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Fast joint inspection kit for human immunodeficiency viruses, hepatitis B viruses and hepatitis C viruses and preparation and application thereof

An immunodeficiency virus and hepatitis B virus technology, applied in the field of biotechnology detection, can solve the problems of being unsuitable for clinical testing and large-scale blood screening, time-consuming, and complicated to operate, so as to reduce the risk of cross-contamination and shorten the Experiment period, the effect of high detection sensitivity

Active Publication Date: 2016-06-22
INSPECTION & QUARANTINE TECH CENT OF GUANGDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there are a variety of single nucleic acid detection / typing products for HCV, HBV, and HIV on the market, but for patients with mixed infections, the total cost of a single detection for each virus is relatively high. The test results take a long time
For the inspectors in hospital laboratories, due to the different genetic markers and amplification procedures used in the detection of each virus, the operation of a single detection of a single virus is cumbersome, time-consuming, and low-throughput, which is not suitable for clinical testing and mass blood screening

Method used

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  • Fast joint inspection kit for human immunodeficiency viruses, hepatitis B viruses and hepatitis C viruses and preparation and application thereof
  • Fast joint inspection kit for human immunodeficiency viruses, hepatitis B viruses and hepatitis C viruses and preparation and application thereof
  • Fast joint inspection kit for human immunodeficiency viruses, hepatitis B viruses and hepatitis C viruses and preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Preparation of kit

[0048] Synthesize HIV detection primers and probes, HBV detection primers and probes, HCV detection primers and probes, and their nucleotide sequences are shown in Table 1 below:

[0049] Table 1

[0050]

[0051] The above primer pairs and probes can be individually packaged, or combined to form a multiplex fluorescent PCR detection mixture. In the multiplex fluorescent PCR detection mixture, the amounts of the above-mentioned primers and probes may be conventional amounts known to those skilled in the art.

[0052] In other words, the kit of the present invention may contain the above-mentioned individually packaged sets of primer pairs and probes, or may contain a configured multiplex fluorescence PCR detection mixture containing each set of primer pairs and probes.

[0053] Further, the kit may also contain dNTP, MgCl 2 , PCR buffer, reverse transcriptase, TaqDNA polymerase, sterile water (DEPC-ddH 2 O), sample genomic DNA / RNA extraction reagen...

Embodiment 2

[0054] Example 2 Sensitivity evaluation of the kit

[0055] Experimental purpose: to determine the detection limit (minimum detection concentration) of the multiple fluorescent PCR kit of the present invention (Example 1)

[0056] experimental method:

[0057] 1. Preparation of positive control substance:

[0058] A pseudovirus containing specific amplified fragments of HIV, HBV and HCV was constructed as a positive control. The construction method was carried out with reference to the document "Construction and expression of virus-like particles containing long chimeric RNA in ribonuclease (2008)".

[0059] The nucleic acid sequence of the constructed pseudovirus was verified by Sanger sequencing, and the amplicon fragment containing the HIV, HBV and HCV 3 sets of detection primer pairs in Example 1 can be identified by the aforementioned 3 primers and probes.

[0060] Among them, the nucleotide sequence of the HIV-specific amplified fragment is shown in SEQ ID NO. 10, specifically:

[0...

Embodiment 3

[0086] Example 3 Evaluation of the detection effect of the kit

[0087] experimental method:

[0088] 1. Sample processing:

[0089] The samples used included 2 serum samples from HIV, HBV, and HCV patients, and 2 serum samples from healthy people.

[0090] The above-mentioned serum samples were all extracted using a ribonucleic acid (RNA) extraction kit (magnetic bead column extraction method) produced by Shanghai Zhijiang Biotechnology Co., Ltd.

[0091] The composition of the magnetic bead extraction virus RNA kit is as follows:

[0092] table 5

[0093]

Ingredients

Volume / person

1

Affinity column

1 tube

2

Binding buffer

500μl

3

Washing liquid A

1ml

4

Washing liquid W

1ml

5

Eluent

50μl

6

Magnetic beads

20μl

7

RNA sedimentation aid

6μl

[0094] The specific method of using the magnetic bead method to extract viral RNA kit is as follows:

[0095] ①Prepare reagents: before using the washing solution for the first time, add ethanol according to the instructions on t...

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Abstract

The invention belongs to the field of biotechnology detection, and particularly relates to a fast joint inspection kit for human immunodeficiency viruses (HIV), hepatitis B viruses (HBV) and hepatitis C viruses (HCV) and preparation and application thereof. The kit comprises an HIV detecting primer and probe, an HBV detecting primer and probe and an HCV detecting primer and probe. A multiplex fluorescent PCR technology is adopted, three kinds of viral nucleic acid of the HIV, the HBV and the HCV are detected in a single PCR reaction tube at the same time, detecting sensitivity is high, good specificity is achieved, the human error rate is low, and time consumed in experiments is short. Fluorescence signals are detected in real time in the amplified reaction process, the whole process is conducted in a sealed mode, the risk of cross infection among samples is reduced, and the kit is suitable for being applied to large-scale blood screening and clinical examinations.

Description

Technical field [0001] The invention belongs to the field of biotechnology detection, and specifically relates to a rapid joint test kit for human immunodeficiency virus, hepatitis B virus, and hepatitis C virus, and preparation and application thereof. Background technique [0002] Human Immunodeficiency Virus (HIV) is the pathogen of AIDS. The virus destroys the immune system of the human body and causes the immune system to lose its resistance, leading to various diseases and cancers to survive in the human body. There is no effective treatment so far. Statistics in 2015: There are 35 million HIV-infected people worldwide, and China has nearly 500,000, ranking 12th in the world. HIV can be divided into two types based on serology and viral nucleic acid sequence: HIV-1 and HIV-2. HIV-1 is prevalent in most parts of the world, and HIV-2 is only prevalent in some parts of Africa. [0003] Hepatitis B virus (HBV) and hepatitis C virus (HCV) are the two main types of viruses that c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/686C12Q1/706C12Q1/707C12Q2600/16C12Q2537/143C12Q2563/107C12Q2561/113
Inventor 黄吉城李小波郑夔师永霞刘燕王宁王凯
Owner INSPECTION & QUARANTINE TECH CENT OF GUANGDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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