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Aptamer for diagnosing rupture of fetal membrane in gynaecology and obstetrics and kit thereof

A kit and technology for premature rupture of membranes, applied in disease diagnosis, biological testing, scientific instruments, etc., can solve problems such as detection tools for premature rupture of membranes that have not yet been discovered, and achieve strong application value, fast separation effect, and cost savings Effect

Inactive Publication Date: 2016-07-20
杨岭燕
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although in the third trimester of pregnancy (gestational weeks 28-40), ICAM-1 levels in amniotic fluid of pregnant women increase due to the expression of ICAM-1 on monocytes in the fetal membranes and amniotic fluid and the activation of neutrophils, but currently There are no reports of premature rupture of membranes detection tool using ICAM-1 as the detection index

Method used

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  • Aptamer for diagnosing rupture of fetal membrane in gynaecology and obstetrics and kit thereof
  • Aptamer for diagnosing rupture of fetal membrane in gynaecology and obstetrics and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1: nucleic acid aptamer screening

[0039] From a random oligo DNA library synthesized in vitro, 5'-ACTACGTGCAAGTCACCACA----N36----TGAACATGACGATTACGACC-3'.

[0040] Primers used:

[0041] F: 5, -ACTACGTGCAAGTCACCACA-3

[0042] R: 5, -GGTCGTAATCGTCATGTTCA-3

[0043] The single-stranded DNA library is amplified into double-stranded DNA, and the product is subjected to 2% agarose gel electrophoresis and then gel-cut to recover and purify; the recovered double-stranded DNA is used as a template to transcribe a single-stranded RNA random library in vitro, and the transcript is purified by PAGE . 80 μg of RNA library was reverse-screened with nitrocellulose membrane to remove membrane-bound RNA molecules, and then incubated with 3ugICAM-1 protein at 37°C for 40 min, the reaction solution was filtered through nitrocellulose membrane, and the filter membrane was washed; then the filter membrane was cut into pieces and placed in Boil in elution buffer (6mol / L urea...

Embodiment 2

[0044] Example 2 Obtaining ICAM-1 Binding Gamete with High Specific Affinity

[0045] Take 1.5 μg of RNA aptamers and digest them with calf intestinal alkaline phosphatase (CIP) at 37°C for 1 hour, purify and recover dephosphorylated RNA; label [γ-32P]ATP with T4 polynucleotide kinase for dephosphorylated end of the RNA molecule. 10nmol radioactively labeled RNA aptamers were incubated with different concentrations (1-200nM) of ICAM-1 protein at 37°C for 30min, the reaction solution of each group was filtered through nitrocellulose membrane, the filter membrane was washed, dried, and liquid scintillation counting The radioactivity remaining on the filter membrane was measured by the instrument, and the same sample was measured twice in parallel. The dissociation constants of each aptamer and ICAM-1 protein were calculated. The result is as follows:

[0046]

[0047]

Embodiment 3

[0048] Adapter specificity analysis and stability analysis described in embodiment 3

[0049] It can be seen from the above results that the 23 aptamers of the present invention have very strong binding properties, and there are no aptamers with such binding properties in the prior art that can bind to ICAM-1 protein.

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Abstract

The invention relates to an aptamer capable of being specifically combined with ICAM-1. The aptamer has the high combining characteristic and the high stability. The aptamer is obtained in the mode that the new combinatorial chemistry technology SELEX is applied, ICAM-1 serves as a target, and the DNA aptamer capable of being specifically combined with the ICAM-1 is screened out from a random library. The invention further relates to a kit for identifying the premature rupture of fetal membranes, and the kit can rapidly identify the premature rupture of fetal membranes, and has quite high application value.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an aptamer and a kit for diagnosing fetal membrane rupture in obstetrics and gynecology. Background technique [0002] Rupture of Fetal Membrane (Rupture of Fetal Membrane, tube called ROM) may occur at any time during pregnancy, the rupture of membranes that occurred before labor is called premature rupture of membranes (PROM). The incidence of PROM after term (37 weeks of pregnancy) is about 10%; the incidence of PROM before term (before 37 weeks of pregnancy) is 2-3.5%. Premature rupture of membranes is the main factor causing prenatal and postpartum complications in obstetric patients, and the main reason for premature birth and neonatal admission to the intensive care unit. Because medical personnel have to balance the prolongation of gestational weeks with the risk of intrauterine and maternal infection and fetal lung development problems, the management of patients with prema...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115G01N33/68
CPCC12N15/115C12N2310/16C12N2320/13G01N33/6854G01N33/689G01N2800/36
Inventor 杨岭燕
Owner 杨岭燕
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