Chlamydia trachomatis nucleic acid detection kit for constant-temperature amplification by using RNA

A technology for Chlamydia trachomatis and detection kits, which is applied to the determination/inspection of microorganisms, microorganism-based methods, microorganisms, etc. high sex effect

Inactive Publication Date: 2009-08-19
SHANGHAI RENDU BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0008] The technical problem to be solved by the present invention is to overcome the shortcomings of prior art specificity, low sensitivity, and difficult control of experimental pollution, and to provide a technology for extracting and purifying target RNA using magnetic bead-RNA enrichment technology and constant temperature nucleic acid synchronous amplification detection technology ( SAT) kit for detection of Chlamydia trachomatis (CT)

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  • Chlamydia trachomatis nucleic acid detection kit for constant-temperature amplification by using RNA
  • Chlamydia trachomatis nucleic acid detection kit for constant-temperature amplification by using RNA

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Embodiment 1

[0039] The extraction of embodiment 1 CT nucleic acid RNA

[0040] The specific steps of nucleic acid extraction are:

[0041] Add 100 μl of nucleic acid extraction solution to the sample processing tube (1.5ml centrifuge tube), add 400 μl of urine sample or swab washing solution, change a tip for each sample, vortex and mix well; keep warm at 60°C for 5 minutes; place at room temperature for 10 minute;

[0042] Place the sample processing tube on the magnetic bead separation device and let it stand for 5 minutes (if some magnetic beads are difficult to adsorb to the tube wall during the magnetic bead adsorption process, the adsorption time should be extended appropriately);

[0043] After the magnetic beads are adsorbed on the tube wall, keep the sample processing tube on the magnetic bead separation device, discard the liquid and keep the magnetic beads;

[0044] Wash twice with washing solution (if there is white flocculent precipitate in the washing solution, heat it at ...

Embodiment 2

[0047] Example 2 SAT nucleic acid amplification detection

[0048] Take 30 μl of the amplification detection solution (including magnetic beads) from the above amplification detection solution after shaking and mixing to a clean micro-reaction tube, incubate at 60°C for 10 minutes, and at 42°C for 5 minutes; at the same time, preheat the SAT enzyme solution to 42°C;

[0049] Add 10 μl of preheated enzyme solution to the micro-reaction tube (do not touch the micro-reaction tube with the sampling tip, and replace the tip if there is any contact), cover the tube after adding enzyme and shake at 1200rpm for 15 seconds to mix well;

[0050] Quickly transfer the micro reaction tube to a suitable constant temperature fluorescence detection instrument, react at 42°C for 40 minutes, set the fluorescence to be detected every 1 minute, and detect 40 times in total;

[0051] After the reaction is over, take out the micro reaction tube and soak it in 10% 84 disinfectant solution. Take the...

Embodiment 3

[0059] The preparation and assembly of each component of embodiment 3 CT kit

[0060] The kits are divided into box A (specimen processing unit) stored at 2-30°C and box B (nucleic acid amplification detection unit) stored at -15-35°C.

[0061] Box A (specimen processing unit) consists of:

[0062] Urine sample preservation solution: 0.1M ammonium sulfate; 0.15M HEPES;

[0063] Nucleic acid extraction solution: 0.15uM capture probe and magnetic particles 250mg / L;

[0064] Washing solution: 0.1% SDS.

[0065] Box B (nucleic acid amplification detection unit) consists of:

[0066] CT reaction solution: 4mM dNTPs and 16mM NTPs;

[0067] SAT enzyme solution: M-MLV2000U; T7RNA polymerase 500U;

[0068] CT detection solution: mainly containing 3.5uM CT-specific primers and 2.7uM specific fluorescent probes;

[0069] CT positive control: mainly containing 10 7 copies / ml CT RNA;

[0070] CT negative control: does not contain any nucleic acid, used to test the effectiveness of ...

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Abstract

The invention relates to a kit for utilizing a magnetic bead-RNA concentrating technology to extract purified target RNA as well as testing chlamydia trachomatis (CT) by using constant temperature nucleic acid simultaneous amplification detection technology (SAT). The kit comprises urine sample preservation solution, nucleic acid extracting solution, cleaning solution, CT reaction solution, CT detection solution, STA enzyme liquid, CT positive control and CT negative control. The kit has high specificity and sensitivity; furthermore, the amplified product RNA is easy for degradation in natural environment with little pollution.

Description

technical field [0001] The invention relates to the technical field of in vitro diagnostic reagents, in particular to a kit for detecting Chlamydia trachomatis (CT) by using magnetic bead-RNA enrichment technology to extract and purify target RNA and constant temperature nucleic acid simultaneous amplification detection technology (SAT). Background technique [0002] Chlamydia trachomatis (Chlamydia trachomatis, CT) is a pathogen that survives in the human body for a long time and spreads widely, with a size of about 250-450nm. The diseases caused by it have a wide range, and can involve the eyes, reproductive tract and other organs, and can cause urethritis, epididymitis in men, cervicitis, pelvic inflammatory disease in women, etc., especially when co-infected with other pathogens such as gonorrhea and other pathogens, the disease is more serious The development and cause other complications, can also lead to mother-to-child transmission. Therefore, the prevention and tre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12R1/01
Inventor 张常娥方亮于明辉王敏居金良
Owner SHANGHAI RENDU BIOTECH
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