Virus sample preservation solution, nucleic acid extraction reagent and viral nucleic acid extraction method

A nucleic acid extraction reagent and viral nucleic acid technology, which is applied in the field of virus sample preservation solution, can solve the problems of reducing the concentration of viral nucleic acid, limited viral nucleic acid content, and the inability to increase the amount of samples, so as to optimize the process, increase the amount of sample input, and reduce pollution. or the effect of the risk of error

Inactive Publication Date: 2020-05-19
上海思路迪医学检验所有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are many factors affecting the detection of viral nucleic acid, for example: (1) the content of viral nucleic acid in the sample is limited; (2) the virus sample preservation solution needs to be mixed with the lysate during the subsequent extraction of viral nucleic acid, which further reduces the concentration of viral nucleic acid; (3) The volume of the o...

Method used

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  • Virus sample preservation solution, nucleic acid extraction reagent and viral nucleic acid extraction method
  • Virus sample preservation solution, nucleic acid extraction reagent and viral nucleic acid extraction method
  • Virus sample preservation solution, nucleic acid extraction reagent and viral nucleic acid extraction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1 test group and control group sample preparation

[0049] Prepare enough virus sample preservation solution with cracking effect: guanidine thiocyanate (3.5mol / L), Tween-20 (volume percentage is 2%), polidocanol (volume percentage is 2%), ethanol (volume percentage Percentage is 30%), trisodium citrate (70mmol / L), citric acid (0.5mmol / L), dithiothreitol (mass volume percentage is 0.3g / 100mL);

[0050] Sample collection: 6 people. Each person collected throat swab samples 3 times and marked them. The 3 repeated samples were marked as -1, -2, -3 in turn.

[0051] Test group sample preparation: Add 19.5mL of freshly prepared virus sample preservation solution with lysing effect to the 50mL centrifuge tube, 10 5 PFU / mL MS2 pseudovirus 97.5μL (virus preservation solution volume: 10 5 MS2 volume of PFU / mL=200:1), mix thoroughly, and add 1.5mL of the mixture to the centrifuge tubes marked -2 ​​and -3 respectively to obtain the test group samples.

[0052] Sample...

Embodiment 2

[0053] Example 2 Virus nucleic acid extraction and purification of control group sample

[0054] Viral nucleic acid extraction reagents and Shanghai Kangyu automatic nucleic acid purification instrument (model: KY-32A) were used for viral nucleic acid extraction and purification, and 6 samples numbered -1 in Example 1 were extracted to obtain corresponding viral nucleic acid samples.

[0055] experiment process:

[0056] Briefly centrifuge the pre-packaged reagent plate, tear off the heat-sealed aluminum film, add 200 μL of the control sample and 20 μL of proteinase K to the 6 wells of A1-F1 in the first row of the 96 deep-well plate, and place the reagent plate in the instrument compartment Put a new magnetic rod cover into the corresponding card slot at the fixed position inside.

[0057] In the virus nucleic acid extraction reagent provided: lysate (columns 1 and 7 of 96-well deep-well plate, 700 μL per well), including guanidine thiocyanate with a final concentration of 3...

Embodiment 3

[0067] Example 3 Test group sample virus nucleic acid extraction and purification

[0068] Viral nucleic acid extraction reagents and Shanghai Kangyu automatic nucleic acid purification instrument (model: KY-32A) were used for viral nucleic acid extraction and purification, and 6 samples numbered -2 ​​in Example 1 were extracted to obtain corresponding viral nucleic acid samples.

[0069] experiment process:

[0070] Briefly centrifuge the pre-packaged reagent plate, tear off the heat-sealed aluminum film, discard the lysate in the 6 wells of A1-F1 in the first column of the 96 deep-well plate, and add 900 μL of test group samples, 20 μL of For proteinase K, place the reagent plate at a fixed position in the instrument compartment, and put a new magnetic rod cover into the corresponding card slot.

[0071] In the virus nucleic acid extraction reagent provided: proteinase K, the protein content is 15-30mg / mL. Cleaning solution I (columns 2 and 8 of 96-well deep-well plate, 90...

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Abstract

The invention discloses a virus sample preservation solution with a lysis function and a method for extracting viral nucleic acid. Since sample preservation and lysis are completed in one step, no additional lysis solution is needed, further diluting of a virus sample is prevented, the adding amount of the sample during the extraction process of viral nucleic acid can be increased, and the sensitivity of subsequent detection is improved. Meanwhile, because the preservation and lysis of the virus sample are completed in one step, the step of nucleic acid detection is removed, the process is optimized, and the efficiency of sample detection is improved. Compared with a conventional virus sample preservation solution such as a PBS buffer solution, the virus sample preservation solution has good preservation effect, more detection samples can be obtained, the detection sensitivity is improved, and the processing flow is simplified.

Description

technical field [0001] The invention relates to molecular biology detection technology, in particular to a virus sample preservation solution, a virus nucleic acid extraction reagent and a virus nucleic acid extraction method. Background technique [0002] There should be normal oral flora in the isthmus of normal people without the growth of pathogenic bacteria. The bacteria in the pharynx all come from the outside world and do not cause disease under normal circumstances, but infections can occur when the body's systemic or local resistance decreases and other external factors cause diseases. Therefore, the isolation of pathogenic bacteria from pharyngeal swabs is helpful for the diagnosis of diphtheria, suppurative tonsillitis, acute pharyngitis, respiratory tract infection and other diseases. [0003] The virus sample preservation solution currently on the market can only protect the stability of the virus. However, when extracting viral nucleic acid, especially for hig...

Claims

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Application Information

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IPC IPC(8): C12Q1/6806C12Q1/6851C12Q1/70C12R1/93
CPCC12Q1/6806C12Q1/6851C12Q1/701C12Q2527/125C12Q2523/308C12Q2521/107C12Q2531/113C12Q2563/107C12Q2545/114
Inventor 冯二环周科隆盛滔熊磊陈才夫
Owner 上海思路迪医学检验所有限公司
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