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Chlamydia trachomatis genetic tag, and detecting method and kit

A kind of chlamydia trachomatis, genetic marker technology, applied in the field of molecular biology

Inactive Publication Date: 2006-10-25
SHANGHAI XINGYAO MED TECH DEV CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with PCR, due to the use of two pairs of primers, the use of LCR to detect Ct infection has higher sensitivity and specificity, and it is less used in China

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Isolation of Chlamydia trachomatis DNA for LCR analysis

[0056]In order to isolate the Chlamydia trachomatis DNA for ligase chain reaction detection from the sample to be tested, a simple method that does not affect the ligase chain reaction must be used. First, take 1ml clinical sample (urine) in a 1.5ml centrifuge tube, centrifuge at 15,000rpm for 15 minutes, discard the supernatant, so that the Chlamydia trachomatis that may be contained in the urine is collected. Add 50ul of DNA lysis solution to the precipitate, pipette evenly, and then boil in a boiling water bath at 100°C for 15 minutes to lyse Chlamydia trachomatis to release DNA. Finally, it was centrifuged at 15,000 rpm for 10 minutes, and the DNA was dissolved in the supernatant, and the supernatant was taken as the Chlamydia trachomatis DNA for LCR analysis.

Embodiment 2

[0057] Example 2 Preparation of detection kit

[0058] A conventional method is used to prepare an LCR kit for rapid detection of Chlamydia trachomatis. The kit includes a) a DNA lysis solution, b) a fluorescent LCR reaction solution, c) a positive control substance, and d) a negative control substance. among them:

[0059] 1) The fluorescent LCR reaction solution contains LCR buffer, NAD solution, BSA, sterile double distilled water, EDTA solution, sodium azide, and two pairs of primer pairs that specifically amplify the genetic markers of the Chlamydia trachomatis MOMP gene (such as A, B The primer sequence is SEQ ID NO: 3 ~ SEQ ID NO: 4, A'and B'primer sequence is SEQ ID NO: 5, SEQ ID NO: 6);

[0060] Specifically, the fluorescent LCR reaction solution contains LCR buffer (50mM EPPS, 20mM KCl, 30mM MgCl 2 , 10uM NAD), 1.7uM dATP, each of the four primers 8×10 11 Each molecule, 5ug / ml BSA, 0.5mM EDTA, 0.02% sodium azide, 18000U Thermus thermophilus DNA ligase (Abbott Laboratorie...

Embodiment 3

[0064] Example 3 Ligase chain reaction with various serotypes of Chlamydia trachomatis

[0065] Take 45 μL each of the fluorescence reaction solution in Example 2 and add them to different reaction tubes, and add 5 μL each of the DNA and the positive control substance obtained in Example 1 to the fluorescence LCR reaction solution for a total volume of 50 μL. Start amplification detection on a real-time fluorescent quantitative PCR machine (ABI, PE7000). The amplification parameters were set to a cycle of denaturation temperature of 95°C for 30 seconds and annealing connection temperature of 63°C for 40 seconds, and 40 cycles of LCR amplification were performed. Set the fluorescence detection program at the end of the annealing step of each cycle, and the detection wavelength is 640nm.

[0066] Sample to be tested (Ct serotype)

Fluorescence value

Qualitative judgment

A

1.41

+

B

0.89

+

Ba

1.12

+

C

...

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PUM

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Abstract

The invention supplies a genetic marking material for testing chlamydi trachomatis and the LCR method and the agent box for testing chlamydi trachomatis using the genetic marking material. It could rapidly, expediently and accurately test the chlamydi trachomatis.

Description

Technical field [0001] The invention relates to the field of molecular biology, in particular to ligase chain reaction. Background technique [0002] Chlamydia (Chalmydiae) is a type of microorganism that specializes in parasitic life in eukaryotic cells. Its growth and reproduction in host cells will cause clinical signs of the host. The genus Chlamydia is divided into three species: Chlamydia trachomatis (Ct below C.trachomatis), Chlamydia psittaci (C.psittaci) and Chlamydia pneumoniae (C.pneumonia). Among them, Chlamydia trachomatis and Chlamydia pneumoniae are pathogens that can cause human diseases, such as trachoma, male non-gonococcal urethritis, female chlamydial cervicitis, and pneumonia. [0003] Various methods have been established for clinical detection of Chlamydia trachomatis. The cell culture method is used to detect Chlamydia trachomatis. A single layer of eukaryotic cells is used. After incubation, it is stained with fluorescently labeled antibodies, enzyme-conj...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53G01N33/563G01N33/58G01N33/68
Inventor 李超夏懿
Owner SHANGHAI XINGYAO MED TECH DEV CO LTD
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