A method for isothermal amplification of nucleic acid segments by using PCR primers

A nucleic acid fragment and isothermal amplification technology, which is applied in biochemical equipment and methods, microbial determination/inspection, DNA preparation, etc. The effect of low false positive rate and easy promotion

Inactive Publication Date: 2013-10-02
王德国
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] However, the loop-mediated isothermal amplification technique needs to design primers for 6-8 regions of the nucleic acid fragment, wherein at least 6 regions of the nucleic acid fragment (F3 C 、F2 C , F1, B1, B2 C and B3 C ) and 2 pairs of primers (essentially 3 pairs of primers: F3, F1 C -F2, B1 C -B2, B3) can onl

Method used

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  • A method for isothermal amplification of nucleic acid segments by using PCR primers
  • A method for isothermal amplification of nucleic acid segments by using PCR primers
  • A method for isothermal amplification of nucleic acid segments by using PCR primers

Examples

Experimental program
Comparison scheme
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Example Embodiment

[0024] Example 1:

[0025] Staphylococcus aureus specific gene femA Isothermal amplification test

[0026] Primer design

[0027] Select the gene fragments of Staphylococcus aureus by consulting literature and analyzing with BLAST software femA , Design isothermal amplification primers for the two regions of the fragment and synthesize them to obtain the following primers; primer design is completed by PrimerPremier5.0 primer design software.

[0028] Upstream primer F 5’- AAA AAA GCA CAT AAC AAG CG-3’

[0029] Downstream primer R 5’- GAT AAA GAA GAA ACC AGC AG -3’

[0030] reaction system (The total reaction volume is 25μl)

[0031] Amplification reaction

[0032] Real-time PCR instrument, ESEQuant Tube Scanner or other constant temperature amplification fluorescence detector 59-65 ℃ for 60 minutes; or 59-65 ℃ water bath for 20-60 minutes.

[0033] Reaction product detection

[0034] A) Real-time fluorescence detection: The supporting software of the gene amplification fluorescence d...

Example Embodiment

[0037] Example 2:

[0038] Isothermal Amplification Detection of Noda Virus Specific Gene RNA-2 in Macrobrachium rosenbergii

[0039] The specific gene fragment RNA-2 of Macrobrachium rosenbergii Noda virus was selected by consulting the literature and analyzing with BLAST software, and isothermal amplification primers were designed and synthesized for the two regions of the fragment, and the following primers were obtained; the primer design was through PrimerPremier5.0 The primer design software is completed.

[0040] Upstream primer F 5’- CCAATATGAACCGGGAGTG-3’

[0041] Downstream primer R 5’- GGGTTCAACCTTGAGTTCC-3’

[0042] reaction system (The total reaction volume is 25μl)

[0043] Amplification reaction

[0044] Real-time PCR instrument, ESEQuant Tube Scanner or other constant temperature amplification fluorescence detector 59-65 ℃ for 60 minutes; or 59-65 ℃ water bath for 20-60 minutes.

[0045] Reaction product detection

[0046] A) Real-time fluorescence detection: the suppo...

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Abstract

The invention discloses a method for isothermal amplification of nucleic acid segments. A pair of PCR primers are complementary with two areas of the nucleic acid segments, and the isothermal amplification of the nucleic acid segments is performed through a chain substitute reaction under the action of DNA polymerase. The method can be used as a rapid amplification and detection method of a specific gene segment.

Description

technical field [0001] The invention relates to a method for isothermally amplifying nucleic acid fragments, in particular to amplifying nucleic acid fragments through a strand substitution reaction using a pair of PCR primers complementary to two regions of a target sequence. The method can be used as a rapid amplification detection method for specific gene fragments . Background technique [0002] With the development of modern molecular biology and molecular technology, many nucleic acid amplification techniques have been researched and developed. Among them, the nucleic acid loop-mediated isothermal amplification technology (patents ZL 00818262.0, ZL01810370.7, ZL01812204.3, ZL02815992.6, ZL200610080379.7), due to its strong specificity, high sensitivity, and fast reaction speed, is widely used in nucleic acid specific The field of sexual amplification and detection has a tendency to replace PCR, and has become one of the research hotspots in the field of life sciences....

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/68
Inventor 王德国
Owner 王德国
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