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Title: Nucleic acid based amplification methods for the detection quantification and characterization of viruses in clinical specimens
Guide(s): H. N. MADHAVAN
Keywords: Nucleic acid based amplification methods, quantification and characterization of viruses
University: Birla Institute of Technology and Science
Completed Date: 
Abstract: Aim: newlineThe aim of the present study is to identify the nature of genotypes and serotypes of viruses causing congenital cataract and epidemic conjunctivitis in Indian patients which may be different from those reported from other parts of the world. newlineMethods: newlineNucleic acid based molecular biological methods like uniplex Polymerase chain reaction; semi-nested PCR, nested PCR, uniplex Reverse Transcriptase Polymerase chain reaction and nested RT-PCR were applied on various clinical specimens for the detection of various viral etiological agents causing congenital cataract and epidemic conjunctivitis. Quantification of the Herpes simplex viral particles were done by the application of standardized Real Time PCR. The genotype identity was established by DNA sequencing and by analyzing the sequences using MultAlin software. The serotype of HSV was confirmed using various techniques like PCR based Restriction Fragment Length Polymorphism (PCR - RFLP) and DNA sequencing. Conventional methods of Indirect Immunofluorescence staining (IIF) and virus isolation in cell cultures were also attempted. newline newline newline newlineResults: newlineThe results of the present study indicated the association of Rubella virus (RV) with 12% of congenital cataract patients in less than one-year age. Twenty RV isolates have been obtained using the conventional cell culture methods. 18 of the twenty isolates have been genotyped as belonging to RGI and two to RGII. Direct evidences of association of RV and HSV 2 were demonstrated in 18 (36 %) lens aspirates with RV in 9 (18 %) and HSV 2 in 9 (18 %) others. VZV was not detected in any of the lens aspirates tested. The results of PCR-RFLP and DNA sequencing for HSV serotype confirmation were in concordance with that of the snPCR. Conjunctival swabs collected from patients of Epidemic conjunctivitis indicated the presence of Adenovirus (Ad) in 15% of the patients and remaining 77.0% of the patients were positive for Enterovirus (EV). 49% of these patients turned positive for Coxsackie A24 variant (CA24v) and DNA sequencing.
Pagination: 51.3 MB
Appears in Departments:Biological Science

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