CN101638694A

CN101638694A - Viral combined detection technology

Info

Publication number: CN101638694A
Application number: CN200810117400A
Authority: CN
Inventors: 张永钢
Original assignee: Individual
Current assignee: Individual
Priority date: 2008-07-30
Filing date: 2008-07-30
Publication date: 2010-02-03

Abstract

The invention relates to a method for preparing a virus detection kit in the field of biological systems, which consists of a tissue buffer solution and an amplification system. The method is characterized in that the amplification system is prepared by partially or entirely pre-mixing heat-resistant DNA polymerase, deoxynucleotide triphosphate, a reaction buffer solution and one or more primers for amplification, and is subpackaged for standby; infested water or a tissue extraction solution is directly added into the amplification system, the mixture is mixed evenly, and then the amplification detection can be immediately performed; and particularly when a sample is RNA viruses, the extraction process of nucleic acid is canceled, which can reduce operation steps, can also improve experimental efficiency, and reduce false negative results. The method is suitable for routine examination and emergency detection of medical treatment and other inspection and quarantine fields.

Description

A kind of viral combined detection technology

Technical field

The present invention is a kind of virus detection kit preparation method of biological technical field.The extraction of Yeast Nucleic Acid, reverse transcription and amplification are finished in same system, can be used for the joint-detection of dna virus or RNA viruses.

Background technology

Since late period in last century, Revertase was found, the research of Yeast Nucleic Acid (RNA) and application were accepted by people gradually, had obtained using widely in the exploitation and the especially viral quarantine and examination field of eukaryote of eukaryote biological products.Mainly by the extraction of Yeast Nucleic Acid, three steps of reverse transcription and gene amplification constitute in the operating process of Yeast Nucleic Acid, because operation steps is more, and the existence of factors such as the unstable chemcial property of Yeast Nucleic Acid, the false recessive probability of the failure of an experiment or detection is higher.Especially at the extraction step of Yeast Nucleic Acid, to environmental factors and human factor require morely, though people can reverse transcription and the gene amplification process be integrated have been finished, the improvement of overall operation effect is also not obvious.

In the DNA operant level, because there is the high hot stage in the PCR operating process, can directly finish breaking of cell, people can increase as direct pair cell of template or in-house gene fragment with bacterium, cell, tissue block even section, but when carrying out the microbe colony amplification, we find that false-positive result can appear in the part microorganism owing to the influence of factors such as cell walls, and do not find above-mentioned phenomenon as yet in virus and phage amplification procedure.And, producer such as NEB have been arranged at present in the DNA operant level, and TAKARA, companies such as sky, Beijing root are prefabricated into the reflection mixture with archaeal dna polymerase, substrate and damping fluid mixing and have obtained success, and we think that this mode is equally applicable to the operation of Yeast Nucleic Acid.Based on this, we have carried out improving with the flow process that simplifies the operation to experimental technique, and detection range is not found relevant report as yet both at home and abroad at present.

Summary of the invention

The present invention relates to a kind of virus detection kit preparation method in living things system field, constitute by tissue buffer solution and amplification system, it is characterized in that: amplification system by hot resistant DNA polymerase, triphosphate deoxy-nucleotide, reaction buffer and amplification primers pre-mixing according to being made for amplification system, standby after the packing; Epidemic disease water or tissue extract directly join in the amplification system can carry out augmentation detection behind the mixing at once.

The virus detection kit preparation method who the present invention relates at object can to make be dna virus or RNA viruses; When detected object is RNA viruses, hot resistant DNA polymerase selects to have the hot resistant DNA polymerase of reverse transcriptase activity in the test kit, comprising being not limited to TaqDNA polysaccharase and segment thereof, TthDNA polysaccharase and segment thereof, the heat-resisting reversed transcriptive enzyme of FD (FD-TRT) and segment thereof, can select wherein one or several mixture, preferred but be not limited to the Taq archaeal dna polymerase, select reverse transcriptase activity and dna polymerase activity reaction system according to the work characteristics of selected enzyme system simultaneously.

Among the virus detection kit preparation method who the present invention relates to, reaction buffer is by buffer system, and inorganic salt, nonspecific proteins matter constitute; When detected object is RNA viruses, can add or not add ribonuclease inhibitor in the reaction system, wherein ribonuclease inhibitor includes but not limited to non-specific Yeast Nucleic Acid, the Yeast Nucleic Acid enzyme antibody.

Among the virus detection kit preparation method who the present invention relates to, amplification primers team can contain one to 4,000 pair right at primer identical or the different virus gene, wherein preferred but be not limited to contain one to five pair right at primer identical or the different virus gene; Each primer differs 1 to 4000bp to the length of amplified production, and is preferred but be not limited to 100 to 500bp.

Embodiment

1, makes up amplification system

1) chooses suitable archaeal dna polymerase system with reverse transcriptase activity.

2) selecting suitable enzyme for use according to related data is buffer system and ion component preparation reaction buffer and the sample buffer that reverse transcription is active and archaeal dna polymerase plays a role, and adds or do not add nonspecific proteins matter, non-specific Yeast Nucleic Acid and ribonuclease inhibitor therein.

3) the triphosphate deoxyribose nucleotide mixture of preparation proper concn

4) amplimer of preparation proper concn is right, and primer is a kind of to 4,000 kinds to having, and is preferred but be not limited to a kind of to five kinds; Each primer differs 1 to 4000bp to the length of amplified production, and is preferred but be not limited to 100 to 500bp

5) in prefabricated reaction mixture mode, above-mentioned toolenzyme, reaction buffer and triphosphate deoxyribose nucleotide are mixed, make 1 times of prefabricated reaction mixture to 10 times of concentration, preferred but be not limited to one to twice; But the freezing preservation of Lee's packing wherein can add an amount of glycerine after low-temperature mixed was finished.

