HepatitisC genotyping in haemodialysis patients and the development of genotyping method based on Taqman probe real-time PCR
Abstract number: P1115
Hamzah H.A., Mustafa M.I., Abdul-M M.S., Nasarudin A., Bin Hasmani M.H.
Objectives: This is a seroprevalence study of HCV genotypes amongst infected haemodialysis patients in Pahang, Malaysia and the development of genotyping method using real-time PCR.
Methods: Patients were screened using ELISA by detecting the anti-HCV in the sera. HCV RNA was detected by RT-PCR technique targeting 5'UTR region. All negative first-round PCR products were re-tested by nested PCR. The base sequence of the PCR products was determined using the same primers as for the RT-PCR. By comparing the obtained nucleotide sequence data with sequences of known genotypes from the NCBI homepage, we deduced that our local isolates could be assigned to genotypes 1, 3, 4 and 6. Based on the data, TaqMan® probes were designed for the simultaneous identification and quantitation of these genotypes. A new batch of blood samples was recollected from patients and one step real-time RT-PCR (Applied Biosystem) assay was conducted for genotyping and viral load estimation.
Results: Out of 472 patients, 43 (26 males) were diagnosed positive for anti-HCV by ELISA. Two seropositive patients were excluded as they refused to give consent. Excluding another two patients who were seroconverted to HCV negative, 66.6% (26/39) were of genotype 3, 23.1% (9/39) of genotype 1, 5.1% (2/39) of genotype 6, followed by 2.6% (1/39) of genotype 4 and one patient gave a discordant result with the sequencing analysis.
Conclusion: Genotype 3 was the most prevalent genotype followed by genotype 1, 6 and 4. TaqMan real-time PCR has the potential as a method for rapid HCV genotyping.
|Session name:||Abstracts 20th European Congress of Clinical Microbiology and Infectious Diseases|
|Location:||Vienna, Austria, 10 - 13 April 2010|
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