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Adenovirus R-Gene® US

Real-Time PCR for the Detection of Adenoviruses in Respiratory Infections

Adenovirus R-gene® US Assay is a Real Time PCR in vitro diagnostic test for the rapid and qualitative detection of Adenovirus viral DNA isolated and purified from nasopharyngeal swab or nasopharyngeal wash/aspirate specimens obtained from individuals exhibiting signs and symptoms of acute respiratory infection.

  • Easy assay set-up
  • Rapid turn-around time (< 1 h post extraction)
  • FDA cleared for extraction with the NucliSENS® easyMAG

Adenovirus R-gene® US will enable the detection of all of the following serotypes:

  • Adenovirus A (AdV 12, 18, 31)
  • Adenovirus B (AdV 3, 7, 11, 14, 16, 21, 34, 35, 50)
  • Adenovirus C (AdV 1, 2, 5, 6)
  • Adenovirus D (AdV 8 to 10, 13, 15, 17, 19, 20, 22 to 30, 32,33, 36-39, 42-49, 51)
  • Adenovirus E (AdV 4)
  • Adenovirus F (AdV 40, 41)
  • Adenovirus G (AdV 52)
  • Unclassified (AdV 53 to 60)

ADENOVIRUS R-GENE® US PERFORMANCE

Assay is a Real Time PCR in vitro diagnostic test for the rapid and qualitative detection of Adenovirus viral DNA isolated and purified from nasopharyngeal swab or nasopharyngeal wash/aspirate specimens obtained from individuals exhibiting signs and symptoms of acute respiratory infection. Easy assay set up and rapid turn around time (< 1 h post extraction).

Swab Specimens

  1. Among the 43 negative samples for rapid culture and positive for Adenovirus, 42 were confirmed as positive for adenovirus using Real Time PCR.
  2. Among the 4 positive samples for rapid culture and negative for Adenovirus, 1 was confirmed as positive for adenovirus using Real Time PCR.

Nasopharyngeal Wash/Aspirate Specimens

 

ADENOVIRUS R-GENE® USES

Human adenoviruses are members of the Adenoviridae family, Mastadenovirus genus. Adenoviruses are medium-sized, non-enveloped DNA viruses. At least seven human adenoviruses species (A-G) have been described, comprising at least 60 types/serotypes [2, 6, 7, 8, 9, 10, 11, 12].

  • Adenoviruses typically infect children and military recruits either during a local outbreak or as endemic infections and can cause respiratory, ocular or gastrointestinal disease.
  • Adenovirus infections are common, have a worldwide distribution and can occur throughout the year.
  • Adenoviruses have been increasingly recognized as significant viral pathogens and can result in high morbidity and mortality among immunocompromised patients.
  • Clinical manifestations in immunocompromised patients include pneumonia, hepatitis, hemorrhagic cystitis, colitis, pancreatitis, meningoencephalitis and disseminated disease, depending on the underlying disease, affected organ system, patient age, and virus serotype [1, 3, 4, 5].

Amplification and detection of the viral genome by Real Time PCR is highly-sensitive, and is especially applicable when viral load is too low to be detected by culture, or when results are needed rapidly.

ADENOVIRUS R-GENE® US PRODUCT INFORMATION

  • 5’ nuclease technology (Patents N°5210015, 5487972), also called TaqMan® probes.
    • The primers allow a very efficient amplification of a fragment located in the Hexon gene region, coding for the hexagonal capsomers. The amplified fragment size is 138 bp. The probes are dual-labelled with a reporter dye attached to the 5’-end and a quencher dye attached to the 3’-end.
  • Sample preparation: The assay requires extraction to be carried out using the NucliSENS®easyMAG® from bioMérieux.
  • Internal control (IC2) introduced before the extraction step to control the whole process of cell lysis and viral DNA amplification.
  • Suitable for use with the SmartCycler® II instrument using the Dx Software.
  • Ready-to-use amplification mixture, including:
    • Primers
    • dNTPs
    • amplification buffer
    • Taq Polymerase
    • specific probes for adenoviruses
    • probe and specific primers for the internal control 2.
  • Results are validated using the negative extraction + inhibition control (IC2 + W0) and the positive amplification control (PC10) provided with the ADENOVIRUS R-gene® US kit.

OVERVIEW OF THE PROCEDURE

  1. Collect specimens as described in the specimen collection section from symptomatic patients using a polyester, rayon, nylon tipped swab or sterile suction catheter for nasopharyngeal washes/aspirates specimen.
  2. Add the Internal Control 2 (IC2) to every sample to detect the presence of inhibitors or lysis failure.
  3. Perform isolation and purification of nucleic acids using NucliSENS®easyMAG® System (bioMérieux) and the automated Magnetic Extraction Reagents (bioMérieux).
  4. Perform real time amplification of extracted samples using the ADENOVIRUS R-gene® US kit on the SmartCycler® II instrument (Cepheid), Dx Software.
  5. The ADENOVIRUS R-gene® US premix is ready-to-use and contains all the reagents necessary for the amplification. Only the extracted sample has to be added.
    1. A positive control enables the amplification run to be properly validated. This positive control is amplified with the same primers as the viral DNA of the Adenovirus potentially present in patient samples.
    2. An internal control (IC2) is included in the ADENOVIRUS R-gene® US kit in order to check if the sample has been lysed and purified efficiently and to verify the absence of amplification inhibitors in the extracted sample.
  6. Interpretation.

