Tags

Type your tag names separated by a space and hit enter

Dual-probe real-time PCR assay for detection of variola or other orthopoxviruses with dried reagents.
J Virol Methods. 2008 Nov; 153(2):190-5.JV

Abstract

A real-time, multiplexed polymerase chain reaction (PCR) assay based on dried PCR reagents was developed. Only variola virus could be specifically detected by a FAM (6-carboxyfluorescein)-labeled probe while camelpox, cowpox, monkeypox and vaccinia viruses could be detected by a TET (6-carboxytetramethylrhodamine)-labeled probe in a single PCR reaction. Approximately 25 copies of cloned variola virus DNA and 50 copies of genomic orthopoxviruses DNA could be detected with high reproducibility. The assay exhibited a dynamic range of seven orders of magnitude with a correlation coefficient value greater than 0.97. The sensitivity and specificity of the assay, as determined from 100 samples that contained nucleic acids from a multitude of bacterial and viral species were 96% and 98%, respectively. The limit of detection, sensitivity and specificity of the assay were comparable to standard real-time PCR assays with wet reagents. Employing a multiplexed format in this assay allows simultaneous discrimination of the variola virus from other closely related orthopoxviruses. Furthermore, the implementation of dried reagents in real-time PCR assays is an important step towards simplifying such assays and allowing their use in areas where cold storage is not easily accessible.

Authors+Show Affiliations

Virology Division, United States Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Frederick, Frederick, MD 21702-5011, USA. mohamed.aitichou@amedd.army.milNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Evaluation Study
Journal Article
Research Support, U.S. Gov't, Non-P.H.S.

Language

eng

PubMed ID

18725245

Citation

Aitichou, Mohamed, et al. "Dual-probe Real-time PCR Assay for Detection of Variola or Other Orthopoxviruses With Dried Reagents." Journal of Virological Methods, vol. 153, no. 2, 2008, pp. 190-5.
Aitichou M, Saleh S, Kyusung P, et al. Dual-probe real-time PCR assay for detection of variola or other orthopoxviruses with dried reagents. J Virol Methods. 2008;153(2):190-5.
Aitichou, M., Saleh, S., Kyusung, P., Huggins, J., O'Guinn, M., Jahrling, P., & Ibrahim, S. (2008). Dual-probe real-time PCR assay for detection of variola or other orthopoxviruses with dried reagents. Journal of Virological Methods, 153(2), 190-5. https://doi.org/10.1016/j.jviromet.2008.07.018
Aitichou M, et al. Dual-probe Real-time PCR Assay for Detection of Variola or Other Orthopoxviruses With Dried Reagents. J Virol Methods. 2008;153(2):190-5. PubMed PMID: 18725245.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Dual-probe real-time PCR assay for detection of variola or other orthopoxviruses with dried reagents. AU - Aitichou,Mohamed, AU - Saleh,Sharron, AU - Kyusung,Park, AU - Huggins,John, AU - O'Guinn,Monica, AU - Jahrling,Peter, AU - Ibrahim,Sofi, Y1 - 2008/09/10/ PY - 2008/03/21/received PY - 2008/06/25/revised PY - 2008/07/17/accepted PY - 2008/8/30/pubmed PY - 2008/12/17/medline PY - 2008/8/30/entrez SP - 190 EP - 5 JF - Journal of virological methods JO - J Virol Methods VL - 153 IS - 2 N2 - A real-time, multiplexed polymerase chain reaction (PCR) assay based on dried PCR reagents was developed. Only variola virus could be specifically detected by a FAM (6-carboxyfluorescein)-labeled probe while camelpox, cowpox, monkeypox and vaccinia viruses could be detected by a TET (6-carboxytetramethylrhodamine)-labeled probe in a single PCR reaction. Approximately 25 copies of cloned variola virus DNA and 50 copies of genomic orthopoxviruses DNA could be detected with high reproducibility. The assay exhibited a dynamic range of seven orders of magnitude with a correlation coefficient value greater than 0.97. The sensitivity and specificity of the assay, as determined from 100 samples that contained nucleic acids from a multitude of bacterial and viral species were 96% and 98%, respectively. The limit of detection, sensitivity and specificity of the assay were comparable to standard real-time PCR assays with wet reagents. Employing a multiplexed format in this assay allows simultaneous discrimination of the variola virus from other closely related orthopoxviruses. Furthermore, the implementation of dried reagents in real-time PCR assays is an important step towards simplifying such assays and allowing their use in areas where cold storage is not easily accessible. SN - 0166-0934 UR - https://www.unboundmedicine.com/medline/citation/18725245/Dual_probe_real_time_PCR_assay_for_detection_of_variola_or_other_orthopoxviruses_with_dried_reagents_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0166-0934(08)00254-1 DB - PRIME DP - Unbound Medicine ER -