Development of real‐time reverse transcriptase polymerase chain reaction methods for human rabies diagnosis

Abstract

To improve timely ante-mortem human rabies diagnosis, methods to detect viral RNA by TaqMan-based quantitative reverse transcriptase polymerase chain reactions (qRT-PCRs) have been developed. Three sets of two primers and one internal dual-labeled probe for each primer set that target distinct conserved regions of the rabies virus N gene were designed and evaluated. Using a collection of 203 isolates representative of the world-wide diversity of rabies virus, all three primers/probe sets were shown to detect a wide range of rabies virus strains with very few detection failures; the RABVD1 set in particular was the most broadly reactive. These qRT-PCR assays were shown to be quantitative over a wide range of viral titer and were 100-1,000 times more sensitive than nested RT-PCR; however, both the standard and real-time PCR methods yielded concordant results when used to test a collection of archived human suspect samples. The qRT-PCR assay was employed to monitor virus load in the saliva of a rabies virus-infected patient undergoing the Milwaukee treatment protocol. However in this case it would appear that reduction of the viral load in the patient's saliva over time did not appear to correlate well with clearance of viral components from the brain.

Susceptibility and Pathogenesis of Little Brown Bats (Myotis lucifugus) to Heterologous and Homologous Rabies Viruses

“…Four of the bats previously inoculated i.m. with EfV2 developed clinical rabies (12,12,16, and 23 days after the challenge inoculation), and one bat initially inoculated s.c. with LnV1 developed rabies 14 days after the challenge inoculation.…”

Role of Nuclear Factor-κB in Oxidative Stress Associated with Rabies Virus Infection of Adult Rat Dorsal Root Ganglion Neurons

“…Total RNA was isolated using the mirVana miRNA isolation kit (Ambion, Foster City, CA), and real-time onestep reverse transcription (RT)-PCR was performed using the QuantiTect Probe RT-PCR kit (Qiagen, Toronto, Ontario, Canada). The RAVD1 primer/probe set, which targets the N gene of CVS-11, was used as described by Nadin-Davis et al (18), and a primer/probe set specific for ␤-actin was used as described previously (34). Both probes were labeled at the 5= end with carboxyfluorescein.…”

Comparative Analysis of Rabies Virus Reverse Transcription-PCR and Virus Isolation Using Samples from a Patient Infected with Rabies Virus

“…Confirmatory assays for DFA comprise the rabies tissue culture infection test (RTCIT) and mouse inoculation test (MIT) but require days (RTCIT) to weeks (MIT) until final diagnosis. In addition to these classical methods, reverse transcription (RT)-PCR and real-time RT-PCR, which can be accomplished in less than 6 to 8 h, have been developed and used in pilot studies (1,10,13,16). Although preliminary data suggest that sensitivity of RT-PCR seems to be higher than that of RTCIT or MIT, this issue has not been systematically analyzed with actual human rabies cases (3,6,16).…”

“…Although nested RT-PCR may be less sensitive than real-time RT-PCR in selected cases (16), it provides simultaneous detection of different rabies virus genotypes and high confidence in exclusion screening for rabies. In selected situations, quantitative realtime RT-PCR data might also prove beneficial for follow-up of treatment (8,10). In addition it can be used for assessment of risks of transmission in the nosocomial setting (9).…”