A Closed-Tube Colorimetric PCR Based on Serial Invasive Reaction Assisted Gold Nanoparticle Assembling for IL28B Genotyping

IL28B gene polymorphism is associated with curative effects of interferon and ribavirin (PegIFN/RBV) therapy for Hepatitis C patients. Therefore, genotyping IL28B gene could predict responses of anti-virus treatment and guide the optimal dosing regimens. Although many conventional DNA detection methods can accurately genotype IL28B gene polymorphism, the requirements of expansive instruments and tedious operation limit their clinical applications. Here, a colorimetric detection method for IL28B genotyping was developed by coupling PCR with serial invasive reaction assisted gold nanoparticle assembling in a closed-tube. The detection limit is as low as 0.05 ng of genomic DNA. The specificity is high enough to pick up 0.1% single-base mutated targets from a large amount of wild-type targets. Twenty seven clinical samples were successfully genotyped with our method, and only one sample was inconsistent with ARMS-PCR. The genotype of the sample was further validated by pyrosequencing, and the result was consistent with our method, indicating that our method is more accurate than ARMS-PCR. Because the detection results can be readout by naked eyes, no expensive instruments are required in our method; and close-tube detection lowers the risk of cross-contamination from PCR amplicons. Therefore, the proposed method is suitable for genotyping IL28B gene polymorphism of clinical samples.