Core antigen fragment for detecting hepatitis C virus and application of core antigen fragment

A technology for hepatitis C virus and core antigen is applied in the field of detecting the core antigen fragment of hepatitis C virus, which can solve the problems of reduced detection sensitivity of specimens, cross-contaminated aerosols, and reduced detection sensitivity, and achieves perfect detection sensitivity and accuracy. , the effect of making up for the lack of sensitivity

Inactive Publication Date: 2015-02-25
PEOPLES HOSPITAL PEKING UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Anti-HCV detection is the most widely used screening test for HCV infection. It is easy to operate, short in time, and low in cost. However, its disadvantage is that it has a long window period and cannot distinguish whether it is an active infection or the virus has been cleared. It is not suitable for immunodeficiency population; HCV RNA is the direct evidence of viral infection, and quantitative detection of HCV RNA is also a key indicator for monitoring the efficacy of CHC patients during antiviral treatment. It is easy to produce cross-contamination and aerosol pollution when operating in a small room space, so it is not suitable for small laboratories
However, in clinical testing practice, some samples of specific genotypes appeared. Although the concentration of HCV RNA was high, indicating that the virus was in the active replication phase, the result of quantification of HCV core antigen using this kit was "negative", which indicated that The current HCV detection kit still has the problem of false negatives for some samples, that is, its detection sensitivity for some positive HCV samples is significantly reduced, but no report has pointed out the reasons for the reduction in detection sensitivity of these samples

Method used

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  • Core antigen fragment for detecting hepatitis C virus and application of core antigen fragment
  • Core antigen fragment for detecting hepatitis C virus and application of core antigen fragment
  • Core antigen fragment for detecting hepatitis C virus and application of core antigen fragment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Comparison of core antigen levels in samples of different HCV genotypes

[0030] 1. Case selection

[0031] From March to June 2011, sera from 203 CHC patients were collected, including 130 males and 73 females, aged 24-76 years, with an average of (37.3±8.6) years old. HCV genotypes were tested using Versant HCV Genotype 2.0 (LiPA, Siemens, Marburg, Germany), among which genotype 1b was 41 cases, 2a was 43 cases, 3a was 31 cases, 3b was 43 cases, and 6a was 45 cases. Covers common genotypes in China. HCV RNA was quantified using Roche Taqman HCV RNA kit, with a minimum detection limit of 15IU / mL. Patients with different HCV genotypes are comparable in age, gender and HCV viral load.

[0032] 2. Method

[0033] Use Abbott Architect HCV Ag Core Antigen Quantitative Detection Kit to perform HCV antigen quantitative detection on Abbott Architect i2000 automatic chemiluminescence analyzer. The linear range of quantitative detection is 3.00fmol / L-20000fmol / L, and the l...

Embodiment 2

[0038] Example 2 Comparison of the sensitivity of Abbott Architect HCV Ag Quantitative Kit for detecting HCV samples of different genotypes

[0039] 1. Sample selection Select 1 high viral load serum of 1b, 2a, 3a, 3a and 6a genotypes.

[0040] 2. Experimental method Use Abbott Realtime HCV RNA quantification kit to detect the viral load of each genotype sample, and then dilute each genotype serum so that the final concentration of HCV RNA is 10000, 7500, 5000, 1000, 750, 500, 250, respectively And 100IU / mL. Dilute samples of different concentration levels were tested 20 times with Abbott Architect HCV Ag quantitative reagent. Using the probability analysis method (Probit method), calculate the lowest HCV RNA concentration when 95% of the samples are “positive” by the Abbott Architect antigen quantitative detection. This concentration is regarded as the sensitivity of the Architect HCV Ag quantitative kit, which is the “lowest” Detection limit".

[0041] 3. Experimental results

[...

Embodiment 3

[0045] Example 3 Alignment of Amino Acid Sequences in the Core Region of HCV Strains of Different Gene Subtypes

[0046] 1. Materials and methods

[0047] (1) Serum HCV RNA extraction

[0048] Use the QIAamp virus RNA extraction kit from QIAGEN, Germany to extract HCV RNA from 140 μl serum.

[0049] (2) Reverse transcription and PCR amplification (RT-PCR) of HCV core region

[0050] Reverse transcription of HCV RNA was performed using AMV reverse transcriptase from Promega, USA. The total volume of one RT reaction system is 10μl, including 6μl HCV RNA, 1×AMV reaction buffer, 1mM each dNTP, 3μM RT primer Core410R and 6U AMV reverse transcriptase. The RT reaction conditions are: 42°C, 60 min. Add 40μl PCR reaction solution to 10μl 5’-UTR reverse transcription system to make the total volume of PCR reaction system 50μl, containing 2.5U Platinum Taq DNA polymerase from Invitrogen, 1×PCR buffer, 1.5mMMg 2+ , 200μM each dNTP, 0.6μM primer Core406F. PCR conditions are: 95°C, 5min for DNA d...

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Abstract

The invention provides a core antigen fragment for detecting hepatitis C virus and an application of the core antigen fragment. The antigen level of different HCV genotype serum samples is detected by using an Abbott Architect HCV quantitative core antigen kit, the sensitivity of different genotypes is compared and detected, a sample of specific genotype which influences the detection sensitivity is analyzed, a polymorphic site of the core antigen which influences the detection sensitivity of the kit is found, and the amino acid sequence of the core antigen is as shown in SEQ ID NO.1-2. The amino acid fragment of the polymorphic site of the core antigen can serve as a molecular marker of HCV core antigen detection and is used for detecting the genotype of hepatitis C virus 3b. Moreover, the existence of the molecular marker can indicate that the sensitivity of the conventional quantitative antigen detection kit is reduced when the kit is used for detecting a sample of the genotype of hepatitis C virus 3b, normalization and standardization of the core antigen detection can be facilitated, and the core antigen fragment can be used for preparing a kit for detecting the HCV 3b genotype.

Description

Technical field [0001] The present invention relates to the field of virus detection, in particular to core antigen fragments used for detecting hepatitis C virus and applications thereof. Background technique [0002] Chronic hepatitis C (CHC) caused by persistent hepatitis C virus (HCV) infection has always been a global public health problem. The onset of CHC is hidden and easily ignored by patients. It can lead to liver cirrhosis, even hepatocellular carcinoma and liver failure, posing a serious threat to human health. Therefore, the screening and early treatment of HCV infection are particularly important. The common hepatitis C genotypes in my country mainly include 1b, 2a, 3a, 3b and 6a genotypes. [0003] Anti-HCV test is the most widely used HCV infection screening test. It is easy to operate, short in time and low in cost, but its disadvantage is that the window period is long and it cannot be distinguished whether it is an active infection or the virus has been cleared...

Claims

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Application Information

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IPC IPC(8): G01N33/576G01N33/569
CPCG01N33/56983G01N33/5767G01N2333/186
Inventor 杨瑞锋
Owner PEOPLES HOSPITAL PEKING UNIV
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