| Summary: | 碩士 === 國立臺灣大學 === 醫學檢驗暨生物技術學研究所 === 97 === Hepatitis C virus, HCV, is classified in the Hepacivirus genus within the Flaviviridae family. There are six major genotypes that differ in their nucleotide sequence. The transmission of HCV has been associated with blood and body fluid HCV infect is one of the leading causes for chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Seroprevalence studies suggest that at least 170 million individuals have been infected worldwide. Many commercial kits of serological assay or nucleic acid assay are available. These methods have disadvantages such as time-consuming and requiring a high-precision instrument. In this study, we intend to setup an economical, rapid, and sensitive assay for HCV detection to provide the information for HCV treatment.
Loop-mediated isothermal amplification (LAMP) is a novel technique for nucleic acid amplification. This simple and rapid method can be finished in one hour under the isothermal condition. We used LAMP assay combined with the fluorescence dye, SYTO-9, for real-time detection. In order to optimize the condition of LAMP assay, we adjusted the key factors of LAMP assay. The best condition of reaction was later carried out in a mixture containing the following factors: 0.3μM SYTO-9, 0.8 M Betaine, 8 mM MgSO4, 1.4 mM dNTP, 0.2 μM F3/B3, 1 μM LF/LB, and 2 μM FIP/BIP, and the mixture was incubated at 65℃. To check the specificity, we tested the DNA of different viruses and only HCV could be amplified. The limit of detection for this assay was 30 copies per reaction. Moreover, the LAMP assay was able to detect different genotypes of HCV.
We used this method to test 10 samples, which previously were quantified by branched DNA (bDNA) method. The sensitivity and specificity were 100％ as compared to real-time RT-PCR method. The scatter plot showed a high correlation coefficient (R2 = 0.956) and the Bland-Altman plot showed that differences between the two methods were less than 1.96 SD. Further, 79 samples, previously determined by real-time RT-PCR, were examined by this method. The sensitivity was 90.19% and the specificity was 71.42% as compared to real-time RT-PCR method. The scatter plot showed R2 = 0.8947 and the Bland-Altman plot showed that the differences between them were mostly less than 1.96 SD. The results by both methods were highly correlated. To confirm if there was any false positive or false negative, we used melting curve analysis to determine the Tm value. The results showed that all the positive samples had the same Tm value, 87.25℃, as the positive control, and the negative samples were the same after repeated testing. Finally, for these 79 samples, genes were sequenced and genotypes were determined by phylogenetic analysis. The results showed that 1b was major genotype in Taiwan, and one sample with genotype 6 was found.
The LAMP assay for HCV detection was established in this study. In the future, we need to improve the sensitivity and to simplify the method by combining reverse transcription and amplification in a one-step reaction. We hope that HCV LAMP assay could be useful for rapid quantification of HCV in the future.
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