Lyssavirus detection and typing using pyrosequencing.
نویسندگان
چکیده
Rabies is a fatal zoonosis caused by a nonsegmented negative-strand RNA virus, namely, rabies virus (RABV). Apart from RABV, at least 10 additional species are known as rabies-related lyssaviruses (RRVs), and some of them are responsible for occasional spillovers into humans. More lyssaviruses have also been detected recently in different bat ecosystems, thanks to the application of molecular diagnostic methods. Due to the variety of the members of the genus Lyssavirus, there is the necessity to develop a reliable molecular assay for rabies diagnosis able to detect and differentiate among the existing rabies and rabies-related viruses. In the present study, a pyrosequencing protocol targeting the 3' terminus of the nucleoprotein (N) gene was applied for the rapid characterization of lyssaviruses. Correct identification of species was achieved for each sample tested. Results from the pyrosequencing assay were also confirmed by those obtained using the Sanger sequencing method. A pan-lyssavirus one-step reverse transcription (RT)-PCR was developed within the framework of the pyrosequencing procedure. The sensitivity (Se) of the one-step RT-PCR assay was determined by using in vitro-transcribed RNA and serial dilutions of titrated viruses. The assay demonstrated high analytical and relative specificity (Sp) (98.94%) and sensitivity (99.71%). To date, this is the first case in which pyrosequencing has been applied for lyssavirus identification using a cheaper diagnostic approach than the one for all the other protocols for rapid typing that we are acquainted with. Results from this study indicate that this procedure is suitable for lyssavirus detection in samples of both human and animal origin.
منابع مشابه
Advances in Diagnostic Methods and Typing of Rabies Virus **
Significant advances in the techniques available for rabies diagnosis have taken place over the past thirty years. The traditional method of histological examination for Negri bodies in the brain sections has been largely replaced by immunofluorescent staining methods for the detection of nucleocapsid antigen in brain smears (fluorescent antibody test: FAT) (Dean and Abelseth, 1973). Neverthele...
متن کاملAmplicon identification using SparsE representation of multiplex PYROsequencing signal (AdvISER-M-PYRO): application to bacterial resistance genotyping
MOTIVATION Pyrosequencing is a cost-effective DNA sequencing technology that has many applications, including rapid genotyping of a broad spectrum of bacteria. When molecular typing requires to genotype multiple DNA stretches, several pyrosequencing primers could be used simultaneously but this would create overlapping primer-specific signals, which are visually uninterpretable. Accordingly, th...
متن کاملMethod Development and Applications of Pyrosequencing Technology
The ability to determine nucleic acid sequences is one of the most important platforms for the detailed study of biological systems. Pyrosequencing technology is a relatively novel DNA sequencing technique with multifaceted unique characteristics, adjustable to different strategies, formats and instrumentations. The aims of this thesis were to improve the chemistry of the Pyrosequencing techniq...
متن کاملMultiPSQ: A Software Solution for the Analysis of Diagnostic n-Plexed Pyrosequencing Reactions
BACKGROUND Pyrosequencing can be applied for Single-Nucleotide-Polymorphism (SNP)-based pathogen typing or for providing sequence information of short DNA stretches. However, for some pathogens molecular typing cannot be performed relying on a single SNP or short sequence stretch, necessitating the consideration of several genomic regions. A promising rapid approach is the simultaneous applicat...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
عنوان ژورنال:
- Journal of clinical microbiology
دوره 49 5 شماره
صفحات -
تاریخ انتشار 2011