Journal
ANALYTICAL CHEMISTRY
Volume 83, Issue 12, Pages 4752-4758Publisher
AMER CHEMICAL SOC
DOI: 10.1021/ac200624f
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Funding
- National Natural Science Foundation of China [20773067, 20833006, 20905036]
- Research Fund for the Doctoral Program of Higher Education of China [20103207110004]
- Foundation of the Jiangsu Education Committee [09KJA150001, 09KJB150006, 10KJB150009, CX09B_307Z]
- Foundation of Jiangsu Provincial Key Laboratory of Palygorskite Science and Applied Technology [HPK201005]
- Priority Academic Program Development of Jiangsu Higher Education Institutions
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This work proposes a new strategy for the electrochemical detection of hepatitis C virus (HCV) RNA level and identification of HCV-1b genotype based on the site-specific cleavage of BamHI endonuclease combined with gold nanoparticles (AuNPs) signal amplification. The assay procedures include the reverse transcription, polymerase chain reaction (PCR) amplification, and electrochemical detection. The samples of 244 mer sequence of HCV RNA from the highly conserved region of HCV-1a, HCV-1b, HCV-1, and HCV-6a, respectively, were first reverse transcribed into complementary cDNA and amplified by PCR. The PCR-amplified samples were then analyzed using a synthetic 21 mer DNA probe, which has been assembled on the electrode surface via a bifunctional molecule of p-aminobenzoic acid (ABA). The results demonstrated that the developed approach can be used for specifically identification of the HCV-1b genotype and selective and sensitive detection of HCV-1b cDNA (244 mer) with a detection limit as low as (3.1 +/- 0.8) x 10(-22) M (less than 200 molecules; the concentration refers to the one before PCR amplification). Moreover, the developed method has an ability to discriminate the HCV-1b cDNA sequence from even single-base mismatched DNA sequence, to assay the HCV-1b cDNA level precisely from the mixture of HCV-1, HCV-1b, HCV-1a, and HCV-6a, and to detect HCV in real clinical samples. The protocol has high potential application in molecular diagnostics of HCV in clinical environments.
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