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A kit and method for detecting alkaline phosphatase

A phosphatase and kit technology, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve problems that do not involve nucleic acid amplification process, low detection sensitivity, etc., to achieve shortened time, high sensitivity, and reduced cost Effect

Active Publication Date: 2021-10-19
SHANDONG NORMAL UNIV
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Problems solved by technology

However, DNA is also used as an efficient substrate for alkaline phosphatase detection in some assays, but none involve nucleic acid amplification processes
It is worth noting that although DNA polymerases are used in molecular beacons and hairpin probe-based alkaline phosphatase detection, they only catalyze a simple extension reaction to open molecular beacons or hairpin probes, and still do not involve Nucleic acid amplification reaction, with low detection sensitivity

Method used

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  • A kit and method for detecting alkaline phosphatase
  • A kit and method for detecting alkaline phosphatase
  • A kit and method for detecting alkaline phosphatase

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Embodiment 1

[0057] First, the phosphorylation reaction is carried out in a 20 microliter reaction system, including 5 units per liter of basic phosphatase, 100 nano-liter double-stranded DNA substrate (T7 promoter - template double strand) 1 × CUT SMART buffer, incubated for 30 minutes at 37 ° C, and then at 65 ° C for 5 minutes. Then, 1 unit of lambda nucleic acid extracormase (λexo) was added to the above mixture, and then incubated for 30 minutes at 37 ° C, and then incubated at 90 ° C for 5 minutes inactivation.

[0058] Second, the transcriptional reaction is carried out in a 30 microliter reaction solution, including 6 microlitulus λ nucleic acid extracoridase (λexo) digestive product, in the T7Ribomax Express large-scale RNA production system at 37 ° C for 60 minutes.

[0059] Third, 5 microliters of transcriptional RNA and 1 × double-stranded nuclease buffer (50 mmol per liter of pH 8.0 trihydroxymethylmethane - hydrochloric acid (Tris-HCl), 5 mmol Each liter of magnesium chloride, 1 ...

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Abstract

The invention relates to a method for sensitively detecting alkaline phosphatase through dual-signal amplification mediated by dephosphorylation-triggered transcription reaction. A 5'-phosphorylated T7 promoter single-chain is designed, and alkaline phosphatase can catalyze The 5'-phosphorylated T7 promoter is dephosphorylated, protecting the T7 promoter chain from digestion by lambda exonuclease, and the remaining T7 promoter can activate the transcription reaction mediated by T7 RNA polymerase, resulting in a large amount of RNA transcripts . Subsequently, these RNA transcripts are complementary paired with Taqman probes to form RNA-DNA duplexes, and duplex-specific nucleases are introduced to trigger cyclic cleavage of Taqman probes, resulting in significantly enhanced fluorescent signals. The method is simple to operate, has high sensitivity and high specificity, and can be applied to the screening of target inhibitors in complex biological samples and the quantitative detection of targets in cervical cancer cells.

Description

Technical field [0001] The present invention belongs to the field of organic matter detection, and more particularly to a kit and method thereof for detecting an alkaline phosphatase. Background technique [0002] Alkaline phosphatase (Alp) is a hydrolase that is generally present in prokaryotic and eukaryotic biological cells, and the catalyzed phosphate group removes from a polyphosphate substrate containing phosphate. In human body, alkaline phosphatase is widely present in tissue (e.g., bone, kidney, liver, etc.), in a variety of normal cell functions such as cell signal transduction, cell division, differentiation, and skeleton calcification. . On the other hand, the decision of alkaline phosphatase activity is closely related to a variety of human diseases, including low phosphatase disorder, primary biliary cirrhosis, diabetes, and various cancers. Therefore, it is quite valuable to develop reliable alkaline phosphatase activity for the diagnosis of clinical diseases and t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/682C12Q1/42
CPCC12Q1/42C12Q1/682C12Q2563/107
Inventor 张春阳马飞刘文静
Owner SHANDONG NORMAL UNIV
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