Loop-mediated isothermal amplification method based on taqman probe and its special lamp primer and kit
A loop-mediated constant temperature and probe technology, applied in the field of molecular biology detection of nucleic acids, can solve the problems of inability to amplify products specific detection, false positive detection results, non-specificity, etc. Simple operation and efficient amplification effect
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Embodiment 1
[0101] Embodiment 1, detect NDM-1 gene with the LAMP method based on Taqman probe
[0102] Taking the NDM-1 gene as an example, the LAMP method based on the Taqman probe of the present invention is described in detail. The specific detection method includes the following steps:
[0103] 1. Primer design for the detection of NDM-1 gene based on Taqman probe for LAMP
[0104] The NDM-1 gene sequence (GenBank number: FN396876.1) was retrieved from the American Gene Database, and the homology analysis was performed by BLAST software to obtain the specific conserved target sequence of the NDM-1 gene (sequence 1 in the sequence table), and then according to The conserved target DNA sequence was designed using the software Primer Explorer V4 (http: / / primerexplorer.jp / e / ) to design primers for the detection of LAMP based on Taqman probes for the NDM-1 gene. The primer sequences are as follows:
[0105] Forward outer primer CJXJ1F3: 5'-GCATAAGTCGCAATCCCCG-3' (designed for the specific...
Embodiment 3
[0128] Embodiment 3, the LAMP detection kit based on Taqman probe
[0129] The reaction solution with the following substance concentrations: 20mM Tris·HCl (pH8.8), 10mM KCl, 10mM (NH 4 ) 2 SO 4 , 0.1% Tween20, 0.8M betaine (betaine), 8mM MgSO 4 L, 1.4mM dNTP each, 8U BstDNA polymerase, and LAMP amplification system (double distilled water) without DNA as a negative control were packaged together to obtain a kit for LAMP detection based on Taqman probes.
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