Human immunodeficiency virus, hepatitis B virus, hepatitis C virus rapid joint detection kit and its preparation and application

A technology of immunodeficiency virus and hepatitis B virus, which is applied in the field of biotechnology detection, can solve the problems of being unsuitable for clinical testing and large-scale blood screening, cumbersome operation, and high total cost, so as to reduce the risk of cross-contamination and detect High sensitivity and the effect of reducing manual errors

Active Publication Date: 2019-04-23
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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  • Human immunodeficiency virus, hepatitis B virus, hepatitis C virus rapid joint detection kit and its preparation and application
  • Human immunodeficiency virus, hepatitis B virus, hepatitis C virus rapid joint detection kit and its preparation and application
  • Human immunodeficiency virus, hepatitis B virus, hepatitis C virus rapid joint detection kit and its preparation and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] The preparation of embodiment 1 kit

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

[0049] Table 1

[0050]

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

[0052] That is to say, the kit of the present invention may contain the aforementioned independently packaged sets of primer pairs and probes, or may contain a configured multiplex fluorescent PCR detection mixture containing each set of primer pairs and probes.

[0053] Further, the kit can also contain dNTP, MgCl 2 , PCR buffer, reverse transcriptase, Taq DNA polymerase, sterile wat...

Embodiment 2

[0054] The sensitivity evaluation of embodiment 2 kit

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

[0056] experimental method:

[0057] 1. Preparation of positive control substance:

[0058] Pseudoviruses containing specific amplified fragments of HIV, HBV and HCV were constructed as positive controls, and the construction method was carried out with reference to "Construction and Expression of Ribonuclease-resistant Virus-Like Particles Containing Long Fragment Chimeric RNA (2008)".

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

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

...

Embodiment 3

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

[0087] experimental method:

[0088] 1. Sample processing:

[0089] The samples used include: HIV, HBV, HCV patient serum samples of 2 cases each, and healthy human serum samples of 2 cases.

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

[0091] The components of the kit for extracting viral RNA by magnetic bead method are shown in Table 5:

[0092] table 5

[0093]

Element

volume / person

1

affinity column

1 tube

2

binding buffer

500μl

3

Washing solution A

1ml

4

Washing liquid W

1ml

5

eluent

50μl

6

magnetic beads

20μl

7

RNA precipitation aid

6μl

[0094] The method of using the magnetic bead method extraction virus RNA kit is as follows:

[0095] ①Prepar...

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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 in particular relates to a rapid joint detection kit for human immunodeficiency virus, hepatitis B virus and hepatitis C virus and its preparation and application. Background technique [0002] Human immunodeficiency virus (Human Immunodeficiency Virus, HIV) is the pathogen of AIDS. The virus destroys the immune ability of the human body and causes the immune system to lose its resistance, thus causing various diseases and cancers to survive in the human body. So far, there is no effective treatment. 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 according to 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 (...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/686C12R1/93
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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