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Hepatitis B virus rapid detection system combining MCDA with biosensor

A biosensor, hepatitis B virus technology, applied in the determination/inspection of microorganisms, methods based on microorganisms, microorganisms, etc., can solve the problems of low detection sensitivity, high cost of detection methods, and long time consumption.

Active Publication Date: 2021-04-30
贵州中医药大学第二附属医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The object of the present invention is a rapid detection system of hepatitis B virus nucleic acid combining MCDA with biosensing technology to solve the problem of high cost, time-consuming and long-term detection of existing detection methods. Technical problems such as complicated operation and low detection sensitivity

Method used

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  • Hepatitis B virus rapid detection system combining MCDA with biosensor
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  • Hepatitis B virus rapid detection system combining MCDA with biosensor

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Embodiment 1

[0053] 1. Materials and Equipment

[0054] Genomic DNA and RNA extraction kits were purchased from Xi’an Tianlong Technology, the colorimetric indicator (Colorimetric indicator) was purchased from Malachite Green Company, the universal constant temperature amplification kit was purchased from Beijing Haitai Zhengyuan, and biotin-labeled BSA (bovine serum albumin) Purchased from Abcam Corporation. LFB materials include backsheets, sample pads, absorbent pads, conjugate pads, and NC membranes, all of which were purchased from Jie-Yi Biotechnology.Co.Ltd. Nanoparticle-coupled streptavidin (dark red, Dye streptavidin-coated polymer nanoparticles) was purchased from Bangs Laboratories. HBV real-time PCR detection kit was purchased from DaAn Gene Company. Nucleic acid purity and concentration were analyzed by Nano-Drop ND-2000 (A260 / 280).

[0055] 2. MCDA primer design

[0056] Design primers according to the S gene of hepatitis B virus (the sequence of the S gene is shown in Ge...

Embodiment 2

[0065] Embodiment 2: MCDA optimal reaction temperature test

[0066] In this embodiment, it is basically the same as in Example 1, except that the difference lies in the setting of temperature. In this experimental example, the optimum reaction temperature of MCDA is detected and confirmed, and the amplification temperature is set at 60-67° C. (every 1° C. is a gradient). Use a real-time turbidimeter (LA-500) to detect amplicons in the system (turbidity greater than 0.1 is considered a positive result). Experimental results such as image 3 and Figure 4 As shown (the turbidity detector uses the judgment mode to mainly examine the amplification time), 63°C is the optimal reaction time.

Embodiment 3

[0067] Embodiment 3: the sensitivity test of MCDA-LFB

[0068] Dilute the HBV template to a nucleic acid content of 5.0 × 10 3 IU, 5.0×10 2 IU, 5.0×10 1 IU, 5IU, 5IU, 0.5IU, 0.05IU, obtain 7 kinds of diluted templates, then carry out MCDA amplification (same as Example 1) with the above templates respectively, and carry out colorimetric indicator detection and LFB detection to MCDA amplification products . Experimental results such as Figure 5 As shown, the detection sensitivity of this scheme is that the amount of HBV in each reaction is 5 IU (ie LoD, limited of detection).

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Abstract

The invention relates to the technical field of biological medicine detection, and relates to a POCT rapid nucleic acid detection system, in particular to a hepatitis B virus rapid detection system combining MCDA with a biosensor. The system comprises an MCDA unit for amplifying an S gene of hepatitis B virus and a detection unit for detecting an MCDA product obtained from the MCDA unit, the MCDA unit comprises a replacement primer pair, a cross primer pair and three pairs of amplification primer pairs. According to the scheme, the technical problems of high cost, long consumed time, complex operation, low detection sensitivity and the like of an existing detection method can be solved. According to the scheme, the MCDA technology and the LFB technology are combined to be used for detecting the HBV, the operation process is simple, the amplification efficiency is high, the cost is low, and the system is very suitable for POCT detection and rapid detection under the condition that detection equipment is lacked in economically underdeveloped areas.

Description

technical field [0001] The invention relates to the technical field of biomedical detection, and relates to a POCT rapid nucleic acid detection system, in particular to a rapid detection system for hepatitis B virus with MCDA combined with a biosensor. Background technique [0002] Hepatitis B virus (Hepatitis B, HBV) is the pathogen that causes hepatitis B (hepatitis B for short), belongs to the hepadnaviridae family, and HBV infection is a global public health problem. In particular, HBV infection is often accompanied by hepatitis C virus (HCV) and human immunodeficiency virus (HIV) infections, posing a significant public health and socioeconomic burden. Statistics from the World Health Organization show that 257 million people in the world suffer from chronic hepatitis B, and about 15-40% of chronic hepatitis B will transform into liver cirrhosis or liver cancer. Therefore, relevant detection in the early stage of HBV occurrence has positive significance for the preventi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12R1/93
CPCC12Q1/706C12Q1/6844C12Q2531/119C12Q2521/101C12Q2563/131C12Q2565/625Y02A50/30
Inventor 陈旭刘正奇李玉洁
Owner 贵州中医药大学第二附属医院