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Quantitative measurement of hepatitis b virus cccdna

A technology of DNA molecules and DNA sequences, applied in the field of quantitative measurement of hepatitis B virus CCCDNA, can solve the problems of high cost and side effects

Inactive Publication Date: 2018-09-28
JBS SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If their cccDNA levels could be measured, this "cured" population could discontinue lifelong antiviral therapy, which is costly and has unknown side effects

Method used

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  • Quantitative measurement of hepatitis b virus cccdna
  • Quantitative measurement of hepatitis b virus cccdna
  • Quantitative measurement of hepatitis b virus cccdna

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] Example 1: Recovery of DNA after bisulfite treatment as detected by PCR.

[0096] The complementary strands of double-stranded DNA are rendered non-complementary by bisulfite treatment, which converts cytosine nucleotides to uracil, as Figure 2B shown.

[0097] Here, the Qiagen Epitect Bisulfite Conversion Kit (Qiagen, CA) and the Zymo Research EZ DNA Methylation Gold Kit (ZymoResearch, Zymo Research Corporation, CA) were used according to the manufacturer's instructions for bisulfite conversion. DNA from human HCC tissue samples was bisulfite-treated and tested with specific assays before and after bisulfite treatment. Regions on the constitutive gene actin were used for comparison by running assays targeting non-bisulfite-treated DNA regions and bisulfite-treated DNA regions (see Figure 8 ).

[0098] Figure 8 It was shown that bisulfite treatment (BS) did not significantly reduce copy detection of DNA target regions. Primers for the amplification of short prod...

Embodiment 2

[0100] Example 2: DNA quantification by real-time polymerase chain reaction (RT-PCR).

[0101] Total HBV DNA was quantified by real-time PCR using the LightCycler PCR instrument (Roche Biochemical, Germany) and LightCycler Probe Master Mix (Roche Biochemical, Germany) according to the manufacturer's instructions. Primers for bisulfite-treated total HBV DNA (forward, 5'-TTATGTTAATGTTAATATGGGTTTAAA-3'; reverse, 5'-TTCTCTTCCAAAAATAAAACAA-3'; probe, 5'-6FAM-TTAGATAATTATTG+TGG+T+ T+T+TA+TA+T-BBQ 3') and serially diluted HBV DNA plasmids were used as quantitative standards for quantification.

[0102] To quantify bisulfite-converted cccDNA, primers within the gap region of the (+) strand (forward, 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTTTGTYGGATYGTGTGTATTT-3′; reverse, 5′-AACRTTCACRATAATCTCCATAC-3′) were selected to prevent rcDNA amplification. TaqMan probe 5'6FAM-TTTTAT+T+T+T+T+G+TAY+G+TA-BBQ-3' was used to detect the amplification product.

Embodiment 3

[0103] Example 3: 1-step BS-cccDNA assay for detection of cccDNA by real-time PCR.

[0104] Figure 7 shows the sensitivity of 100 copies of BS-cccDNA template in a one-step cccDNA assay, and the sensitivity at 10 5 10 copies of rcDNA background 8 Linearity of synthetic BS-cccDNA to 100 copies. Figure 5 The 10-fold serial dilutions of the synthetic HBV template described in range from 100 to 10 for the sensitivity and linearity of the assay. 8 copies. Data are presented as amplification curve (top) and standard curve (bottom).

[0105] One-step real-time PCR was performed using the primer set of SEQ ID NO: 11 and SEQ ID NO: 24. Detection of the amplified product was performed using SEQ ID NO: 29 as a probe. Human methylated bisulfite-treated DNA was used as a negative control at 10,000 copies and showed no observable amplification. Note, 10 4 HMBS are amplified at cp values ​​similar to 10 copies of cccDNA, so at 10 4 The sensitivity or limit for detection of 1-step ...

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Abstract

Hepatitis B Virus (HBV) infection results in the entry of viral genomic DNA into host liver cells. This viral relaxed circular DNA (rcDNA) is transported into the nucleus and converted into covalent closed-circular DNA (cccDNA), which serves as a template for viral transcription. Elimination of cccDNA is needed to cure HBV infection, which remains a major therapeutic challenge. A robust and sensitive method to measure cccDNA described here is useful to facilitate drug development and to monitor efficacy of therapy. A set of primers were designed in combination with sodium bisulfite treatment of viral DNA, allowing specific amplification of cccDNA without interfering amplification of rcDNA. This method can be used to further guide therapeutic development, and to provide a non-invasive alternative to monitoring of HBV-infected patients undergoing antiviral treatments.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to US Provisional Application No. 62 / 295,481, filed February 15, 2016, the contents of which are hereby incorporated by reference in their entirety. [0003] References to Sequence Listings Submitted Electronically [0004] The contents of the electronically submitted Sequence Listing accompanying the submission, file name cccDNA_SeqListing_PCT.txt, size 11,609 bytes; creation date February 15, 2017, is hereby incorporated by reference in its entirety. technical field [0005] The present disclosure relates generally to methods and kits for detecting or quantifying closed covalent circular DNA (cccDNA) (e.g., of hepatitis B virus (HBV), and more particularly to biological methods for detecting or quantifying HBV infection. A method to distinguish cccDNA from relaxed circular DNA (rcDNA or RC-DNA) in a sample. Background technique [0006] HBV infection is a global public health proble...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/686C12Q1/70
CPCC12Q1/686C12Q1/706C12Q2527/125C12Q2565/1015
Inventor 苏比希·詹恩雅明·D·斯蒂芬宋尉
Owner JBS SCI
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