利用迴路恆溫增幅法快速偵測C型肝炎病毒

Abstract

C型肝炎病毒屬於黃熱病毒科，肝炎病毒屬，為單股正向RNA病毒，依基因序列主要可分為6種基因型。C型肝炎病毒主要由血液或體液傳染，其感染易轉變成慢性肝炎，或甚至惡化為肝硬化或肝細胞癌，現今估計全球至少有一億七千萬人感染C型肝炎病毒。目前現有的檢測試劑包含了免疫法偵測及核酸偵測，但其缺點如耗時與需高度精密儀器等，本研究希望能夠發展一套成本低、速度快、敏感度高的C型肝炎病毒定量方法來偵測C型肝炎病毒，以提供用藥的依據。 2000年日本Notomi等人發展出迴路恆溫增幅法(loop-mediated isothermal amplification, LAMP)，其反應只需在單一溫度下，一個小時內，就能完成訊號放大。本研究嘗試運用此原理偵測C型肝炎病毒，並利用SYTO-9螢光物質於即時偵測系統測試。本實驗比對各基因型的較保守區5’NCR區域並設計六條特殊引子，再以TA cloning建立病毒標準品，針對影響反應關鍵因子，進行一系列最適化條件測試，測試各反應成分之最適濃度。SYTO-9物質所使用的最適濃度為0.3μM；Betaine為0.8 M；MgSO4為8 mM；dNTP為1.4 mM；F3/B3為0.2 μM；LF/LB為1.0 μM；FIP/BIP為2.0 μM；反應溫度則以65℃最佳。再進行特異性分析，測試不同的病毒核酸，結果顯示只有C型肝炎病毒能成功放大訊號。測試定量標準曲線結果，每次反應偵測極限則可至30 copies。為了解此方法是否針對不同基因型的C型肝炎病毒偵測具有良好的穩定性，本實驗測試了不同基因亞型，結果顯示其穩定性良好。 系統建立後與目前已商業化的定量系統比較其相關程度。與分枝DNA方法比較10支臨床檢體結果，敏感性與特異性皆可達100％，散佈圖比較結果其相關程度為R2 = 0.956，Bland-Altman plot分析兩種方法差異皆小於0.5 log也呈現高度一致性。另外再與即時反轉錄聚合酶連鎖反應比較79個臨床檢體，敏感度為90.2％，而特異性則為71.4％。其散佈圖也顯示有R2 = 0.8947的良好相關性，Bland-Altman plot分析顯示差異皆小於0.5 log，呈現高度一致性。為確定此方法是否有偽陽性或偽陰性發生，本實驗取了可能發生偽陽性及偽陰性的臨床檢體，進一步以melting curve測試，測試結果，增幅的產物與陽性控制組的Tm值皆位於87.25℃。而原未增幅的檢體也維持未增幅狀態。最後再將檢體以演化樹狀圖分析，其結果顯示分析的檢體中，其基因型大部分以1b亞型為主；而有一支檢體較為特殊，為第6型。 本研究成功建立迴路恆溫增幅法來偵測C型肝炎病毒。而後續的研究中，希望能再進一步改良偵測的敏感度，並且將反轉錄與增幅反應合為同一步驟進行，期待未來能應用於C型肝炎病毒的快速定量檢測。

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.