Seven RNA virus nucleic acid detection quality control products and preparation method thereof

A technology of RNA virus and quality control products, which is applied in the fields of clinical laboratory science and applied molecular biology, can solve the problems of reduced yield of virus-like particles, difficulty in sharing quality control products, and limited application range, etc., to overcome low preparation efficiency, High safety, simple and efficient preparation method

Pending Publication Date: 2021-11-02
BEIJING HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the virus-like particles prepared by conventional MS2 phage virus-like particle packaging technology have limitations in the size of the inserted foreign sequence. When the length exceeds 500bp, the packaging efficiency drops rapidly, and the yield of virus-like particles is greatly reduced. , the product concentration is low, only about 10 6 copies / mL
At the same time, due to the different detection target areas of different kits, the application range of existing quality control products is limited. A quality control product can only be applied to its corresponding detection kit, resulting in the quality control of various kits hard to share

Method used

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  • Seven RNA virus nucleic acid detection quality control products and preparation method thereof
  • Seven RNA virus nucleic acid detection quality control products and preparation method thereof
  • Seven RNA virus nucleic acid detection quality control products and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0103] Example 1 Construction of pACYC-MS2-virus vector construction

[0104] 1. Construction of pACYC-MS2 recombinant plasmid

[0105] 1) Obtain the gene sequence encoding MS2 phage capsid protein and mature enzyme protein from the GenBank database, and design and add BamHI and NotI restriction enzyme site sequences at both ends of the sequence. The sequence information is shown in SEQ ID No.1; The design sequence was artificially synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. and inserted into the PUC-SP vector.

[0106] 2) Use the restriction endonucleases BamHI and NotI to double-digest the pUC-57 and pACYCDuet-1 plasmids respectively, and the restriction enzyme digestion system is as follows:

[0107] Table 2

[0108]

[0109]

[0110] Digest at 37°C for 1 hour, inactivate restriction enzymes at 80°C for 5 minutes, perform 1% agarose gel electrophoresis on the digested target products, and recover and purify by cutting the gel. The products are named MS...

Embodiment 2

[0126] Embodiment 2 Preparation of MS2 virus-like particles containing seven kinds of RNA virus nucleic acids

[0127] 1) The pACYC-MS2-virus recombinant plasmids of the seven viruses successfully sequenced were transformed into expressive BL21(DE3) E. Coli competent cells.

[0128] 2) Positive clones were inoculated into 5 mL of LB liquid medium containing chloramphenicol (final concentration 34 μg / mL), cultured for about 4-6 hours, and 2 mL of bacterial liquid was added to 200 mL of LB liquid medium for expansion, 37 ° C, 250 rpm Shake culture for about 2-3 hours, when the OD 600 value of the bacterial solution is about 0.6-0.8 with a spectrophotometer, add isopropylthio-β-D-galactoside with a final concentration of 1mmol / L to the bacterial solution (IPTG), 37° C., 250 rpm and continue shaking culture for about 12-16 hours to induce the expression of MS2 virus-like particles.

[0129] 3) Centrifuge at 9500rpm for 3min at 4°C, discard the supernatant, retain the bacterial pe...

Embodiment 3

[0134] Embodiment 3 MS2 virus-like particle verification and quantification

[0135] 1. DNase Ⅰ and RNase A enzyme digestion verification

[0136] The MS2 virus-like particle solution obtained in Example 2 was digested with DNase I and RNase A with final concentrations of 200 U / mL and 500 U / mL respectively, and the sample loading system was as follows:

[0137] Table 6

[0138]

[0139] Digest for 2 hours in a water bath at 37°C to remove residual nucleic acid, and perform 1% agarose gel electrophoresis analysis.

[0140] Taking the EV71 virus as an example, through electrophoresis analysis (as attached to the instruction manual) image 3 As shown), it can be seen that the EV71MS2 virus-like particles purified in Example 2 are a bright single band after being digested by DNase I and RNase A, while the virus-like particles that have not been digested by DNase I and RNase A are due to the residual nucleic acid adhesion on the surface. The "tailing phenomenon" appears, indi...

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Abstract

The invention discloses seven RNA virus nucleic acid detection quality control products and a preparation method thereof, and belongs to the field of clinical laboratory science and application molecular biology. The invention has nucleotide sequences as shown in SEQ ID NO.3 to SEQ ID NO.13 in a sequence table. According to the invention, an optimized MS2 bacteriophage virus-like particle packaging technology is adopted, recombinant conventional detection region sequences of seven kinds of RNA viruses are respectively wrapped in MS2 capsid protein, and the MS2 virus-like particle nucleic acid detection quality control product of the seven kinds of viruses is prepared. The quality control product fully simulates a natural virion structure, is stable in performance, resistant to nuclease and free of any infection risk, and realizes quality monitoring and evaluation of the whole process of nucleic acid extraction, reverse transcription and amplification detection. The quality control product is good in universality, wide in detection area and suitable for quality control of a conventional detection method and various detection kits, and the problem that different kits cannot share the quality control product is solved.

Description

technical field [0001] The invention relates to seven RNA virus nucleic acid detection quality control products and a preparation method thereof, belonging to the fields of clinical laboratory science and applied molecular biology. Background technique [0002] Patients with acute respiratory infection often have symptoms such as fever, cough, and runny nose. Some patients (such as children, patients with underlying diseases, and immunocompromised patients) often cause severe complications such as pneumonia and bronchitis. leading cause of death among children. Clinically, there are many kinds of pathogens causing acute respiratory infection, 80% of which are viral infections, mainly including influenza virus (Influenza virus, IFV), respiratory syncytial virus (Respiratory syncytial virus, RSV), parainfluenza virus (Parainfluenza virus, PIV) , Rhinovirus (Human rhinovirus, HRV) and so on. Coxsackievirus A16 (Coxsackievirus A16, CA16) and enterovirus 71 (Enterovirus71, EV71...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12N15/70C12N7/01C12N15/11C12R1/93C12R1/92
CPCC12Q1/701C12N15/70C12N7/00C12N2795/18021C12N2795/18052
Inventor 李金明
Owner BEIJING HOSPITAL
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