Title : A qPCR-HRM assay (high-resolution melting-curve analysis) to discriminate between pathotypes of Synchytrium endobioticum
Synchytrium endobioticum (Shilb.) Perc. is an obligate biotrophic, soil-borne fungus which causes the potato wart disease. S. endobioticum originated from Andean zone in South America and is currently present almost all over the world. The thick-walled winter sporangia are the dormant structures of the fungus and can survive for more than 40 years in the absence of host even under adverse environmental conditions. Since the discovery of pathotype 2(G1) in Germany, more than 40 pathotypes have been reported in Europe. HRM is a recently developed molecular technique. It is a cost-efficient, closed-tube system that allows high-throughput analysis without any post-PCR processing. PCR amplification and HRM were performed using specific primers of SNP sites (L1, L2, L3, L4, L5, L8 & L9) and DNA extracts of 10 different pathotypes of S. endobioticum [1(D1), 2(G1), 2(Ch1), 3(M1), 6(O1), 8(F1), 18(T1), 39(P1), #PL5/2010 & #PL2/20015]. Testing the primers set L1 on all pathotypes showed that HRM discriminates clearly between pathotype-1(D1) and non pathotype-1(D1). The second PCR test (L3) showed that HRM discriminates clearly between pathotype 1(D1)/8(F1)/18(T1) and other pathotypes of S. endobioticum. The third PCR test (L4) showed that HRM discriminates clearly between pathotypes 1(D1)/3(M1) and 2(G1)/6(O1)/ 8(F1)/18(T1) and 39(P1)/ #PL5/2010/#PL2/20015. This study presents the first results on HRM method to discriminate different pathotypes of S. endobioticum and confirms results obtained by Bonants et al. (2015) by the use of TaqMan PCR assays. HRM is more cost-effective than other genotyping technologies, such as TaqMan SNP typing, because no additional probes are required.
Take Away Notes:
• The results presented on the poster can be used by other conference participants. Especially in countries where this quarantine fungus occurs?
• HRM technique is much cheaper in comparison to TaqMan method. The results provide practical solution for scientists who for scientists who work with S. endobioticum.