIN REACTION. EC 2.7.7.-. Taq DNA Polymerase,Taq1 Polymerase,Thermus aquaticus Polymerase,DNA Polymerase, Taq,Polymerase, Taq,Polymerase, Taq DNA,Polymerase, Taq1,Polymerase, Thermus aquaticus,aquaticus Polymerase, Thermus
D031541 Fluorescence Resonance Energy Transfer A type of FLUORESCENCE SPECTROSCOPY using two FLUORESCENT DYES with overlapping emission and absorption spectra, which is used to indicate proximity of labeled molecules. This technique is useful for studying interactions of molecules and PROTEIN FOLDING. Forster Resonance Energy Transfer
D036022 Capsid Proteins Proteins that form the CAPSID of VIRUSES. Procapsid Protein,Procapsid Proteins,Viral Coat Protein,Viral Coat Proteins,Viral V Antigens,Viral V Proteins,Capsid Protein,Viral Outer Coat Protein,Antigens, Viral V,Coat Protein, Viral,V Antigens, Viral,V Proteins, Viral

Related Publications

Narayanan Jothikumar, and Theresa L Cromeans, and Vincent R Hill, and Xiaoyan Lu, and Mark D Sobsey, and Dean D Erdman
November 2016, Journal of virological methods,
Narayanan Jothikumar, and Theresa L Cromeans, and Vincent R Hill, and Xiaoyan Lu, and Mark D Sobsey, and Dean D Erdman
January 2016, Water science and technology : a journal of the International Association on Water Pollution Research,
Narayanan Jothikumar, and Theresa L Cromeans, and Vincent R Hill, and Xiaoyan Lu, and Mark D Sobsey, and Dean D Erdman
April 2013, Journal of clinical microbiology,
Narayanan Jothikumar, and Theresa L Cromeans, and Vincent R Hill, and Xiaoyan Lu, and Mark D Sobsey, and Dean D Erdman
July 2008, Journal of virological methods,
Narayanan Jothikumar, and Theresa L Cromeans, and Vincent R Hill, and Xiaoyan Lu, and Mark D Sobsey, and Dean D Erdman
January 1988, Intervirology,
Narayanan Jothikumar, and Theresa L Cromeans, and Vincent R Hill, and Xiaoyan Lu, and Mark D Sobsey, and Dean D Erdman
January 2004, Methods in molecular biology (Clifton, N.J.),
Narayanan Jothikumar, and Theresa L Cromeans, and Vincent R Hill, and Xiaoyan Lu, and Mark D Sobsey, and Dean D Erdman
September 2006, Journal of clinical microbiology,
Narayanan Jothikumar, and Theresa L Cromeans, and Vincent R Hill, and Xiaoyan Lu, and Mark D Sobsey, and Dean D Erdman
May 2018, Virology journal,
Narayanan Jothikumar, and Theresa L Cromeans, and Vincent R Hill, and Xiaoyan Lu, and Mark D Sobsey, and Dean D Erdman
April 2017, Journal of microbiology and biotechnology,
Narayanan Jothikumar, and Theresa L Cromeans, and Vincent R Hill, and Xiaoyan Lu, and Mark D Sobsey, and Dean D Erdman
October 2003, Journal of clinical microbiology,
Copied contents to your clipboard!