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HBV nucleic acid quantitative detection system based on micro-droplet digital PCR technology

A technology of nucleic acid quantification and detection system, applied in the field of digital PCR, which can solve the problems of difficult judgment of results and insufficient accuracy of qPCR

Inactive Publication Date: 2018-07-31
TARGETINGONE CORP +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] qPCR has certain technical limitations: first, qPCR relies on external DNA calibrators and Cq values ​​to determine the original concentration of target DNA, and Cq values ​​depend on threshold settings, which is subjective and empirical; second, qPCR is not accurate enough, especially When the Cq value of an unknown sample is close to the critical point, the result is often difficult to judge; in addition, the expression difference of HBV surface antigen and the low copy of HBV often lead to frequent false negative results in qPCR

Method used

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  • HBV nucleic acid quantitative detection system based on micro-droplet digital PCR technology
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  • HBV nucleic acid quantitative detection system based on micro-droplet digital PCR technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Quantitative detection of HBV DNA by micro-droplet digital PCR

[0026] Sample selection: Use high-value clinical samples (quantified by methods traceable to national reference materials), and perform serial dilution with HBV-negative plasma to obtain 10 6 、10 5 、10 4 、10 3 , 100 and 10IU / ml concentrations were tested, and the plasma volume was 500μl.

[0027] DNA extraction: nucleic acid extraction was performed using a virus nucleic acid extraction system (Thermo Fisher, 37011D), and the operation steps were performed according to the system manual. Extracted nucleic acids should be stored at -20°C and long-term stored at -80°C.

[0028] PCR reaction system preparation: the system includes 1×PCR reaction buffer (containing 50mmol / L Tris.HCl (PH8.3), 10mmol / L potassium chloride, 5mmol / L ammonium sulfate, 2.5mmol / L magnesium chloride); Glycoside triphosphate dATP, dCTP, dGTP and dUTP, the final concentration is 200nmol / L, and the final concentration of dU...

Embodiment 2

[0065] Embodiment 2: PCR system anti-pollution ability

[0066] After the dU-containing PCR product was purified, it was quantified by a UV spectrophotometer and the copy number was calculated, and the dilution was quantified to about 1×10 10 copies / μl, then serially diluted 10 times to 10 -10 dilution. Take dU-containing PCR products of various dilutions as templates for simulating pollution sources, and use this system for PCR amplification. The amplified products were detected by agarose gel electrophoresis, and the experimental results were as follows: figure 2 shown.

[0067] The experimental results show that 10 3 copy / reaction (corresponding to 10 -7 dilution) and below simulated pollution sources, no amplified product bands were seen; while for the control PCR amplification system without dUTP and UNG, it was as low as 10 1 The simulated pollution source of the copy / reaction can still see clear amplification product bands, and the experimental results are as fol...

Embodiment 3

[0068] Embodiment 3: the detection of clinical sample

[0069] Collect clinical HBV positive samples (with high, medium and low concentration distribution) and HBV negative samples, a total of 130 samples (30 HBV positive samples, 100 HBV negative samples), carry out micro-droplet digital PCR reaction, and compare with the current HBV The gold standard in the DNA quantification industry - Roche Cobas Amliprep / Cobas TaqMan products are compared, and the data are statistically analyzed. The detection sensitivity of the HBV quantitative detection system based on micro-droplet digital PCR technology in the present invention is 100% (30 / 30), The specificity was 100% (100 / 100). The quantitative results are highly consistent with Roche Cobas Amliprep / Cobas TaqMan products. This shows that the detection system based on the digital PCR system of the present invention has high consistency with Roche products, and can realize accurate absolute quantification of HBV DNA.

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Abstract

The invention provides a HBV nucleic acid quantitative detection system based on a micro-droplet digital PCR technology, the HBV nucleic acid quantitative detection system comprises an upstream primerand a downstream primer for amplifying hepatitis B virus DNA, a specific probe for detecting the hepatitis B virus DNA; a non-infected linearizing double-stranded plasmid quality control standard product and a probe for detecting the quality control standard product; and a buffer system comprising deoxyribonucleoside triphosphates dATP, dCTP, dGTP, and dUTP, and UNG. The HBV nucleic acid quantitative detection system is based on the micro-droplet digital PCR technology to realize the accurate absolute quantification of HBV DNA, faces the clinical actual demand, and has an important application prospect.

Description

technical field [0001] The invention relates to the technical field of digital PCR, in particular to an HBV nucleic acid quantitative detection system based on micro-droplet digital PCR technology. Background technique [0002] Hepatitis B virus (HBV) is a worldwide prevalent partial double-stranded deoxyribonucleic acid (DNA) virus, which is the causative agent of acute and chronic hepatitis and hepatocellular carcinoma, and seriously endangers the safety of human public health. The concentration of HBV DNA in plasma (serum) is related to the number of viral particles in the circulation. Quantitative detection of HBV DNA concentration in plasma (serum) of HBV-infected patients is a reliable indicator for understanding the level of HBV replication in the liver. An important basis for guiding medication. Standardized antiviral treatment can effectively block or slow down disease progression and reduce the incidence of end-stage liver disease. HBV DNA detection has important ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686
CPCC12Q1/686C12Q1/706C12Q2600/166C12Q2563/159C12Q2545/114C12Q2563/107
Inventor 雷孟平祝令香朱宏艳郭永杨文军王勇斗
Owner TARGETINGONE CORP
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