Low-cost genotyping method based on allele-specific recombinase polymerase amplification and colorimetric microarray detection

Abstract

The costs of current genotyping methods limit their application to personalized therapy. The authors describe an alternative approach for the detection of single-point-polymorphisms (SNPs) using recombinase polymerase amplification (RPA) as an allele-specific technique. The use of short and chemically modified primers, locked nucleic acids (LNA), allowed the selective isothermal amplification of wild-type or mutant variants at 37 C in 40 min. An amplification chip containing 100 wells was manufactured with a 3D printer and using thermoplastic polylactic acid (PLA). The platform reduces costs, reagent consumption, sample volume and allows assay parallelization compared to other amplification formats. As proof of concept, the method was applied to the genotyping of four SNPs that are related to the treatment of tobacco addiction. The target polymorphisms were rs4680 (COMT gene), rs1799971 (OPRM1 gene), rs1800497 (ANKK1 gene), and rs16969968 (CHRNA5 gene). The capabilities of the method are demonstrated detecting the reaction products using a colorimetric technique in microarray format. The genotype populations can be well discriminated.

Recombinase polymerase amplification: Basics, applications and recent advances

“…Whilst it was initially believed that specifically designed primers of 30e35 bases in length were necessary for RPA, there are several reports demonstrating that normal PCR primers can be used and efficient amplification achieved [5,6]. Longer primers (up to 45 nucleotides) can be used, but they could lead to secondary structures and potential primer artifacts.…”

“…In solution-phase, due to the unimpeded diffusion of primers and reaction reagents, amplification kinetics are favoured and the achieved limit of detection is subsequently usually better and amplification is achieved in a faster time than solid-phase. Nevertheless, solidphase and bridge amplification present some advantages, such as the potential for spatially resolved multiplexed amplification or the possibility to couple the amplification with diverse detection techniques including ring resonators [27e29], electrochemical [30e36] and colorimetric detection [5,6,30,33,37,38]. Several methods have been developed with solid phase amplification with performances usually inferior to that achieved with solution phase amplification [5,27e30,39] as primer accessibility is more restricted impeding amplification efficiency, and future work will need to focus on strategies to decrease amplification time.…”

“…Other reports detail pseudomultiplexing platforms through parallelised single reactions, using foil based centrifugal microfluidic cartridges with stored reagents [49,58], digital versatile discs (DVD) [41,42,44], vacuum degassed microfluidic cartridges [59] or polylactic acid/polycarbonate chips [6].…”

“…Furthermore, similar to the use of PNA, the use of natural dNTPs vs locked nucleic acids was compared. However, a loss of specificity was observed when multiplexing in the same reaction mixture was pursued, which was attributed to a competition between primers and amplicon [6].…”

An isothermal method for sensitive detection ofMycobacterium tuberculosis complexesusing CRISPR/Cas12acis-andtrans-cleavage

“…Recently, the RPA based methods have been used for detection of MTB and other pathogen, while it always had high false positive results in clinical samples 19 . To further improve specificity, long oligonucleotide probe with fluorophores and quenchers or other modifications of probe, such as biotin, tetrahydrofuran, etc, have been designed to recognize RPA amplicon for specific cleavage by nucleases, and these newly developed methods have been successfully applied for pathogen diagnosis 20–22 , liquid biopsy 23 , and SNP/genotyping 24 . However, the synthesis of long oligonucleotide probes with multiple modifications is cost, and different probes with the same amplified region may also affect the efficiency of RPA.…”