Multiplex PCR-ASE functionalized microfluidic diagnostic platform for the detection of clarithromycin resistance mutations in Helicobacter pylori

Abstract

With the application of clarithromycin for Helicobacter pylori treatment, the number of resistant strains has increased greatly and has become a problem for treatment. Here, a novel multiplex PCR- allele-specific extension (PCR-ASE) functionalized microfluidic diagnostic platform with advantages of low-cost, easy-to-use and high sensitivity was firstly fabricated and proposed. A biphasic amplification multiplex PCR–ASE assay was integrated and used for the rapid detection of different mutations at positions 2142/2143 in the 23S rRNA gene in a single tube, and the results can be read with the naked eye using a nucleic acid detection strip (NADS). Our data revealed good performance for the multiplex diagnostic platform, which had an analytical sensitivity of approximately 50 copies/reaction and the ability to detect hetero-resistance at proportions as low as 0.5 %. Furthermore, it had no cross-reactivity with 10,000 copies of the wild-type template. The performance of platform for detection of clarithromycin resistance in 25 tissue biopsies was compared to that of Sanger sequencing and found to be in complete concordance. Due to its efficiency and simplicity, the multiplex PCR-ASE functionalized detection platform has great advantages for clinical application using a conventional thermal cycler for the detection of clarithromycin resistance within 2 h.