CRISPR/Cas13輔助乙型肝炎病毒共價閉合環狀DNA檢測

StephenW

CRISPR/Cas13輔助乙型肝炎病毒共價閉合環狀DNA檢測

    張向英、袁天、徐玲、范子豪、曹雅玲、馬英敏、李浩、任豐

Hepatology International 第 16 卷，第 306–315 頁（2022 年）引用這篇文章

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抽象的
背景和目標

肝臟中共價閉合環狀 DNA (cccDNA) 核內池的形成是持續性乙型肝炎病毒 (HBV) 感染的主要原因。在這裡，我們建立了基於 CRISPR-Cas13a 技術的高靈敏度和特異性檢測 cccDNA 的方法。
方法

我們使用質粒安全的 ATP 依賴性 DNase (PSAD) 酶和 HindIII 消化散環 rcDNA 和雙鏈線性 DNA，通過滾環擴增 (RCA) 和 PCR 擴增特定的 HBV cccDNA 片段，並使用 CRISPR- Cas13a 技術。使用 HBV 相關肝組織、血漿、全血和外周血單核細胞 (PBMC) 進一步臨床驗證了基於 CRISPR-Cas13a 的 cccDNA 檢測方法。
結果

在樣品預處理步驟、擴增步驟和檢測步驟的基礎上，我們建立了一種新的基於 CRISPR-Cas13a 的檢測方法，用於檢測 cccDNA。 RCA和PCR擴增後，CRISPR/Cas13輔助熒光讀數可檢測到1拷貝/μl HBV cccDNA。我們使用 ddPCR、qPCR、RCA-qPCR、PCR-CRISPR 和 RCA-PCR-CRISPR 方法分別檢測了來自 40 名 HBV 相關患者的肝組織樣本中的 20、4、18、14 和 29 份陽性樣本。上述5種方法在HBV患者的20份血液樣本（包括血漿、全血和外周血單核細胞）中幾乎完全未檢出HBV cccDNA。
結論

我們開發了一種基於 CRISPR 的新型檢測方法，用於高靈敏度和特異性檢測 HBV cccDNA，為準確檢測 HBV 感染、抗病毒治療評估和治療指導提供了一種有前途的替代方法。

StephenW

CRISPR/Cas13-assisted hepatitis B virus covalently closed circular DNA detection

    Xiangying Zhang, Yuan Tian, Ling Xu, Zihao Fan, Yaling Cao, Yingmin Ma, Hao Li & Feng Ren

Hepatology International volume 16, pages 306–315 (2022)Cite this article

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Abstract
Background and aims

The formation of an intranuclear pool of covalently closed circular DNA (cccDNA) in the liver is the main cause of persistent hepatitis B virus (HBV) infection. Here, we established highly sensitive and specific methods to detect cccDNA based on CRISPR-Cas13a technology.
Methods

We used plasmid-safe ATP-dependent DNase (PSAD) enzymes and HindIII to digest loose circle rcDNA and double-stranded linear DNA, amplify specific HBV cccDNA fragments by rolling circle amplification (RCA) and PCR, and detect the target gene using CRISPR-Cas13a technology. The CRISPR-Cas13a-based assay for the detection of cccDNA was further clinically validated using HBV-related liver tissues, plasma, whole blood and peripheral blood mononuclear cells (PBMCs).
Results

Based on the sample pretreatment step, the amplification step and the detection step, we established a new CRISPR-Cas13a-based assay for the detection of cccDNA. After the amplification of RCA and PCR, 1 copy/μl HBV cccDNA could be detected by CRISPR/Cas13-assisted fluorescence readout. We used ddPCR, qPCR, RCA-qPCR, PCR-CRISPR and RCA-PCR-CRISPR methods to detect 20, 4, 18, 14 and 29 positive samples in liver tissue samples from 40 HBV-related patients, respectively. HBV cccDNA was almost completely undetected in the 20 blood samples of HBV patients (including plasma, whole blood and PBMCs) by the above 5 methods.
Conclusions

We developed a novel CRISPR-based assay for the highly sensitive and specific detection of HBV cccDNA, presenting a promising alternative for accurate detection of HBV infection, antiviral therapy evaluation and treatment guidance.

StephenW

https://link.springer.com/conten ... 072-022-10311-0.pdf