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Design method for realtime fluorescent quantitative PCR experiment interior label

A technology for real-time fluorescence quantification and method design, which is applied in biochemical equipment and methods, and the determination/inspection of microorganisms. The effect of a negative result

Inactive Publication Date: 2009-07-08
戴立忠
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage is that the reverse transcription efficiency of the internal standard and the target nucleic acid may be different, and it is possible that even for the same target nucleic acid, the reverse transcription efficiency will vary greatly
For high-concentration samples, due to competitive effects, the role of internal standards cannot be reflected

Method used

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  • Design method for realtime fluorescent quantitative PCR experiment interior label
  • Design method for realtime fluorescent quantitative PCR experiment interior label
  • Design method for realtime fluorescent quantitative PCR experiment interior label

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1: Example of real-time fluorescence quantitative PCR detection of low-concentration hepatitis B virus deoxyribonucleic acid (HBV-DNA) in serum

[0020] Reference figure 1 (1) Use Genbank to search for homologous segments. With the aid of primer design software, design two pairs of hepatitis B primers, namely upstream primer P1 and downstream primer P2. The sequence of upstream primer P1 is 5'-GTG TCT GCGGCG TTT TAT C-3', the downstream primer P2 sequence is 5'-ACA AAC GGG CAA CAT ACC T-3', the selected synthetic gene is an internal standard gene, and its base sequence is 5'-ACGGGAGCGGTTGGTGGTGGAAATCGTGCGTGACATTAAGA-3', which The primer sequence is the same as the target gene (same as P1, P2); (2) Design the target gene probe, the probe sequence is: 5'FAM-CAT CCTGCT GCT ATG CCT CAT CTT CTT-3', 5'end labeled FAM fluorescent report Set the probe concentration to 15pM; (3) Design two probes for the internal standard gene, the probe concentration for the internal stand...

Embodiment 2

[0022] Example 2: Example of real-time fluorescent quantitative PCR detection of high concentration of HBV-DNA in serum

[0023] The design of the target gene, internal standard, and primer probe is the same as in Example 1. The high-concentration serum sample (containing the HBV-DNA target gene, the concentration is higher than 500IU / ml, gradient dilution) and 0.3ul 10 5 IU / ml internal standard (including internal standard gene) was added to 100ul sample extract, probes for target gene and internal standard gene (concentrations of 15pM and 10pM respectively) were added to 50ul PCR reaction solution, internal standard and sample Synchronous processing, through nucleic acid extraction and purification steps to obtain high-purity nucleic acid, and perform absolute quantitative experiments on a fluorescent quantitative PCR machine (ABI7300, produced by Applied Biosystems, Inc.).

[0024] The same sample adopts the non-competitive internal standard method currently used in the market ...

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Abstract

A design method of real-time fluorescence quantitative PCR internal standard includes the following steps: (1) designing a primer sequence according to the activating genes to be tested, select the corresponding internal standard genes, whose primer sequence is same to that of the activating genes; (2) designing the probe of the activating genes, determining the concentration, and labeling a fluorescent reporting group at the 5' end of the probe; (3) designing two probes of the internal standard genes, one probe corresponding to the internal standard genes, the other probe corresponding to another sequence of the activating genes, and respectively labeling a same fluorescent reporting group at the 5' end of the two probes; (4) extracting and purifying the activating nucleic acid, and performing real-time fluorescence quantitative PCR amplification. The present invention can effectively monitor errors occurring in the nucleic acid extraction, amplification and product analysis processes, thus avoiding false-negative results.

Description

Technical field [0001] The invention relates to a method for designing an internal standard in a real-time fluorescent quantitative PCR experiment. Background technique [0002] At present, the relative or absolute content of the target DNA or gene of a specimen can be detected by real-time fluorescent quantitative PCR. According to whether an internal standard substance is added to the specimen, it can be divided into external standard quantitative PCR and internal standard quantitative PCR. Due to the differences in the amplification efficiency of different specimens in the external standard method and the small changes in specimen extraction (especially RT-PCR) will lead to huge differences in the amplified products, in order to eliminate this effect, the internal standard method is highly recommended at home and abroad. . Adding competitive or non-competitive internal standards to reagents can effectively monitor errors in nucleic acid extraction, amplification and product an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 戴立忠蔡鸣镝熊晓燕
Owner 戴立忠
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