Specific gene segment suitable for real-time fluorescent quantitative PCR detection of pseudomonas PPZ-1 as well as primers thereof and applications

A real-time fluorescence quantification, Pseudomonas technology for DNA/RNA fragments, microbe-based methods, microbial assay/inspection, etc.

Active Publication Date: 2017-12-05
ECOLOGY INST SHANDONG ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, at present, quantitative methods for microorganisms in organically polluted soil and other environmental samples based on real-time fluorescent quantitative PCR technology mostly use 16S / 18SrDNA or functional genes as the target genes, and most of the research focuses on certain types or types of indigenous degrading bacteria. Concerns about the quantity distribution of specific exogenous degrading bacteria after entering the environment

Method used

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  • Specific gene segment suitable for real-time fluorescent quantitative PCR detection of pseudomonas PPZ-1 as well as primers thereof and applications
  • Specific gene segment suitable for real-time fluorescent quantitative PCR detection of pseudomonas PPZ-1 as well as primers thereof and applications
  • Specific gene segment suitable for real-time fluorescent quantitative PCR detection of pseudomonas PPZ-1 as well as primers thereof and applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Screening and primer design of embodiment 1 specific gene sequence

[0034] According to the genome sequencing results of the strain PPZ-1, the coding genes were compared in major general functional databases, and the unannotated gene sequences were screened. These sequences are the possible specific gene sequences of the strain; the sequences are respectively shown as SEQ ID Shown in NO.4, SEQ ID NO.1, SEQ ID NO.5 and SEQ ID NO.6.

[0035] Primers were designed respectively for the above gene sequences, and the primer of SEQ ID NO.4 was recorded as primer set 1, and the nucleotide sequence was as follows:

[0036] F: TCTCCGCCGCCCAGATAA; R: TGGATGTCCCGTTGAAGC

[0037] The primer of SEQ ID NO.1 is recorded as primer set 2, and the nucleotide sequence is as follows:

[0038] F: GCGGGAGGTGTTGCTGTT; R: CCTATTGCCCTTGCGTTC

[0039] The primer of SEQ ID NO.5 is recorded as primer set 3, and the nucleotide sequence is as follows:

[0040] F: CGGGTTATGGGCAGCAGA; R: GGTTGGCAA...

Embodiment 2

[0044] The above primers were used to amplify Pseudomonas (Psedomonassp.) PPZ-1 and two kinds of polycyclic aromatic hydrocarbon contaminated soil sample DNA by PCR, and the amplified products were observed by agarose gel electrophoresis.

[0045] Genomic DNA was extracted using a soil genomic DNA extraction kit to prepare template DNA.

[0046] The PCR amplification system is as follows, the total system is 25 μl:

[0047] Template DNA 0.5μl; Primer F (10μM) 0.5μl; Primer R (10μM) 0.5μl; dNTP (10mM) 0.5μl; TaqBuffer (10×) 2.5μl; MgCl 2 (25mM) 2μl; Taq enzyme (5U / μl) 0.2μl; H 2 O 18.3 μl;

[0048] The PCR reaction conditions were: pre-denaturation at 95°C for 3 min; denaturation at 94°C for 30 s, annealing at 57°C for 30 s, extension at 72°C for 30 s, 35 cycles; repair extension at 72°C for 8 min.

[0049] Electrophoresis conditions are: 1.5wt% agarose gel, 1×TAE, 150V, 100mA, 20min.

[0050] The result is as figure 1 As shown, 1, 2, 3, and 4 correspond to the above prime...

Embodiment 3

[0052] Example 3 Cloning and sequencing

[0053] Cut off the target band with a scalpel, recover it with the kit, connect it with the T carrier, transform it into Escherichia coli competent cells, spread it on an ampicillin-resistant plate, and culture it at 37°C for 24 hours. The clone was cultured in liquid LB medium for 24 hours, and the plasmid was extracted; PCR amplification was performed using the plasmid as a template to obtain a positive target plasmid. After the plasmid was sequenced and identified correctly, the OD of the plasmid was measured with an ultraviolet spectrophotometer 260 The value of is converted into copy number by formula.

[0054] The specific calculation is as follows:

[0055] (1): Plasmid concentration conversion formula (copies / μl)=(mol number / μl)×6.02×10 23 =[mass (g) / molecular weight] / μl×6.02×10 23 = [mass (ng) x 10 -9 / molecular weight] / μl×6.02×10 23 =Concentration (ng / μl)×6.02×10 14 / molecular weight

[0056] (2): Molecular weight = (...

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Abstract

The invention relates to a specific gene segment suitable for real-time fluorescent quantitative PCR detection of pseudomonas PPZ-1 as well as primers of the specific gene segment and applications of the primers. For the specific gene segment suitable for real-time fluorescent quantitative PCR detection of pseudomonas PPZ-1, the nucleotide sequence is as shown in SEQ ID NO.1; the invention further relates to detection primers designed by utilizing the specific gene segment and applications of the detection primers. According to the technical scheme provided by the invention, the distribution of pseudomonas PPZ-1 can be tracked in real time, and thus the demand of biological repair monitoring is satisfied; according to actual detection, background noises can not be detected in a polluted sample in which pseudomonas PPZ-1 is not fed.

Description

technical field [0001] The invention relates to a specific gene fragment suitable for real-time fluorescent quantitative PCR detection of Pseudomonas PPZ-1 and its primers and application, in particular to the quantification of exogenous degradation bacteria Pseudomonas PPZ-1 in polycyclic aromatic hydrocarbon-contaminated soil and environment The invention relates to gene fragments for specific detection, primers and applications thereof, belonging to the technical field of soil microorganism detection. Background technique [0002] Polycyclic aromatic hydrocarbons (PAHs) are a typical class of persistent organic pollutants that widely exist in the environment. The scope of pollution is large, involving a wide range of areas, and it is showing a tendency of aggravation. It is imminent to control the pollution of polycyclic aromatic hydrocarbons. Scholars at home and abroad have carried out a lot of research on PAH pollution remediation technology, mainly including physical,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/38
CPCC12Q1/6851C12Q1/689C12Q2531/113C12Q2563/107C12Q2545/114
Inventor 张闻黄玉杰王加宁高永超郭书海陈贯虹郑立稳王磊磊季蕾
Owner ECOLOGY INST SHANDONG ACAD OF SCI
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