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Primer set and probe for apple Actin gene real-time fluorescent quantitative PCR (polymerase chain reaction) detection and detection method by using same

A real-time fluorescent quantitative and detection method technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problem that primers cannot produce specific amplification and primer dimers, and cannot use multiple Target multiple detection, specificity (poor specificity, etc.), to achieve the effect of high sensitivity, high detection specificity, and strong specificity

Inactive Publication Date: 2015-12-16
HEBEI NORMAL UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] (1) The specificity (specificity) of detection is poor, and non-specific amplification will also generate fluorescent signals;
[0014] (2) The requirements for the designed primers are high, and the designed primers are required not to produce specific amplification and primer dimers;
[0015] (3) It can only be used for single detection, and cannot be used for multiple detection of multiple targets in the same reaction

Method used

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  • Primer set and probe for apple Actin gene real-time fluorescent quantitative PCR (polymerase chain reaction) detection and detection method by using same
  • Primer set and probe for apple Actin gene real-time fluorescent quantitative PCR (polymerase chain reaction) detection and detection method by using same
  • Primer set and probe for apple Actin gene real-time fluorescent quantitative PCR (polymerase chain reaction) detection and detection method by using same

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] 1. Design and synthesis of primers and probes

[0072] Log on to the Genbank website, search for the published apple Actin gene sequence (AB6368619.1), and use Primer5.0 to design primers Act-sta-F1 and Act-sta-R1 that can amplify the full-length sequence of the Actin gene (Table 1).

[0073] Then according to the sequencing results of the amplified Actin gene, use the software ClustalX (1.81) to compare it with the Actin gene sequences of other plants, find a relatively conserved sequence as the detection target region, and use PrimerExpressv3.0 and Primer5.0 software TaqMan probe Act-Probe and specific amplification primers (Table 1) were designed. The 5' end of the probe was labeled with the fluorescent group FAM, and the 3' end was labeled with the quencher group BHQ1, which were synthesized by Dalian Bao Biological Engineering Co., Ltd.

[0074] Table 1 Primer and probe sequences

[0075]

[0076]

[0077] 2. Extraction of total RNA and synthesis of cDNA

...

Embodiment 2

[0154] Make standard curve

[0155] The concentration of in vitro transcribed cRNA was measured by micro-ultraviolet spectrophotometer to be 9.4ng·μl -1 , according to the formula: copy number concentration (copies·μl -1 ) = mass concentration (ng·μl -1 )×10 -9 ×6.02×10 23 / (RNA base number×330), the mass concentration was converted to the copy number concentration (Tao Zhenzhen et al., 2015). The calculated cRNA copy number concentration was 1.48×10 10 copies μl -1 .

[0156] Use EASYDilution to dilute the standard cRNA by 10 times to 1.48×10 8 ...1.48×10 2 copies μl -1 Eight gradient concentrations are used as templates for real-time fluorescent quantitative RT-PCR reactions.

[0157] (1) The RT system is:

[0158]

[0159] The reaction program was: incubate at 42°C for 30 minutes, inactivate at 85°C for 5 seconds, and store at 4°C.

[0160] (2) The real-time fluorescent quantitative PCR system is:

[0161]

[0162] The reaction program was: pre-denaturati...

Embodiment 3

[0166] 1. Comparison of sensitivity between real-time fluorescent quantitative PCR and conventional PCR

[0167] After diluting the standard cRNA by 10 times, real-time fluorescent quantitative RT-PCR detection and conventional RT-PCR detection were carried out respectively. The results are as follows: Figure 7 and Figure 8 shown. Figure 7 and Figure 8 Among them, 1~8: the concentration diluted by 10 times ratio is 1.48×10 8 ...1.48×10 1 copies μl -1 cRNA; 9: negative control. Figure 7 showed that real-time fluorescent quantitative RT-PCR can detect 1.48×10 1 copies μl -1 cRNA, Figure 8 showed that conventional RT-PCR could only detect 1.31×10 4 copies μl -1 cRNA, indicating that real-time fluorescent quantitative RT-PCR is 1000 times more sensitive than conventional RT-PCR.

[0168] 2. Reproducibility of real-time fluorescent quantitative PCR

[0169] Statistical analysis was performed on the Ct values ​​of different concentrations of cRNA in the same test a...

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Abstract

The invention relates to the field of molecular detection, particularly a primer set and probe for apple Actin gene real-time fluorescent quantitative PCR (polymerase chain reaction) detection and a detection method by using the same. The apple Actin gene real-time fluorescent quantitative PCR detection method provided by the invention comprises the following step: carrying out real-time fluorescent quantitative PCR detection on a sample to be detected by using Act-Probe-F and Act-Probe-R as primers and Act-Probe as a probe. The apple Actin gene real-time fluorescent quantitative PCR detection method provided by the invention has the advantages of high specificity, favorable repetitiveness and high sensitivity; the lower detection limit is 1.48*10<1>copies.mul<-1>; and the sensitivity is 1000 times of that of the conventional RT-PCR (reverse transcription-polymerase chain reaction) technique. The invention lays the foundation for quantitative analysis on expression of apple functional genes and pathogenetic genes by using the real-time fluorescent quantitative PCR method.

Description

technical field [0001] The invention relates to the field of molecular detection, in particular to a primer set, a probe and a detection method for real-time fluorescent quantitative PCR detection of apple Actin gene. Background technique [0002] In recent years, quantitative analysis of mRNA transcript level, as an important aspect of understanding gene function, has become a hot spot in the field of gene research. Real-time fluorescent quantitative RT-PCR (Real-timeRT-PCR) is a new technology developed in recent years. It can not only detect the qualitative detection of the target gene, but also accurately quantify its expression. It has been widely used in gene in the field of research. [0003] However, in actual research, the quantitative analysis of the target gene is mostly carried out in different tissues, so the experimental conditions need to be as consistent as possible. However, due to the possible differences in the yield, quality and reverse transcription eff...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6851C12Q2545/113C12Q2561/101C12Q2561/113
Inventor 秦子禹王娜项殿芳
Owner HEBEI NORMAL UNIVERSITY OF SCIENCE AND TECHNOLOGY
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