Hello
multiplex PCR assay is good , also In these links a lot of methods you can chose the best for you .
Good Luck
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC100152/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1248436/
http://jcm.asm.org/content/40/6/2046.full
http://hepatmon.com/6012.fulltext
If you are going to diagnostic and therapeutic management purpose, then I strongly suggests Abbott Real time PCR for HCV-Genotyping is best, which is FDA, IVD approved. The best way to detect HCV genotyping by kit based approach is useful because, minimum chances of contamination, avoiding different measurements errors, and every kit contains calibrators and controls for calibration and quality checking. However, for research purpose many other systems and methods used and published literature, as above @Prof Saleh AlKarim suggestions.
DNA sequencing based methods are considered to be Gold standard for genotyping. In HCV 5'UTR region sequencing based genotyping has been found to be most reliable hence you can use PCR primers for this region and consider RT-PCR mediated amplification and sequencing. Although its not economical as that of multiplex PCR it surely is less expensive than using commercially available ready made kits as Abbott or using Line probe assays, etc
Thank you so much Prof Saleh AlKarim, Abdul Hafeez Kandhro and Yashwant Chavan or your valuable suggestions.
@Shelly Sehgal one more thing, from my experience in multiplex pcr, sometimes HCV genotypes in a sample more than 1 (Mixed genotypes), here multiplex pcr only detect higher concentration genotype, but fails to lower concentration genotype. For example: Genotype 1a and Genotype 3a; if Genotype 3a is higher in concentration, it is detected, while Genotype 1a has lower concentration, so might be missed due to low signals to dye or not detected at specific dye. Although it is expensive than other methods, but this is major obstacle of multiplex pcr.
Sir then what alternatives you suggest to overcome this obstacle? And are RFLP based approaches helpful in its genotyping?
First answer from Saleh Alkarim explains it all. Depends on budget. We normally use whole genime sequencing for complete information.
@Shelly Sehgal, I think you talk to Abbott company for HCV-Genotyping or consult with any good standard commercial molecular laboratory for performing samples on Abbott system.
Another option, which type of instruments are available in your department? Based on instrument option I will suggest you alternative.
Sir, we have illumina miseq and Applied biosystems Real time PCR 7500 make.
Well, You can take a look of this article, might be you can get an idea based on instrument availability:
"HCV Genotyping from NGS Short Reads and Its Application in Genotype Detection from HCV Mixed Infected Plasma"
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0122082
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