PrimeSeqMDR (Multi-Drug Resistance)
There is an urgent need to rapidly characterize drug resistant MTB strains so that personalized drug therapies can be implemented.
Standard whole-genome analysis using next-generation sequencing (NGS) has enabled rapid genome analysis with minimal sample preparation time (2-4 days) and at moderate costs when multiple samples are analyzed per run. The Illumina MiSeq platform can sequence up to 24 whole MTB genomes per run with an average reproducible coverage depth of ~30 times. However, the standard NGS workflow requires the utilization of expensive equipment and reagents for library preparation which are cost prohibitive in many areas of the world.
The Longhorn PrimeSeqMDR targeted gene sequence kit (patents pending) circumvents the use of this expensive methodology by utilizing a highly sensitive and specific ‘targeted’ amplification approach that employs hybrid oligonucleotide primers that perform two critically important steps: 1) Targeted amplification of the region of interest of key TB genes (such as rpoB, katG, gyrA, pncA) in specific regions where drug resistance-conferring mutations are known to arise. 2) Through PCR the targeted oligonucleotides generate amplicons of the desired length which circumvents the need for size-selection using the Agilent system or cumbersome excision using gel electrophoresis. 3) The target oligos contain oligonucleotide sequences that enable a subsequent downstream amplification for ‘indexing’ multiple patient samples on a single run. This reduces the costs associated with NGS and enables a unified analysis of genomic data for rapid screening of drug resistance-conferring mutations in MTB genes. Thus, the developed methodology reduces the workflow, the need for ancillary equipment, and the cost for Illumina or other NGS library preparation kits and reagents.