A Probe Directed Recombinase Amplification Assay for Detection of MTHFR A1298C Polymorphism Associated with Congenital Heart Disease

Abstract

Single nucleotide polymorphisms (SNPs) play an important role in susceptibility to complex diseases, treatment efficacy and adverse drug responses. Conventional methods to detect SNPs are usually based on PCR or DNA sequencing, which are typically time-consuming and require sophisticated equipment. In this proof-of-concept study, a probe-directed recombinase amplification (PDRA) assay was developed to detect the A1298C polymorphism of 5,10-methylenetetrahydrofolate reductase (MTHFR). The PDRA assay included two real-time reactions to detect the A and C nucleotides of A1298C polymorphism. Each reaction contained only one primer and one probe and was finished at 39°C within 35 min. The results of genotyping of 150 clinical samples using PDRA were completely consistent with those by direct sequencing. Additionally, when the 1000 Genomes Project HCB frequencies were used as the control group, MTHFR A1298C was found to be associated with congenital heart disease. In conclusion, the proposed novel PDRA assay is a valuable tool for the detection of SNPs and demonstrates significant potential to be widely applicable in both research and clinical settings.

Development of a duplex reverse transcription recombinase-aided amplification assay for respiratory syncytial virus incorporating an internal control

“…Thus, a reliable detection method is critical for clinical diagnosis. In our previous studies, the development of RAA assays for the detection of several pathogens, such as the RSV-A and -B [27], coxsackievirus A10 and coxsackievirus A6 [28], and single-nucleotide polymorphisms (SNPs) [29] only focused on detection of pathogens but did not successfully apply an internal control to monitor the reaction system. Since the RAA reaction is an enzymatic reaction controlled by a variety of enzymes, it is more susceptible to the influence of inhibitors in the specimen.…”

A duplex probe-directed recombinase amplification assay for detection of single nucleotide polymorphisms on 8q24 associated with prostate cancer

“…The duplex-PDRA assay for dual detection of SNPs was established in this study on the basis of previous PDRA study, in which the classification of MTHFR A1298C was successfully performed in 45 min at 39°C (29). PDRA technique utilizes an oligonucleotide probe to fully match the template strand under the action of a recombinase, a single-stranded binding protein, and a DNA polymerase to initiate amplification, and the mismatched probe initiates a later amplification or non-expansion.…”

“…A novel isothermal amplification technique -recombinant aided amplification (RAA) -was originally designed to rapidly amplify target sequences at 37-42°C in less than 30 min. In our previous report, RAA was modified and named as probe-directed recombinase amplification (PDRA) for a single SNP detection (MTHFR A1298C) (29). In order to further save time, money, and be more efficient, in the present study, we aimed to establish a duplex real-time fluorescent PDRA method for simultaneous detection of two SNP sites (rs6983267 and rs1447295) located on 8q24, which are considered to be highly correlated with the susceptibility to PCa (3)(4)(5).…”

A Look Back at 2018

“…The authors propose a novel PDRA assay as a valuable tool for the detection of SNPs and demonstrate significant potential to be widely applicable in both research and clinical settings [3]. Interestingly, to our knowledge, the proposed method is the first report to detect SNPs using one probe and one primer.…”