Highly multiplex PCR assays by coupling the 5′-flap endonuclease activity of Taq DNA polymerase and molecular beacon reporters

Abstract

Real-time PCR is the most utilized nucleic acid testing tool in clinical settings. However, the number of targets detectable per reaction are restricted by current modes. Here, we describe a single-step, multiplex approach capable of detecting dozens of targets per reaction in a real-time PCR thermal cycler. The approach, termed MeltArray, utilizes the 5′-flap endonuclease activity of Taq DNA polymerase to cleave a mediator probe into a mediator primer that can bind to a molecular beacon reporter, which allows for the extension of multiple mediator primers to produce a series of fluorescent hybrids of different melting temperatures unique to each target. Using multiple molecular beacon reporters labeled with different fluorophores, the overall number of targets is equal to the number of the reporters multiplied by that of mediator primers per reporter. The use of MeltArray was explored in various scenarios, including in a 20-plex assay that detects human Y chromosome microdeletions, a 62-plex assay that determines Escherichia coli serovars, a 24-plex assay that simultaneously identifies and quantitates respiratory pathogens, and a minisequencing assay that identifies KRAS mutations, and all of these different assays were validated with clinical samples. MeltArray approach should find widespread use in clinical settings owing to its combined merits of multiplicity, versatility, simplicity, and accessibility.

Programmable Analysis of MicroRNAs by Thermus thermophilus Argonaute-Assisted Exponential Isothermal Amplification for Multiplex Detection (TEAM)

“…The reporter is a guide-complementary DNA hairpin labeled with a 6-FAM fluorophore at the 5′-end and a BHQ1 quencher at the 3′-end. The hairpin helps reduce the background fluorescence signal relative to a linear reporter (Figure S2) and simultaneously enhances the detection specificity. , Real-time fluorescence monitoring was used to investigate the TEAM detection system. As shown in Figure A, as the EXPAR products of miRNA-21 (2 μL) were created in the TEAM cleavage reaction (20 μL) at 80 °C, a strong fluorescence signal was quickly produced because of the highly efficient cleavage of the miRNA-21 reporter by TtAgo, which, being a multiple-turnover enzyme, is able to cleave multiple targets .…”

“…The reporter is a guide-complementary DNA hairpin labeled with a 6-FAM fluorophore at the 5′-end and a BHQ1 quencher at the 3′-end. The hairpin helps reduce the background fluorescence signal relative to a linear reporter 47 (Figure S2) and simultaneously enhances the detection specificity. 48,49 Real-time fluorescence monitoring was used to investigate the TEAM detection system.…”

Accessible and Adaptable Multiplexed Real-Time PCR Approaches to Identify SARS-CoV-2 Variants of Concern

“…The Omicron BA.1 variant has 32 mutations in the spike protein, far more than the number found in the Alpha and Delta variants. To examine all 32 mutations in one reaction, we used MeltArray, a multiplex mini-sequencing approach previously developed in our laboratory ( 16 ). In MeltArray, the Taq DNA polymerase cleaves the first base of the probe-binding region during the extension stage of PCR.…”

“…For this purpose, we developed a program that can generate variant-representative mutations by analyzing the SARS-CoV-2 sequences in the GISAID database. We then established assays to detect the chosen mutations that can represent the Alpha, Delta, and Omicron lineages, using either multicolor melting curve analysis (MMCA) ( 15 ) or MeltArray ( 16 ) approaches depending on lineage mutation count. These assays were used to dynamically monitor the prevalence of local VOCs.…”

Reprisal of Schima superba to Mn stress and exploration of its defense mechanism through transcriptomic analysis

“…The 7500 Real-Time PCR System was used to conduct PCR analysis in three replicates on an optical 96-well plate (Applied Biosystems). The PCR mixture was made using the Huang et al, 2022 technique. Thermal cycling started with denaturation at 95°C for 1 minute, then 40 cycles of 95°C for 15 seconds, 57°C for 15 seconds, and 72°C…”