Heminested PCR assay for detection of six genotypes of rabies and rabies-related viruses

Heaton, Paul R.; Johnstone, P.; McElhinney, Lorraine M.; Cowley, Robin Neil; O'Sullivan, Eithne; Whitby, J. E.

doi:10.1128/jcm.35.11.2762-2766.1997

Cited by 249 publications

(132 citation statements)

References 21 publications

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“…Virus isolates (n= 106) from the six established genotypes of rabies and rabies-related viruses and one Australian bat Lyssa6irus isolate were selected for this study. The rabies virus isolates were propagated and passaged in mice or wherever possible in BHK-21 cells as described (King, 1996;Heaton et al, 1997;Black et al, 2000). The first 405 bases of the N gene had previously been sequenced to confirm the genotype of the strain.…”

Section: Cells and 6irusesmentioning

confidence: 99%

“…Total RNA was extracted directly from rabiesinfected BHK-21 monolayers or infected mouse brain tissue using TRIzol ® (Gibco BRL) according to manufacturer's instructions. Complementary DNA was produced using the RNA extracted from each of the infected cultures or mouse brains and the messenger sense RT primer JW12 as described (Heaton et al, 1997).…”

Section: Rna Extraction and Cdna Synthesismentioning

confidence: 99%

“…The primer sets for PCR amplification were based on those of Heaton et al (1997) although a new messenger sense primer BB6 was found to be more efficient than JW12 for PCR amplification. The TaqMan™ probes were designed by eye using multiple alignments (MegAlign, Dnastar) following the recommended criteria (Applied Biosystems, UK).…”

Section: Primers and Taqman™ Probesmentioning

confidence: 99%

“…The resultant data can then be used for phylogenetic analyses that enable a highly accurate characterisation of virus isolates. A hemi-nested reverse transcriptase PCR (hnRT-PCR) assay that uses a cocktail of primers capable of detecting the six established genotypes of rabies and rabies-related viruses has been described (Heaton et al, 1997). The hnRT-PCR offers a higher level of sensitivity than the FAT for normal and decomposed tissues (Heaton et al, 1997), but it does not distinguish between the genotypes.…”

Section: Introductionmentioning

confidence: 99%

“…A hemi-nested reverse transcriptase PCR (hnRT-PCR) assay that uses a cocktail of primers capable of detecting the six established genotypes of rabies and rabies-related viruses has been described (Heaton et al, 1997). The hnRT-PCR offers a higher level of sensitivity than the FAT for normal and decomposed tissues (Heaton et al, 1997), but it does not distinguish between the genotypes. However, the resultant N-terminal 405 bp sequence data is sufficient to distinguish between the various genotypes ( Fig.…”

Section: Introductionmentioning

confidence: 99%

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A rapid RT-PCR method to differentiate six established genotypes of rabies and rabies-related viruses using TaqMan™ technology

Black

Lowings

Smith

et al. 2002

Journal of Virological Methods

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A rapid and sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) assay incorporating TaqMan probes has been developed that can distinguish among the six established rabies and rabies-related virus genotypes. TaqMan probes were designed and validated against 106 rabies and rabies-related virus isolates, one isolate of the Australian bat Lyssaviruses (genotype 7), and 18 other non-rabies viruses important in the veterinary field. The N gene was used as the target for the probes as it is well conserved and has been intensively used to genotype rabies isolates. Additionally, it was found to contain regions specific to each genotype conducive to probe design. The RT-PCR assay described amplifies a portion of the nucleoprotein gene of all 107 rabies and rabies-related viruses, but none of the other viruses tested. Inclusion of TaqMan-genotype-specific probes in the RT-PCR assay permits rapid identification of the virus present. By combining RT-PCR with TaqMan genotyping probes suspect rabies virus isolates can be identified in a single closed tube system that prevents potential PCR-product carry over contamination.

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Section: Cells and 6irusesmentioning

confidence: 99%

Section: Rna Extraction and Cdna Synthesismentioning

confidence: 99%

Section: Primers and Taqman™ Probesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

A rapid RT-PCR method to differentiate six established genotypes of rabies and rabies-related viruses using TaqMan™ technology

Black

Lowings

Smith

et al. 2002

Journal of Virological Methods

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Case report: Rapid ante‐mortem diagnosis of a human case of rabies imported into the UK from the Philippines

Smith¹,

McElhinney²,

Parsons³

et al. 2002

Journal of Medical Virology

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The United Kingdom is free from rabies, with the last human death from indigenous rabies recorded in 1902. However, between 1946 and 2000, 20 deaths were reported in the United Kingdom in people who were bitten and infected while abroad in rabies endemic areas. The rapid diagnosis of suspected human rabies cases influences the use of anti-rabies post-exposure prophylaxis for potential contacts with the victim. In addition, the occurrence of a human rabies case requires urgent investigation to support patient management policies. In May 2001, a case of human rabies imported into the United Kingdom from the Philippines was identified. A 55-year-old man was admitted to University College Hospital, London, with clinical symptoms and a history consistent with exposure to rabies. Saliva, cerebrospinal fluid), and skin biopsies (from the wound site and nape of the neck) were submitted for conventional ante-mortem diagnostic techniques. Established diagnostic techniques, including the fluorescent antibody test (FAT), mouse inoculation test, (MIT) and the rabies tissue culture inoculation test (RTCIT), failed to detect the virus. In contrast, hemi-nested reverse transcription-polymerase chain reaction (RT-PCR), followed by automated sequencing confirmed the presence of classical rabies virus (genotype 1) in both the saliva and skin specimens within 36 hr of sample submission. Subsequent phylogenetic analysis demonstrated that this isolate was closely related to that of canine variants currently circulating in the Philippines.

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Case report: Isolation of a European bat lyssavirus type 2a from a fatal human case of rabies encephalitis

Fooks¹,

Pounder

Finnegan³

et al. 2003

Journal of Medical Virology

146

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A 55-year-old bat conservationist was admitted to Ninewells Hospital, Dundee, Scotland, on November 11, 2002, with an acute haematemesis. He gave a 5-day history of pain and paraesthesia in the left arm, followed by increasing weakness of his limbs with evidence of an evolving encephalitis with cerebellar involvement. The patient had never been vaccinated against rabies and did not receive postexposure treatment. Using a hemi-nested reverse transcriptase-polymerase chain reaction (RT-PCR), saliva samples taken intravitam from different dates proved positive for rabies. A 400-bp region of the nucleoprotein gene was sequenced for confirmation and identified a strain of European bat lyssavirus (EBLV) type 2a. The diagnosis was confirmed using the fluorescent antibody test (FAT) and by RT-PCR on three brain samples (cerebellum, medulla, and hippocampus) taken at autopsy. In addition, a mouse inoculation test (MIT) was performed. Between 13 and 17 days postinfection, clinical signs of a rabies-like illness had developed in all five inoculated mice. Brain smears from each infected animal were positive by the FAT and viable virus was isolated. This fatal incident is only the second confirmed case of an EBLV type-2 infection in a human after exposure to bats.

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Heminested PCR assay for detection of six genotypes of rabies and rabies-related viruses

Cited by 249 publications

References 21 publications

A rapid RT-PCR method to differentiate six established genotypes of rabies and rabies-related viruses using TaqMan™ technology

A rapid RT-PCR method to differentiate six established genotypes of rabies and rabies-related viruses using TaqMan™ technology

Case report: Rapid ante‐mortem diagnosis of a human case of rabies imported into the UK from the Philippines

Case report: Isolation of a European bat lyssavirus type 2a from a fatal human case of rabies encephalitis

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