47 Emerging NATs for malaria diagnosis seek to be appropriate for the POC through a variety of methods, including reducing the cost and difficulty of the amplification step and generating a quick and easy to use detection schemes.
Although 'brute force' DNA genotyping by dideoxy sequencing has been the method of choice for de novo SNP detection, it suffers from difficulty of DNA template amplification, limitations of PCR multiplexing per reaction, and increasing equipment costs.
The difficulty of obtaining PCR amplification of additional sequences, such as the GroEL and 47-kD protein genes, would be consistent with the identification of a new lineage divergent from Asian forms of O. tsutsugamushi.
Some studies have demonstrated difficulty in amplification of multiple alleles, absence of PCR amplification products, varying data interpretation, and lack of reproducibility among laboratories (7 ).
Because of amplification difficulties, some data were missing and partial sequences were present in some species.
Nevertheless, because of amplification difficulties for samples from the five northernmost populations, a new set of degenerated primers was designed using the PRIMER3 software [ 87]: amF 5' GTTTTRGGTGGATGCATGTC 3' and amR: 5' TGRTAYAARATTGGRTCTCAAC 3'.
As already mentioned, even though QUB 3232 was the most discriminative locus, difficulties with the reproducibility of amplification and gel band previously reported [ 9, 20, 24] lead us to not recommending this locus for routine use.
Due to the difficulty in amplification of larger amplicons from paraffin embedded tissue DNA, we performed methylation specific bisulfite sequencing of a slightly smaller region (−603 to −1297 positions) of the ACVR2 promoter in the clinical specimens.
We sampled all non-monotypic tribes with multiple genera although only a single representative of Gonichthyini is included due to technical difficulties in amplification of DNA.