ADENOVIRUS R-GENE® US ORDERING INFORMATION

US Item #

Product

Quantity (Tests)

69-010B-US

Adenovirus r-gene® US

90

MATERIALS PROVIDED – ADENOVIRUS R-GENE® US

Reagent

Description

Quantity/tube

Number of Tubes

Number of reactions

Rx10

Adenovirus and IC2 Amplification premix

450 µL

3

90

IC2

Internal Control 2

1 mL

1

100

W0

Water for extraction (Molecular grade)

1.8 mL

2

18

PC10

Adenovirus Positive Amplification Control

300 µL

1

30

Ready-to-use amplification mixture, including: 

  • Primers
  • dNTPs
  • amplification buffer
  • Taq Polymerase
  • specific probes for adenoviruses
  • probe and specific primers for the internal control 2.

ADENOVIRUS R-GENE® US PUBLICATIONS

[1] HEIM A., EBNET C., HARSTE G., PRING-AKERBLON P. Rapid and Quantitative Detection of Human Adenovirus DNA by Real-Time PCR. Journal of Medical Virology 70 : 228-239, 2003.

[2] TATE JE, BUNNING ML, LOTT L, LU X, SU J, METZGAR D, BROSCH L, PANOZZO CA, MARCONI VC, FAIX DJ, PRILL M, JOHNSON B, ERDMAN DD, FONSECA V, ANDERSON LJ, WIDDOWSON MA. Outbreak of Severe Respiratory Disease Associated with Emergent Human Adenovirus Serotype 14 at a US Air Force Training Facility in 2007. J Infect Dis. 15;199(10):1419-1426, May 2009.

[3] VABRET A., GOUARIN S., JOANNES M., BARRANGER C., PETITJEAN J., CORBET S., BROUARD J., DUHAMEL JF., GUILLOIS B. and FREYMUTH F. Development of a PCR and hybridization-based assay (PCR ADENOVIRUS CONSENSUS) for the detection and the species identification of adenoviruses in respiratory specimens. J. Clin virology 31(2):116-22, Oct 2004.

[4] ECHAVARRÍA M. Adenoviruses in Immunocompromised Hosts. Clin. Microbiol. Rev. 21: 704-715, 2008.

[5] ECHAVARRÍA M. Principles and practice of clinical virology, 6th ed. John Wiley and Sons, San Diego, CA. In A. J. Zuckerman, J. E. Banatvala, B. D. Schoub, P. D. Griffiths, and P. Mortimer (ed.) 2. Adenovirus, p. 463–488, 2009.

[6] ISHIKO, H., Y. SHIMADA, T. KONNO, A. HAYASHI, T. OHGUCHI, Y. TAGAWA, K. AOKI, S. OHNO, AND S. YAMAZAKI. Novel human adenovirus causing nosocomial epidemic keratoconjunctivitis. J. Clin. Microbiol. 46:2002–2008, 2008.

[7] JONES II, M.S., HARRACH, B., GANAC, R.D., GOZUM, M.M., DELA CRUZ, W.P., RIEDEL, B., PAN, C., DELWART, E.L., SCHNURR, D.P. New adenovirus species found in a patient presenting with gastroenteritis. J. Virol. 81 (11), 5978–5984, 2007.

[8] LIU EB, FERREYRA L, FISCHER SL, PAVAN JV, NATES SV, ET AL. Genetic Analysis of a Novel Human Adenovirus with a Serologically Unique Hexon and a Recombinant Fiber Gene. PLoS ONE 6(9): e24491. doi:10.1371/journal.pone.0024491, 2011.

[9] ROBINSON C.M., SINGH G., HENQUELL C., WALSH M.P., PEIGUE-LAFEUILLE H., SETO D., JONES M.S., DYER D.W., CHODOSH J. Computational analysis and identification of an emergent human adenovirus pathogen implicated in a respiratory fatality. Virology 409 (2011) 141–147, 2011.

[10] WALSH, M.P., CHINTAKUNTLAWAR, A., ROBINSON, C.M., MADISCH, I., HARRACH, B., HUDSON, N.R., SCHNURR, D., HEIM, A., CHODOSH, J., SETO, D., JONES, M.S. Evidence of molecular evolution driven by recombination events influencing tropism in a novel human adenovirus that causes epidemic keratoconjunctivitis. PLoS ONE 4 (6), e5635, 2009.

[11] WALSH, M.P., SETO, J., JONES, M.S., CHODOSH, J., XU, W., SETO, D. Computational analysis identifies human adenovirus type 55 as a re-emergent acute respiratory disease pathogen. J. Clin. Microbiol. 48 (3), 991–993, 2010.

[12] ISHIKO, H., AND K. AOKI. Spread of epidemic keratoconjunctivitis due to a novel serotype of human adenovirus in Japan. J. Clin. Microbiol. 47:2678–2679, 2009

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