Development of new IL28B genotyping method using Invader Plus assay

Abstract

IL28B polymorphism is associated with the response to pegylated interferon-α with ribavirin (PEG-IFN-α/RBV) treatment in chronic hepatitis C patients. As a genotyping assay for IL28B single nucleotide polymorphisms (SNPs) in clinical practice, the Invader Plus assay was developed. The accuracy, intra-assay, inter-assay precision, and the limit of detection of the Invader Plus assay were evaluated. Two SNPs (rs8099917 and rs12979860) associated with IL28B were genotyped by the Invader Plus and TaqMan assay in 512 Japanese patients. In comparison with direct sequencing, the Invader Plus assay showed 99% accuracy in rs8099917 and 100% accuracy in rs12979860. Intraassay and inter-assay precision were sufficient to use in clinical practice and the detection limit was 1ngDNA/assay. Genotyping by rs8099917 showed that 361 (71%), 144 (28%) and seven (1%) of the patients were major homozygous, heterozygous and minor homozygous types, respectively. Five of the 512 cases (1%) had haplotype differences, but none showed differences between the two genotyping methods. For patients with HCV genotype 1, the prevalence of responders in the major homozygous type was 83.3%, and that of non-responders in the minor heterozygous/homozygous type was 72.5%. A convenient IL28B genotyping method using the Invader Plus assay could be useful to predict the treatment outcome in clinical practice.

Flap Endonuclease-Induced Steric Hindrance Change Enables the Construction of Multiplex and Versatile Lateral Flow Strips for DNA Detection

“…However, the signal amplification by FEN1 was not enough for DNA detection; various assays were thus constructed to increase the sensitivity by coupling another enzymatic reaction, such as hairpin probe-based invasive reaction (invader assay), 31 nicking endonuclease-catalyzed reaction, 32 rolling circle amplification, 33 strand-displacement signal amplification, 34 and nicking endonuclease-assisted nanoparticles amplification. 35 Because of the good performance of FEN1 in recognizing single-base variation, these FEN1derived assays were successfully employed for genotyping, 36 mutation detection, 37 pathogen detection, 38 and nucleic acid quantification. 39 To achieve a sensitive, specific, and inexpensive POCT for multiplex DNA detection, we proposed to employ FEN1 to generate target-specific flaps from PCR amplicons for constructing a versatile LFS.…”

Interleukin-28B Polymorphisms and Response of Chronic Hepatitis C Patients from Indonesia to Pegylated Interferon/Ribavirin Treatment

“…To determine the interleukin 28B (IL-28B) single nucleotide polymorphisms (SNPs), PCRs amplified a short fragment containing rs12979860, rs11881222, rs8103142, and rs8099917 using specific primer pairs (4,5). PCR amplification was performed as previously described (4, 5) using Hot Star Taq master mix (Qiagen).…”