First International Quality Assurance Study on the Rapid Detection of Viral Agents of Bioterrorism

Niedrig, Matthias; Schmitz, Herbert; Becker, Stephan; Günther, Stephan; Meulen, Jan ter; Meyer, Herman; Ellerbrok, Heinz; Nitsche, Andreas; Gelderblom, Hans R.; Drosten, Christian

doi:10.1128/jcm.42.4.1753-1755.2004

Cited by 48 publications

(34 citation statements)

References 8 publications

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“…In addition, we detected 50 copies/ml of MPXV (Lam87 strain), 60 copies/ml of CPXV, 81/01 strain, 240 copies/ml of CPXV, Brighton strain, and 160 copies/ml of VACV (modified virus Ankara strain), calculated by amplifying a 10-fold dilution of extracted DNA from P7, P9, P11, P12, and P15 samples from the first ENIVD quality control panel (15).…”

Section: Resultsmentioning

confidence: 99%

Use of Internally Controlled Real-Time Genome Amplification for Detection of Variola Virus and Other Orthopoxviruses Infecting Humans

et al. 2006

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Smallpox, once a devastating disease caused by Variola virus, a member of the Orthopoxvirus genus, was eradicated in 1980. However, the importance of variola virus infections has been stressed widely in the last few years, particularly following recent social events in the world. Today, variola virus is considered to be one of the most significant agents with potential use as a biological weapon. In this study we developed an internally controlled real-time PCR assay for rapid detection and simultaneous differentiation of variola virus from other orthopoxviruses. The assay is based on TaqMan 3-minor groove binder (MGB) chemistry and uses generic primers, designed in highly conserved genomic regions of the crmB gene, and three TaqMan MGB probes designed to identify orthopoxviruses, variola virus, and an internal control. The results obtained suggest that the assay is rapid, sensitive, specific, and suitable for the generic detection of orthopoxviruses and the identification of variola virus and avoids false-negative results in a single reaction tube.Variola virus (VARV) is the etiological agent of smallpox, a severe and deadly disease that is considered one of the most serious human infections in the history of mankind. Humans are the only known reservoir of VARV, and no animal or insect reservoirs have been identified. VARV is a member of the family Poxviridae, subfamily Chordopoxvirinae, and genus Orthopoxvirus. Its genome consists of 186 kb of linear doublestranded DNA (25). The Orthopoxvirus genus includes other human pathogen viruses: Vaccinia virus (VACV) (used for immunization against smallpox disease), Monkeypox virus (MPXV), Cowpox virus (CPXV), and Buffalopox virus. Following the eradication of smallpox through a global immunization effort, there are still stocks of VARV held in the United States and the former Soviet Union (7). The importance of smallpox has been stressed widely in past years; moreover, VARV is currently considered to be a major biological weapon (2,14,26).Several molecularly based diagnostic methods to detect the DNA of VARV and differentiate it from other orthopoxviruses were published before September 2001. These methods were based on the analysis of the pattern lengths of the restriction endonuclease digestion products of previously amplified genomes (13,22).Soon after this date, as an alternative to these time-consuming methods, several real-time PCR protocols for the detection and identification of orthopoxvirus DNA were described (5,8,17,18,19,24).In this study we describe a real-time PCR based on TaqMan 3Ј-minor groove binder (MGB) chemistry (Applied Biosystems) to simultaneously detect VARV and differentiate it from other orthopoxviruses in a single reaction tube. Moreover the method uses an internal control to detect false negatives.A careful review of previously published methods for the real-time amplification of variola virus is also described. MATERIALS AND METHODSCell culture and viral strains. VACV, Western Reserve strain, was grown for 2 days on CV1 cell monolayers and...

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Section: Resultsmentioning

confidence: 99%

Use of Internally Controlled Real-Time Genome Amplification for Detection of Variola Virus and Other Orthopoxviruses Infecting Humans

et al. 2006

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“…This correlates with the increasing number of reports of molecular technologies to identify and characterize multiple infectious agents and diseases affecting humans, plants, and animals. The above mentioned justifies the establishment of clear regulations and statistical models for evaluation and adoption of these protocols in laboratories of diagnosis [81]. Despite the continuing evolution of molecular biology, future efforts should continue to increase understanding of advantages and disadvantages of molecular methods in diagnosis, and its interpretation within the clinical context.…”

Section: Discussionmentioning

confidence: 99%

Polymerase Chain Reaction: Types, Utilities and Limitations

Hernández-Rodríguez¹,

Ramírez²

2012

Polymerase Chain Reaction

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“…The efficacy of the method is proved on samples sent by the European Network for the Diagnostics of Imported Viral Diseases (ENIVD), as part of external quality control (EQC) assessments. 11 Detection and identification of orthopoxviruses using a generic nested PCR followed by sequencing …”

Section: Introductionmentioning

confidence: 99%

Detection and identification of orthopoxviruses using a generic nested PCR followed by sequencing

Sánchez-Seco

Hernández²,

Eirós

et al. 2006

British Journal of Biomedical Science

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Some orthopoxviruses are considered to be potential biological weapons. After the smallpox eradication campaign ended, routine vaccination was stopped around the world. Consequently, a significant portion of the population is now completely unprotected from infection by variola virus and related orthopoxviruses. Some of the symptoms associated with non-variola infections can be similar to smallpox, causing alert and panic situations. These infections should be considered as real public health concerns, so suitable tools for their differential diagnosis are needed. This study aims to devise a simple and easy-to-perform method that is able to detect and identify any orthopoxvirus that might cause infection in humans. In addition, the similarity of the different genes in the genomes of several species of orthopoxviruses is investigated, and orthopoxvirus-universal primer pairs in the tumour necrosis factor receptor II homologue gene are designed, taking full account of nucleotide similarity. A strategy is devised for their sensitive, rapid and cost-effective detection and identification, based on a nested PCR followed by sequencing. The efficacy of the method is tested with samples sent by the European Network of Imported Viral Diseases as part of two external quality control assays. All human orthopoxviruses assayed were detected and identified.

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First International Quality Assurance Study on the Rapid Detection of Viral Agents of Bioterrorism

Cited by 48 publications

References 8 publications

Use of Internally Controlled Real-Time Genome Amplification for Detection of Variola Virus and Other Orthopoxviruses Infecting Humans

Use of Internally Controlled Real-Time Genome Amplification for Detection of Variola Virus and Other Orthopoxviruses Infecting Humans

Polymerase Chain Reaction: Types, Utilities and Limitations

Detection and identification of orthopoxviruses using a generic nested PCR followed by sequencing

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