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Chlamydia trachomatis nucleic acid rapid detection kit

A technology of Chlamydia trachomatis and a kit, which is applied in the field of biochemistry, can solve the problems of false negative results of PCR inhibitor detection, and achieve the effects of ensuring detection specificity, simple and rapid operation, and avoiding misdiagnosis.

Active Publication Date: 2016-08-10
SHANGHAI XINGYAO MED TECH DEV CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, through searching, it is found that the current domestic applications or authorized patents for fluorescent PCR detection of CT (ZL20048005196.8, ZL200610018766.8, ZL200610018768.7, 2007100797288.8, 200710093933.X, 200810236989.0, 64977) do not use internal PCR to monitor False Negative Test Results Caused by Inhibitors

Method used

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  • Chlamydia trachomatis nucleic acid rapid detection kit
  • Chlamydia trachomatis nucleic acid rapid detection kit
  • Chlamydia trachomatis nucleic acid rapid detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1: the preparation of Chlamydia trachomatis nucleic acid rapid detection kit

[0046] The components in the kit are self-prepared, and the preparation process of each component of the 32-person kit is as follows:

[0047] (1) CT PCR buffer 672μl, containing Tris-HCl (pH8.3) 14.2mM, KCl 71.4mM, gelatin 0.14mg / ml, dATP, dGTP, dCTP, dUTP each 0.43mM, MgCl 2 5 mM, 5 μM each of upstream and downstream primers (SEQ ID No: 2 and 3).

[0048] (2) 96 μl of fluorescent probe, containing 1.5 μM each of CT fluorescent probe (SEQ ID No: 4) and internal reference fluorescent probe (SEQ ID No: 5).

[0049] (3) 64 μl of Taq enzyme, containing 40 U of Taq DNA polymerase and 16 U of uracil-DNA glycosylase (UNG enzyme).

[0050] (4) Internal reference DNA 160μl, containing 10 5 copy / ml internal reference DNA (SEQ ID NO: 1).

[0051] (5) Negative control 200μl, containing 10 4 bacteria / ml E. coli culture.

[0052] (6) Positive control 200μl, containing 10 3 inclusion bodi...

Embodiment 2

[0055] Example 2: Application of the kit in the identification of Chlamydia trachomatis in male urethral swab samples

[0056] (1) Sample processing

[0057] Select 7 cases of male urethral swab samples whose two channels were negative for the initial detection of this reagent, and dilute the extract obtained by the sample processing method in the summary of the invention 5 times with DNA extract for amplification detection.

[0058] (2) Amplification detection

[0059] According to the method in the summary of the invention, take the kit components CT PCR buffer 21μl×9, fluorescent probe 3μl×9, Taq enzyme 2μl×9 and put them in a 1.5ml centrifuge tube, mix well and centrifuge briefly, each amplification tube Aliquot 26 μl, and add 4 μl each of the negative control and positive control extracts of the kit treated in the same way, and the above-mentioned 7 sample templates that were diluted. The volume of each reaction amplification tube is 30 μl. Put the above-mentioned ampli...

Embodiment 3

[0062] Example 3: Application of the kit in the identification of Chlamydia trachomatis in female cervical swabs

[0063] (1) Sample processing

[0064] Select 4 cases of female cervical swab samples whose two channels were negative in the initial detection of this reagent, take 200 μl of swab cleaning solution again, use QIAamp DNA mini kit from QIAGEN Company to process according to its instructions, and perform PCR detection.

[0065] (2) Amplification detection

[0066] According to the method in the summary of the invention, take the kit components CT PCR buffer 21μl×6, fluorescent probe 3μl×6, Taq enzyme 2μl×6 and put them in a 1.5ml centrifuge tube, mix them and centrifuge briefly, each amplification tube Aliquot 26 μl, and add 4 μl each of the negative control and positive control extracts of the kit treated according to the same method, and the above-mentioned 4 sample templates processed by the QIAGEN column method extraction kit. The volume of each reaction amplif...

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Abstract

The invention is a kit for rapidly detecting chlamydia trachomatis (CT) and internal reference nucleic acid by using fluorescent PCR technology. It uses a pair of primers to amplify CT-specific nucleic acid sequences, and detects CTDNA through fluorescent probes; at the same time, using the artificially constructed DNA template containing the above primer sequences as an internal reference, through the above primers and other biological sequences that are different from the existing known biological sequences Fluorescently labeled probes detect internal reference DNA. The present invention simultaneously detects the presence of CT and internal reference nucleic acid through single-tube dual-wavelength fluorescent PCR technology, and can judge whether a sample is infected by CT or whether there is a PCR inhibitor. The kit is easy and fast to operate. It can not only avoid false positives caused by the contamination of self-amplified products, but also solve the problem of misdiagnosis caused by false negatives caused by PCR inhibitors. It can be widely used in the rapid screening of CT for high-risk groups of sexually transmitted diseases. Identification.

Description

technical field [0001] The invention belongs to the technical field of biochemistry, and in particular relates to a single-tube dual-wavelength fluorescence PCR rapid detection kit for chlamydia trachomatis (CT) and an internal reference. Background technique [0002] Sexually transmitted diseases (STDs) are widespread infectious diseases in the world today, among which genitourinary tract diseases caused by CT infection are not only the most common sexually transmitted diseases in western countries, but also present an increasing trend year by year in my country. It is one of the main infectious diseases that affect the physical and mental health of our people, especially women and children. Because CT pathogen infection often lacks specific symptoms, it is easy to form occult infection, which brings difficulties to clinical diagnosis; at the same time, Chlamydia is easily mixed with other microbial pathogens during transmission, resulting in the complexity of diagnosis and ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 吴大治夏懿
Owner SHANGHAI XINGYAO MED TECH DEV CO LTD
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