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Kit for detecting chlamydia trachomatis and application of kit

A Chlamydia trachomatis and kit technology, applied in the biological field, can solve the problems of cumbersome operation and volume limitation of the reaction system, and achieve the effects of simple and efficient operation, high-throughput and sensitive detection, and low price

Inactive Publication Date: 2018-09-14
NANHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The more test samples, the more times of transfer, the more cumbersome the operation
And the size of the cuvette will also limit the volume of the reaction system

Method used

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  • Kit for detecting chlamydia trachomatis and application of kit
  • Kit for detecting chlamydia trachomatis and application of kit
  • Kit for detecting chlamydia trachomatis and application of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Template Selection and Probe Design

[0047] A specific C. trachomatis conserved sequence was selected as a template. The template was designed by Primer-BLAST with an upstream primer 5'-GTGCGGGACAACATGGAGTA-3' (SEQ ID NO: 3), and a downstream primer 5'-AAGGTCCTAAGGCCGATCCT-3' (SEQ ID NO: 4).

[0048] Design the probe with oligo7.0 according to the template:

[0049] Biotin Capture Probe 5'-Biotin(A)10TGTTACAGAAACGACTTCAGCACC-3'

[0050] Sulfhydryl signaling probe 5'-GCGCATTTTCTCTACATTTGGTTT(A)10-(CH 2 ) 3 -SH-3'

Embodiment 2

[0051] Example 2: Chlamydia trachomatis DNA extraction and target sequence preparation

[0052] Chlamydia trachomatis DNA was extracted according to the instructions of the OMEGA nucleic acid extraction kit, and PCR amplification was performed on the extracted DNA. The total system is 50 μL, 2×Taqmix 25 μL, 10 μmol / L upstream and downstream primers 1 μL each, DNA template 2 μl, ddH 2 O 21 μL. PCR conditions: Pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 45 s, extension at 72°C for 90 s, a total of 35 cycles; extension at 72°C for 10 min. The obtained product was treated with a DNA purification kit, and the nucleic acid concentration was measured by an ultraviolet spectrophotometer for use.

Embodiment 3

[0053] Embodiment 3: gold nano-synthesis and gold nano-probe preparation

[0054] Add 50mL of 0.01% chloroauric acid into the Erlenmeyer flask, turn on the magnetic stirring and heat to boiling. Add 1.3mL of 1% trisodium citrate quickly, and react in the dark until the color does not change, and then continue to react for 5 minutes. Turn off the heat and stir to room temperature. After characterization by ultraviolet-visible absorption spectrum and TEM, 500 μL of gold nanometer solution was centrifuged at 13200 rpm for 30 min. After the supernatant was discarded, 46 μL of phosphate buffer (10 mmol / L PB, pH 7.0) was added to resuspend, and then 4 μL of 100 μmol / L sulfhydryl probe was added. Shake at 80rpm at 37°C for more than 16h in the dark. PBS (0.2mol / L NaCl, 10mmol / L PB, pH 7.0) was slowly added every 16h for three times, so that the final concentration of NaCl in the system was 0.1mol / L NaCl. Centrifuge at 13200rpm for 30min, discard the supernatant, add resuspension ...

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Abstract

The invention relates to the technical field of biology, and discloses a kit for detecting chlamydia trachomatis and an application of the kit. The kit comprises a capture probe, a sulfydryl signal probe, a carrier, a silver staining initiating liquid and silver enhancement liquid, the capture probe is modified by a first modifier, the sulfydryl signal probe is marked by gold nano-particles, and asecond modifier wraps the carrier. According to the kit, specific conserved sequences in the chlamydia trachomatis are selected as templates to design the sulfydryl signal probe marked by the gold nano-particles and the capture probe modified by the first modifier, the sulfydryl signal probe, the capture probe and target sequence specificity are combined to form a sandwich compound, the sandwichcompound can be anchored on the carrier through the second modifier capable of being combined with the first modifier, silver shells are formed on the surfaces of the gold nano-particles under matching of the silver staining initiating liquid and the silver enhancement liquid through catalytic actions of the gold nano-particles, different developing signal differences are achieved, and efficient,special, high-throughput and sensitive detection of the chlamydia trachomatis can be achieved by naked eyes and micro-plate readers.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a kit for detecting chlamydia trachomatis and application thereof. Background technique [0002] Chlamydia trachomatis (Ct) is a kind of prokaryotic microorganisms strictly living intracellular parasitism, which mainly infects urogenital epithelial cells, and is one of the most common pathogens of sexually transmitted diseases at home and abroad in recent years. [0003] Clinical detection of Chlamydia trachomatis infection includes cell culture method, direct immunofluorescence method, polymerase chain reaction method, enzyme-linked immunosorbent assay, colloidal gold immunochromatography and so on. In recent years, gold nano-labeled nucleic acid probe technology has developed rapidly. This method has the characteristics of simplicity, rapidity, low cost, and high throughput, such as the isothermal amplification method mediated by nano-gold particle rings. However, the above method...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/04
CPCC12Q1/689
Inventor 陈丽丽周扑帆柏勤勤陈胜华陈曦唐婷
Owner NANHUA UNIV
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