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Detection primer, probe and detection method of human astrovirus nucleotide

An astrovirus and primer detection technology, which is applied in biochemical equipment and methods, microorganism-based methods, and microbial determination/inspection, etc., can solve the problems of low virus sensitivity, easy missed detection, time-consuming and labor-intensive, etc. High, high accuracy, good primer specificity

Inactive Publication Date: 2013-04-03
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The virus sensitivity of electron microscopy is low, and it is prone to missed detection; the specificity of immunological methods needs to be improved, and they are all time-consuming and laborious
Traditional RT-PCR is also used for human astrovirus, but the detection time takes 6 hours

Method used

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  • Detection primer, probe and detection method of human astrovirus nucleotide
  • Detection primer, probe and detection method of human astrovirus nucleotide
  • Detection primer, probe and detection method of human astrovirus nucleotide

Examples

Experimental program
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Effect test

Embodiment 1

[0026] The design of embodiment 1 primer and probe

[0027] Download all known human astrovirus genome sequences from the NCBI database, and perform comparative analysis, select a segment with no secondary structure and a high degree of conservation, and design primers and probes. The sequences are as follows:

[0028] Forward primer: 5'-GCCAGACTCACAGAAGAGCAAC-3';

[0029] Reverse primer: 5'-GGACTTGCTAGCCATCACACTTC-3'.

[0030] The sequence of the probe is: 5'-CCTCCCCTCCAAATGCGATGGAG-3'; the 5' end of the probe is labeled with the reporter fluorescent dye VIC, and the 3' end is labeled with the quencher fluorescent dye BHQ2.

Embodiment 2

[0031] The optimization of embodiment 2 human astrovirus nucleic acid fluorescent RT-PCR conditions

[0032] The inactivated human astrovirus was used as the sample to be tested, and the viral genome RNA was extracted with a commercial RNA extraction kit, and stored at -20°C for later use.

[0033] (1) Optimization of primer concentration: under the same conditions in the reaction system, serially dilute the concentration of primers in Example 1 from 0.1 μmol / L to 1.6 μmol / L, and analyze and compare the test results , to determine the optimal final primer concentration of 0.4μmol / L.

[0034] (2) Optimization of magnesium ion concentration: under the same conditions in the reaction system, the MgCl 2 The concentration of the magnesium ion is increased by 1mmol / L from 1mmol / L to 10mmol / L, and the optimum concentration of magnesium ion is determined to be 5mmol / L.

[0035] (3) Optimization of the amount of reverse transcriptase (AMV RnaseXL): After comparing the test results us...

Embodiment 3

[0046] Example 3 Establishment of Real-time Fluorescent RT-PCR Detection Method for Human Astrovirus Nucleic Acid

[0047] (1) Preparation of the template to be tested: RNA extraction of virus samples was performed using a Roche High Pure viral RNA kit.

[0048] 1) Take 0.5 g of feces, add 500 μL of TE to make a 10-20% suspension, and centrifuge at 8000 rpm for 5 minutes;

[0049] 2) Take 200 μL sample supernatant and add 400 μL binding solution, mix well, add to purification filter tube, 1000rpm, 15s;

[0050] 3) Discard the filtrate, replace with a new collection tube, add 500 μL inhibitor removal buffer, and centrifuge at 10,000 rpm for 1 minute;

[0051] 4) Discard the filtrate, replace with a new collection tube, add 450 μL of washing solution, and centrifuge at 10,000 rpm for 1 minute;

[0052] 5) Rewash once; finally centrifuge at 13000rpm for 10s, discard the residual washing solution;

[0053] 6) Remove the collection tube, replace it with a clean 1.5mL Eppendrof c...

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Abstract

The invention discloses a detection primer, a probe and a detection method of human astrovirus nucleotide, belonging to the technical field of biological detection. The detection primer of the human astrovirus nucleotide comprises a forward primer and a reverse primer, the nucleotide sequences of which are shown as SEQ NO.1 and SEQ NO.2. A nucleotide sequence of the probe which is matched with the detection primer is shown as SEQ NO.3; and one end of the probe is signed with a report fluorescent dye, and the other end of the probe is signed with a quenching fluorescent dye. The detection method of the human astrovirus nucleotide comprises the following steps of: carrying out real-time fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) amplification by taking a sample RNA (Ribonucleic Acid) to be detected as a template and utilizing the forward primer, the reverse primer and the probe, collecting data after every one circulation is finished, and judging a result according to an amplification curve after the reaction is finished. The primer designed according to a human astrovirus genomic sequence is good in specificity and is high in sensitivity when being used for real-time fluorescent RT-PCR detection.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a human astrovirus nucleotide detection primer, probe and detection method. Background technique [0002] Human astrovirus (HAstV) is a non-enveloped single-stranded positive-sense RNA virus belonging to the mammalian genus of the family Astroviridae. HAstV is an important pathogen that causes diarrhea in young animals. Astrovirus infection has been reported all over the world. It can be distributed, cause outbreaks, and cause hospital-acquired infections. Similar to rotavirus, astrovirus infection mostly occurs in infants and young children under 2 years old, especially under 1 year old. At present, human astrovirus is believed to be the second cause of viral gastroenteritis in infants and young children, second only to rotavirus. In addition, the elderly and immunocompromised patients are also high-risk groups for astrovirus infection. Astrovirus infection has be...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
Inventor 肖性龙余以刚吴晖李晓凤唐语谦袁琨
Owner SOUTH CHINA UNIV OF TECH
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