Multiple liquid phase gene chip method and reagent for rapidly detecting guinea pig LCMV, SV, PVM and Reo-3 viruses

A type 3 virus, mouse pneumonia virus technology, applied in microorganism-based methods, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of high cost and difficulty, and achieve lower detection costs, high sensitivity, Guaranteed effect of renaturation temperature and hybridization efficiency

Active Publication Date: 2016-12-07
GUANGDONG LAB ANIMALS MONITORING INST
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with conventional PCR, fluorescent quantitative PCR has advantages in sensitivity, specificity, and speed. However, real-time fluorescent PCR technology is limited by the type of fluorescence and the instrument itself. It can only detect up to 5 targets, and the success of the experiment is extremely difficult. big, expensive

Method used

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  • Multiple liquid phase gene chip method and reagent for rapidly detecting guinea pig LCMV, SV, PVM and Reo-3 viruses
  • Multiple liquid phase gene chip method and reagent for rapidly detecting guinea pig LCMV, SV, PVM and Reo-3 viruses
  • Multiple liquid phase gene chip method and reagent for rapidly detecting guinea pig LCMV, SV, PVM and Reo-3 viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0099] Example 2 Multiplex liquid-phase gene chip method for rapid detection of guinea pig lymphocytic choriomeningitis virus (LCMV), Sendai virus (SV), murine pneumonia virus (PVM), reovirus type 3 (Reo-3) Reagents

[0100] Reagents include the following components:

[0101] (1) The primers designed in Example 1;

[0102] (2) 4 kinds of fluorescently encoded microspheres encoding different fluorescent colors, the 4 kinds of fluorescently encoded microspheres respectively contain fluorescently encoded microspheres with different anti-tag sequences, and the anti-tag sequences can be correspondingly combined with multiple fluorescent immunoassay primers The tag sequences in are complementary paired; the four microspheres were all purchased from luminex, and the numbers of the fluorescently encoded microspheres corresponding to LCMV, SV, PVM and Reo-3 were MTAG-A056, MTAG-A062, MTAG-065 and MTAG- 077.

[0103] (3) Streptavidin-phycoerythrin complex.

Embodiment 3

[0104] Example 3 Establishment of a multiplex liquid-phase gene chip detection method for rapid detection of guinea pig lymphocytic choriomeningitis virus, Sendai virus, murine pneumonia virus, and reovirus type 3

[0105] (1) Viral RNA extraction and reverse transcription

[0106] Use the Tiangen OSR-M202 virus nucleic acid extraction kit to extract the RNA of the above four viruses on the TGuide M16 automatic nucleic acid extraction instrument, and use Takara PrimeScript TM RT Master Mix (Perfect Real Time) for reverse transcription

[0107] The reaction system for reverse transcription is:

[0108]

[0109]The reaction program for reverse transcription is: 37°C for 15min; 85°C for 5s, and store at 4°C.

[0110] (2) Multiplex PCR amplification

[0111] Preparation of primer mixture: mix the primers of LCMV, SV, PVM and Reo-3 at a ratio of 4:1:1:1; the multiplex PCR reaction system is:

[0112]

[0113] The amplification reaction program was as follows: pre-denatur...

Embodiment 4

[0130] Embodiment 4: The detection sensitivity experiment of the multiple liquid phase gene chip of LCMV, SV, PVM and Reo-3

[0131] The reverse-transcribed cDNA in Example 3 was quantified, diluted to 0.7 fg / μL by the 10-fold dilution method, and detected by the multiple liquid-phase gene chip method established above. The test results of the multiplex liquid-phase gene chip detection sensitivity of LCMV, SV, PVM and Reo-3 are shown in Table 2. The experimental results show that the sensitivity of LCMV, SV, PVM and Reo-3 is higher, and the detection limit is 7ng respectively. / μL, 0.07ng / μL, 0.07ng / μL, 7ng / μL.

[0132] Table 2 Sensitivity test results of multiplex liquid-phase gene chip detection for LCMV, SV, PVM and Reo-3

[0133] cDNA concentration

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Abstract

The invention discloses a multiple liquid phase gene chip method and a reagent for rapidly detecting guinea pig LCMV, SV, PVM and Reo-3 viruses. The operation is simple. A target amplified fragment is acquired through PCR, and then the amplified product, fluorescent coding micro-balloon and streptavidin-phycoerythrin are hybridized, MFI value is read through a tester and different viruses are distinguished. According to the method provided by the invention, the rat lymphocyte choriomeningitis virus, sendai virus, rat pneumonia virus and Reo-3 virus can be accurately detected at the same time; the specificity is strong, the sensitivity is high and the repeatability is excellent; compared with the traditional detection method, the method has the advantages that different target molecules in a same sample can be detected, the detection flux is high, the sample dosage is less, the operation is simple and quick, the detection cost is greatly reduced and the detection efficiency is increased.

Description

technical field [0001] The invention belongs to the field of virus detection of experimental animals, and in particular relates to a rapid detection of guinea pig lymphocytic choroid plexus meningitis virus (LCMV), Sendai virus (Sendai Virus, SV), mouse pneumonia virus (PVM) and reovirus type 3 (Reo-3) Multiple liquid phase gene chip method and reagents. Background technique [0002] The quality of experimental animals directly affects the accuracy and reliability of experimental results, the welfare of experimental animals and the health of staff. It is very important to establish and implement a complete health monitoring program for experimental animals to ensure and improve the quality of experimental animals. With the development of my country's experimental animal business, the improvement of various laws and regulations, the increase in international exchanges, and the strict requirements of large multinational companies on the quality of domestic experimental animals...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/6816C12Q1/70C12Q1/701C12Q1/706C12Q2600/16C12Q2531/113C12Q2537/143C12Q2563/107
Inventor 郭鹏举颜丙峰黄韧张钰
Owner GUANGDONG LAB ANIMALS MONITORING INST
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