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Primer, probe set and reagent kit for double fluorescent quantitative PCR detection of copA and czcD genes

A dual-fluorescent, kit-based technology, applied in the determination/inspection of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of lack of tolerance genes, etc., and achieve high sensitivity, high accuracy, and good amplification efficiency Effect

Pending Publication Date: 2020-11-17
ZHEJIANG UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Soil itself is also an important library of heavy metal tolerance genes. However, traditional quantitative detection methods are mainly based on microbial culture and sequencing, and lack relatively powerful means to determine the number of tolerance genes.

Method used

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  • Primer, probe set and reagent kit for double fluorescent quantitative PCR detection of copA and czcD genes
  • Primer, probe set and reagent kit for double fluorescent quantitative PCR detection of copA and czcD genes
  • Primer, probe set and reagent kit for double fluorescent quantitative PCR detection of copA and czcD genes

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Experimental program
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Effect test

Embodiment

[0043] 1. Preparation of pre-configured reagents and equipment:

[0044] (1) PCR reaction solution system: take the PCR detection buffer solution and melt it on ice or 4℃; then take out the mixed enzyme solution; shake all the components evenly and centrifuge briefly at low speed; each 30μL PCR reaction solution system contains PCR Detection buffer 29.5μL and mixed enzyme solution 0.5μL.

[0045] The PCR reaction solution contains 0.3mM dNTP (dNTP contains dUTP) and 4.0mM Mg 2 SO 4 , 5% trehalose, 3% DMSO, 0.5% betaine, 1% BSA, 0.5nM gold nanoparticles (5nm diameter), 0.6μM upstream primer copA F, 0.6μM downstream primer copA R, 0.3μM upstream Primer czcD F, 0.4μM downstream primer cz cD R, 0.3μM probe czcD P;

[0046] among them:

[0047] Upstream primer copA-F: 5’ATYGAGCTKCATYTGACCGG 3’;

[0048] Downstream primer copA-R: 5’ATYTTCMAGKTCRCTCCACATGCC 3’;

[0049] Upstream primer czcD-F: 5’CGACGCCGCGCACATG 3’;

[0050] Downstream primer czcD-R: 5’TANCCSSCGACGCCGA 3’;

[0051] Probe copA-P...

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Abstract

The invention discloses a primer, probe group and reagent kit for double fluorescent quantitative PCR detection of copA and czcD genes. The primer set comprises an upstream primer for detecting the copA gene, as shown in SEQ ID NO. 1; a downstream primer, as shown in SEQ ID NO. 2; a probe which is shown in SEQ ID NO. 3; an upstream primer for detecting the czcD gene, as shown in SEQ ID NO.4; a downstream primer, as shown in SEQ ID NO. 5; and a probe, as shown in SEQ ID NO. 6. The 5'end of the probe is marked with a fluorophore, and the 3 'end of the probe is marked with a quenching group; andthe reagent kit comprises a PCR reaction solution, a mixed enzyme solution, a positive standard substance, a weak positive quality control substance and a strong positive quality control substance. The reagent kit and the positive standard substance are subjected to synchronous determination through specific primers and probes under the monitoring of PCR reaction negative control, weak positive quality control substances and strong positive quality control substances, and the determined result has the characteristics of high sensitivity and high accuracy.

Description

Technical field [0001] The invention relates to a primer and probe set and a kit for double fluorescent quantitative PCR detection of copA and czcD genes, and belongs to the technical field of biological detection. Background technique [0002] At present, environmental heavy metal pollution has attracted more and more attention. Compared with organic pollution, soil heavy metal pollution lasts for a long time and cannot be degraded by the natural process of the soil. The harm to the environment is more lasting, thereby affecting the composition of soil microorganisms. [0003] Copper and cadmium are two typical heavy metal elements in soil heavy metal pollution in my country, and sometimes they cause joint pollution. Heavy metal pollution will exert selective pressure on soil bacteria, which will affect the composition and quantity of soil bacteria. copA is a gene encoding multiple anti-copper oxidases. It is widely present in copper-resistant bacteria and can be used as a genet...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6851C12N15/11
CPCC12Q1/6851C12Q2531/113C12Q2545/114C12Q2537/143C12Q2561/101C12Q2563/107
Inventor 汪华王群孙达宋亚丽陈勇民薛向东
Owner ZHEJIANG UNIVERSITY OF SCIENCE AND TECHNOLOGY