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Kit for detecting alkaline phosphatase and method thereof

A phosphatase and kit technology, applied in biochemical equipment and methods, microorganism determination/inspection, etc., can solve problems such as no nucleic acid amplification process, low detection sensitivity, etc., to avoid complex design and synthesis, and shorten time. , the effect of easy operation

Active Publication Date: 2018-09-28
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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  • Kit for detecting alkaline phosphatase and method thereof
  • Kit for detecting alkaline phosphatase and method thereof
  • Kit for detecting alkaline phosphatase and method thereof

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Embodiment 1

[0057] First, the dephosphorylation reaction was performed in a 20 µl reaction containing 5 units per liter of alkaline phosphatase and 100 nmoles per liter of double-stranded DNA substrate (T7 promoter-template duplex) , 1×Cut Smart buffer, incubate at 37°C for 30 minutes, then inactivate at 65°C for 5 minutes. Then, 1 unit of lambda exonuclease (λexo) was added to the above mixture, incubated at 37°C for another 30 minutes, and then incubated at 90°C for 5 minutes to inactivate lambda exonuclease (λexo).

[0058] Second, the transcription reaction was carried out in 30 μl of reaction solution, including 6 μl of lambda exonuclease (λexo) digestion product, and reacted at 37°C for 60 minutes in a T7RiboMAX Express large-scale RNA production system.

[0059] Third, mix 5 μl of transcript RNA with 1× double-strand-specific nuclease buffer (50 mmol per liter of Tris-HCl, pH 8.0, 5 mmol Magnesium chloride per liter, 1 mmol per liter of dithiothreitol, 0.1 unit of double-strand sp...

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Abstract

The invention relates to a method for sensitively detecting alkaline phosphatase by double-signal amplification mediated by a transcription reaction initiated by dephosphorylation. The method comprises the steps that a 5'-phosphorylated T7 promoter single chain is designed, alkaline phosphatase can catalyze 5'-phosphorylated T7 promoter to be dephosphorylated to protect the T7 promoter chain frombeing digested by Lambada exonuclease, the remaining T7 promoter can activate transcription reaction mediated by T7 RNA polymerase, and thus a large number of RNA transcripts are produced; and subsequently, the RNA transcripts complement and pair with Taqman probes to form a RNA-DNA duplex, duplex-specific nucleases is introduced to initiate circular cutting of the Taqman probes, and a significantly enhanced fluorescence signal is produced. The method is simple and convenient 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 invention belongs to the technical field of organic matter detection, and in particular relates to a kit for detecting alkaline phosphatase and a method thereof. Background technique [0002] Alkaline phosphatase (ALP) is a ubiquitous hydrolase in prokaryotic and eukaryotic cells that catalyzes the removal of phosphate groups from phosphate-containing polyphosphate substrates. In the human body, alkaline phosphatase is widely present in tissues (such as bone, kidney, liver, etc.), and plays an important role in various normal cell functions such as cell signal transduction, cell division, differentiation, and bone calcification . On the other hand, the downregulation of alkaline phosphatase activity is closely related to a variety of human diseases, including hypophosphatasia, primary biliary cirrhosis, diabetes, and various cancers. Therefore, the development of a reliable detection method for alkaline phosphatase activity is quite valuable for ...

Claims

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Application Information

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Patent Type & Authority Applications(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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