2, sample detection method

1) preparation of template: virus vector can join in the amplification system and increase directly as carrier; The zooblast carrier of vitro culture can hot deactivation after or directly join in the amplification system and increase; After the vegetable cell of vitro culture or animal tissues need add sample buffer homogenate, join in the amplification system after hot deactivation or direct the pulverizing and increase.

2) amplification of goal gene: add an amount of sterilized water and template at prefabricated reaction mixture; Setting working routine with reference to the operating characteristic of toolenzyme increases.

3) content that utilizes gel electrophoresis or make the phosphoric acid detection architecture determine amplified production, preferred but be not limited to the agarose gel electrophoresis detection architecture.

Virus detection kit technology of preparing involved in the present invention is an improved product on conventional P CR detection technique.After the improvement, the user can carry out augmentation detection as long as template joined in the prefabricated reaction system to mix; Especially when sample is RNA viruses, because cancelled the nucleic acid extraction process, not only can reduces operation steps, and can improve conventional efficient, reduce the generation of false negative result.

Embodiment

Below with in the suggestion process, two kinds of gene segments of the gene segment of certain RNA viruses length 1000bp and certain RNA viruses length 1500bp as goal gene, are utilized the Taq archaeal dna polymerase that this fragment is increased and describe for the mode of operation of example to the related detection technique of this patent.Description is certain specific working method of this patent, and its content does not constitute the restriction to the prescription of this patent institute.

1, the gene order and the design of primers mode of amplification object

The 1000bp gene segment sequence of certain RNA viruses is as follows:

5’-ATGCGCAAATTTGGCG......CGCCGGCGTTCAATAA-3’

Upstream primer 5 '-ATGCGCAAATTTGGCG-3 '

Downstream primer 5 '-TTATTGAACGCCGGCG-3 '

The sequence of certain dna virus 1500bp gene fragment is as follows:

5’-GCTACCTGAGTAATG......GATTGTTGAGCTATG-3’

Upstream primer 5 '-ATGCGCAAATTTGGCG-3 '

Downstream primer 5 '-CATAGCTCAACAATC-3 '

2, make up prefabricated reaction system

Prefabricated reaction mixture (1 times):

10mM Tris-HCl (pH8.3), 50mM KCl, 1.5mM MgCl ₂, 0.1g/L BSA, 0.02mM dNTP, 50u/mL Taq enzyme, the concentration of every kind of primer is respectively after 1pM mixes each component according to aforementioned proportion, and per 100 microlitres are a freezing preservation of unit packing.

3, using method

Obtain template

Tissue leaches damping fluid 10mM Tris-HCl (pH8.3), 50mM KCl, 3mM MgCl ₂

To organize and leach damping fluid and pathological tissues tissue mixing back homogenate, the centrifuging and taking supernatant liquid is both for organizing leach liquor.

Make up reaction system

Prefabricated reaction mixture, 100 microlitres

Organize leach liquor, 2 microlitres are to 5 microlitres

The abundant mixing of low temperature

Reaction conditions is provided with

1. 94 degrees centigrade of pre-sex change are 200 seconds

2. 94 degrees centigrade of sex change are 60 seconds

3. 50 degrees centigrade of renaturation are 30 seconds

4. 68 degrees centigrade were extended 60 seconds

5. 68 degrees centigrade are continued to extend 200 seconds

6. 4 degrees centigrade of preservations

Wherein step 2 is to step 4 circulation 30 times

4 detection methods

After the conventional agarose electrophoresis, ultraviolet lamp detects amplification down.

Claims

1 the present invention relates to a kind of viral combined detection technology in living things system field, be made of tissue buffer solution and amplification system, it is characterized in that: amplification system is made amplification system by hot resistant DNA polymerase, triphosphate deoxy-nucleotide, reaction buffer, amplification primers to some or all of pre-mixing; Epidemic disease water or tissue extract directly join in the amplification system can carry out augmentation detection behind the mixing at once.

2 virus detection techniques according to claim 1 is characterized in that: this test kit at object can make dna virus or RNA viruses; When detected object comprises RNA viruses, hot resistant DNA polymerase selects to have the hot resistant DNA polymerase of reverse transcriptase activity in the test kit, comprising being not limited to TaqDNA polysaccharase and segment thereof, TthDNA polysaccharase and segment thereof, the heat-resisting reversed transcriptive enzyme of FD (FD-TRT) and segment thereof.

3 virus detection techniques according to claim 1 is characterized in that: reaction buffer is by buffer system, and inorganic salt, nonspecific proteins matter constitute; When detected object comprises RNA viruses, can add or not add ribonuclease inhibitor in the reaction system, comprising but be not limited to non-specific Yeast Nucleic Acid, the Yeast Nucleic Acid enzyme antibody.

4 virus detection techniques according to claim 1, it is characterized in that: amplification primers team can contain one to 4,000 pair right at primer identical or the different virus gene, wherein preferred but be not limited to contain one to five pair right at primer identical or the different virus gene; Each primer differs 1 to 4000bp to the length of amplified production, and is preferred but be not limited to 100 to 500bp.

5 virus detection techniques according to claim 1 is characterized in that: the pre-mixing system can wherein some or all of component premix, and is preferred but be not limited to all mix packing; The concentration of pre-mixing system can be to ten times of working concentration, and is preferred but be not limited to one to twice.