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Method for inspecting hepatitis and AIDS virus nucleic acid by synchronous amplification and its reagent kit

An HIV, kit technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., to avoid missed detection

Active Publication Date: 2007-04-04
SHANGHAI KEHUA BIO ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The object of the present invention is to provide a kind of kit that can synchronously extract the nucleic acid of HBV, HCV, HIV virus, synchronous real-time amplification detection HIV, HBV, HCV virus nucleic acid, to overcome the inherent defects and deficiencies of traditional protein detection, while making up for the existing There are many shortcomings of similar products at home and abroad, and even fatal flaws that cannot be solved in methodology

Method used

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  • Method for inspecting hepatitis and AIDS virus nucleic acid by synchronous amplification and its reagent kit
  • Method for inspecting hepatitis and AIDS virus nucleic acid by synchronous amplification and its reagent kit
  • Method for inspecting hepatitis and AIDS virus nucleic acid by synchronous amplification and its reagent kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Rapid preparation of internal control and positive control by biotin-modified primer plus terminal PCR method (the basis of the green environmental protection kit),

[0045] 1. The preparation of HIV internal control and positive control is as follows

[0046] 1.1 Rapid construction of positive control: commission Shanghai Sangon to synthesize the following primers, the sequence is:

[0047] 5’BIOTIN-AAT TCT AAT ACG ACT CAC TAT AGG GAG gac atc aag cag cca tgc aaa t-3’

[0048] (The italic part at the 5' end is the T7 RNA pol promoter region, and the 3' end is the upstream primer of the HIV GAG region)

[0049] (SEQ.ID.NO.10)

[0050] 5'BIOTIN-cta tgt cac ttc ccc ttg gtt ctc tc-3' (downstream primer of HIV GAG region)

[0051] (SEQ.ID.NO.11)

[0052] Take one specimen (or one plasmid containing HIV GAG gene) that is clinically positive for HIV nucleic acid, and...

Embodiment 2

[0113] Example 2 Specimen processing unit: magnetic bead method for nucleic acid extraction and reagent preparation

[0114] The reagent composition of the specimen processing unit based on the principle of hybridization includes:

[0115] Lysis solution: 4.8M guanidine thiocyanate, 50mM Tris, PH7.0, NP-40, Triton X-100, SDS, biotinylated probe, polymer, etc. where the capture probes are:

[0116] 5'BIOTIN-acc acc aaa tgc ccc tat-3'(HBV)

[0117] 5'BIOTIN-agt acc aca agg cct ttc g-3'(HCV)

[0118] 5'BIOTIN-cta tgt cac ttc ccc ttg gtt ctc tc-3'(HIV)

[0119] Magnetic bead suspension: 1mg / ml streptavidin-coated magnetic beads, containing preservatives and dispersants such as Tween 20

[0120] Washing liquid A: LiCI, Tris PH7.0, lithium dodecylsulfonate, preservatives and dispersants such as Tween 20, pigments, etc.

[0121] Washing liquid B: LiCI, Tris PH7.0, preservatives and dispersants such as Tween 20, pigments, etc.

[0122] Washing liquid C: LiCI, Tris PH7.0, preservat...

Embodiment 3

[0134] Example 3 Nucleic acid amplification detection unit: single-tube dual-enzyme one-step RT-PCR TaqMan probe multi-detection system mode and reagent preparation

[0135] The nucleic acid amplification detection unit adopts fluorescent TaqMan PCR, which is required to be used with a fluorescent PCR instrument. Its components include the following:

[0136] RT-PCR reaction solution: buffer containing 1.8Mm d NTPs (including d UTP), 3.6mM DTT, 3.5mM MgCl2, primers BF, BR, CF, CR, IF, IR 0.4μM each.

[0137] Enzyme mixture: 0.6U / μl AMV reverse transcriptase, 0.6U / μl hot-start Taq DNA polymerase, 1U / μl RNasin; 0.1U / μl UNG (heat-labile), etc.

[0138] Probe: 10μM probe HBV Probe, HCV Probe, HIV Probe, 5μM internal standard probe, stabilizer and buffer.

[0139] Reagent ratio and preparation: According to the ratio of RT-PCR reaction liquid:enzyme mixture:probe=8:6:1 for each test, 15 μl / test is prepared, and 15 μl of nucleic acid template is added to the machine for amplificat...

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Abstract

A method for inspecting hepatitis and AIDS nucleic acid by synchronized amplification and its reagent knit are disclosed. The process is carried out by taking magnetic ball as automatic medium, specific synchronized capturing HBV, CHV and HIV nucleic acid, accelerating biotin primer construction and purification by RNA external label and internal label, real-time synchronized inspecting and T-PCR amplifying based on Tagman probe. The reagent knit consists of dis-inhibitor, cracking liquid, magnetic ball suspension, washing liquor, internal check, RT-PCR reactive liquor, enzyme mixture, fluorescent mixture, positive check and negative check. It's accurate and automatic, has single-tube operation, closed inspection AND synchronized extraction, and it has better sensitivity and specific performance and can be used for large-scale blood screening and large-capacity clinical inspection.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid diagnosis of diagnostic reagents, in particular to a highly sensitive, specific and suitable for synchronous extraction and synchronous real-time detection of hepatitis (including hepatitis B and hepatitis C) and HIV nucleic acid for routine blood nucleic acid screening methods and kits. Background technique [0002] According to the statistics of the World Health Organization (WHO), more than half of the countries do not conduct thorough testing on the blood donated by blood donors, resulting in the spread of diseases such as AIDS, hepatitis, malaria and syphilis in many places. Among the 5.6 million HIV-infected people who increase globally every year, 5% to 10% of them are infected through blood transfusion or blood products. Among African children and women, the proportion is as high as 20%-25%. Since the 1980s, France, Japan, Romania, the United States, Germany, and my country have all...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/70
Inventor 周科隆华锦彪王缦
Owner SHANGHAI KEHUA BIO ENG
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