A novel multiplex polymerase chain reaction assay for detection of both HLA‐A*31:01/HLA‐B*15:02 alleles, which confer susceptibility to carbamazepine‐induced severe cutaneous adverse reactions

Abstract

HLA-A*31:01 and HLA-B*15:02 have been widely reported to confer genetic susceptibility to carbamazepine (CBZ)-induced severe cutaneous adverse reactions (SCARs). Accordingly, the screening for these alleles has been highly recommended to prevent SCAR prior to introducing CBZ therapy. Although a number of methods are available for screening of HLA-A*31:01 or HLA-B*15:02 alleles separately, developing an assay that can detect both these alleles would be more clinically practical, cost-effective and less time-consuming. Therefore, in this study, a multiplex polymerase chain reaction (PCR) using TaqMan Probe was designed and validated to be able to detect HLA-A*31:01 and HLA-B*15:02. In comparison with Luminex-SSO/SBT/SSB, the multiplex PCR assay for detection of HLA-A*31:01 and HLA-B*15:02 had a perfect agreement in the validation group of 125 samples. The method was able to detect the target genes at the DNA concentration of 0.037 ng/μL. The unit cost of this assay is less than $5 USD with total time of 110 minutes.

circMAN1A2 could serve as a novel serum biomarker for malignant tumors

“…There are 2 approaches that use qPCR: 1 based on fluorescent dyes and 1 that is targeted at specific fluorescent‐labeled DNA sequences, called probes . The invention of TaqMan probes has overcome the limitations of SYBR Green I, that is, its lack of specificity owing to its ability to bind to all DNA double‐stranded structures . During the extension stage of TaqMan‐PCR, the DNA polymerase removes the TaqMan probe by hydrolysis, resulting in a fluorescent signal.…”

“…[60][61][62] The invention of TaqMan probes has overcome the limitations of SYBR Green I, that is, its lack of specificity owing to its ability to bind to all DNA double-stranded structures. [63][64][65][66] During the extension stage of TaqMan-PCR, the DNA polymerase removes the TaqMan probe by hydrolysis, resulting in a fluorescent signal. Moreover, multiple fluorescence signals can be used to detect the expression of several genes simultaneously using multiple channels.…”

Developing pharmacogenetic screening methods for an emergent country: Vietnam

“…In comparing the variety of published methods (Table 4), real-time PCR (RT-PCR) (Fig. 3) appears to be the most suitable approach for screening for susceptible HLA alleles 63, 64, 65, 66, 67, 68, 69, 70, 71. RT-PCR utilizes specific dyes to detect PCR amplification instead of the post-PCR procedures of the conventional method [DNA binding dye (SYBR ® green) and Fluorophore-labelled oligonucleotides (TaqMan ® probe)] 72 .…”

“…No internal control. HLA-B*15:02/HLA-A*31:01 RT-PCR (TaqMan ® Probe)Both HLA-B*15:02 and HLA-A*31:01Nguyen et al. 2017 67 110$5The continuous need for sequence updates to avoid any allele drop off due to any polymorphism originating at the binding sites of primers or probe. HLA-B*58:01 PCR-SSPExons 2 and 3 of HLA-B*58:01Virakul et al. 2010 89 RapidCheapUsing touch down PCR.…”

An SSP‐PCR method for the rapid detection of disease‐associated alleles HLA‐A*29 and HLA‐B*51

“…As a consequence, detection of these alleles is requested from diagnostic laboratories and is also of interest in a research context. While commercial diagnostic assays or published methods that detect the alleles of interest in a single sequence‐specific priming PCR (SSP‐PCR) reaction are available for other disease‐ or ADR‐associated HLA alleles, this is not the case for HLA‐A*29. Similarly, laboratory methods for typing of HLA‐B*51 are very limited .…”