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A real-time fluorescent quantitative PCR primer, probe and kit for detecting tree shrew adenovirus

A technology of real-time fluorescence quantification and kits, applied in biochemical equipment and methods, microorganisms, and methods based on microorganisms, etc., can solve the problems of inability to quantify the copy number of DNA or RNA, and achieve cost saving, high sensitivity, and high accuracy sexual effect

Active Publication Date: 2021-01-29
INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since there is no linear relationship between the amount of end product of PCR and the amount of starting template, it is not possible to quantify the starting DNA or RNA copy number

Method used

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  • A real-time fluorescent quantitative PCR primer, probe and kit for detecting tree shrew adenovirus
  • A real-time fluorescent quantitative PCR primer, probe and kit for detecting tree shrew adenovirus
  • A real-time fluorescent quantitative PCR primer, probe and kit for detecting tree shrew adenovirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Design of special primers and probes for tree shrew adenovirus

[0047] Designed and synthesized according to the tree shrew whole genome sequence (GenBank: MF780605) which was isolated, identified and sequenced by our laboratory. The amplicon size is 122bp. The 5' end of the probe is connected with a fluorescent reporter group, and the 3' end is connected Has a fluorescent quencher. Using the primer for PCR amplification can realize qualitative and quantitative detection with good specificity, high sensitivity and strong accuracy. Wherein the preferred primer and probe sequences are:

[0048] Upstream primer: 5′-AAAGAATGCCCACCTCCCAT-3′; (SEQ ID NO.1)

[0049] Downstream primer: 5′-CAGTGGCAGGCCATTAAGC-3′; (SEQ ID NO.2)

[0050] Probe primer: 5′-Fam-CTCGCCGCCGCCGGTTTCAC-Tamra-3′. (SEQ ID NO.3)

[0051] After homology comparison with the designed primers and probes, it was found that it was only highly conserved against tree shrew adenovirus, and there was ...

Embodiment 2

[0052] Example 2 Preparation of standard positive template

[0053] The positive tree shrew adenovirus DNA preserved in our laboratory was used as a template to amplify the target gene fragment.

[0054] Reaction system: Gold Mix (green) 20 μL, 10 μM upstream primer 1 μL, 10 μM downstream primer 1 μL, 50ng / μL DNA template 3 μL, final volume 25 μL.

[0055] The reference annealing temperature of the kit is 55-60°C, and the optimized reaction conditions are: 98°C for 2 min; 98°C for 10 s, 58°C for 15 s, 72°C for 15 s, a total of 35 cycles; 72°C for 5 min.

[0056] The amplified product was identified by agarose gel electrophoresis with a volume fraction of 1.5%; the gel was recovered and purified; the target gene fragment was ligated with the PUC57 vector; the ligated product was transformed into DH5α competent cells; the sequencing verification was the standard positive template. The absorbance values ​​of A260 and A280 were measured by ultraviolet spectrophotometer, the ratio...

Embodiment 3

[0058] Example 3 Establishment of Real-time Fluorescent Quantitative PCR Amplification Method

[0059] 1. Real-time fluorescence quantitative PCR template preparation - sample total DNA extraction:

[0060] 1) Add 500 μL of lysate (100 mmol / L Tris-Cl pH=8.5, 0.1mol / L EDTA, 0.5% SDS) to 200 μL of the solution to be tested (if the sample is a tissue that needs to be ground and homogenized), mix well by inverting , 5min.

[0061] 2) Add 6 μL of proteinase K, bathe in water at 55°C for 15 minutes, and cool down.

[0062] 3) Add 700 μL of a mixed solution of phenol, chloroform and isoamyl alcohol (25:24:1 by volume), and shake to mix.

[0063] 4) Centrifuge at 10000rpm / min for 5min to take the supernatant.

[0064] 5) Repeat steps 3 and 4, then pool the supernatants.

[0065] 6) Add 2 times the volume of the combined supernatant, pre-cooled to 4°C absolute ethanol and 1 / 10 volume of the combined supernatant 3mol / L sodium acetate solution.

[0066] 7) Precipitate at -20°C for 1...

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Abstract

The invention relates to real-time fluorescent quantitative PCR primers, a probe and a kit for detecting tupaia adenoviruses and belongs to the field of biotechnology. The primers are designed and synthesized according to the end 3' conserved sequence of the tupaia whole genome isolated and identified in this laboratory. The primers do not undergo a cross reaction with a tupaia source and common viruses belonging to other species. The end 5' of the probe is connected to a fluorescent reporting group and the end 3' of the probe is connected to a fluorescence quenching group. The kit produces accurate and reliable results, has good stability, good repeatability and high sensitivity, can fast and accurately quantify, has a wide detection range, can be used for epidemiological investigation caused by tupaia adenoviruses, provides the technical support for related basic research and has a wide application prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a real-time fluorescent quantitative PCR primer, a probe and a kit for detecting tree shrew adenovirus, more specifically to a TaqMan real-time fluorescent quantitative PCR primer and a probe for detecting tree shrew adenovirus, At the same time, it also involves the TaqMan real-time fluorescent quantitative PCR detection kit for tree shrew adenovirus. The TaqMan real-time fluorescent quantitative PCR kit can efficiently and conveniently carry out quantitative detection and quality control of tree shrew-derived adenoviruses in various biological materials, and can also conduct The epidemiological investigation of tree shrew adenovirus can also provide technical support for related basic research, and has a wide application prospect. Background technique [0002] Tree shrew adenovirus (Tree Shrew Adenovirus, TAV) belongs to Adenoviridae, mammalian adenovirus genus. Adenovirus has no ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/6851C12Q1/701C12Q2531/113C12Q2561/101C12Q2545/113
Inventor 孙晓梅宋庆凯李晓飞代解杰罕园园
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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