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Chlamydia trachomatis detection colloidal gold chromatography kit and application thereof

A technology for Chlamydia trachomatis and a kit, which is applied to the determination/inspection of microorganisms, biochemical equipment and methods, microorganisms, etc., can solve problems such as being unsuitable for processing a large number of samples, time-consuming, and low sensitivity.

Active Publication Date: 2020-03-27
武汉中帜生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In the experimental diagnosis of genitourinary tract CT infection, although the classic cell culture method is specific, it is time-consuming, costly, requires high technical equipment, is not suitable for processing a large number of samples, and has low sensitivity; the application of immunological methods for Chlamydia trachomatis antigen or Antibody detection has the advantages of simple operation and strong specificity, but the disadvantage is low sensitivity, and there is a window period for antibody detection, which is difficult to detect in the early stage of Chlamydia trachomatis infection; PCR method can directly detect CT nucleic acid, with high sensitivity and strong specificity , the detection speed is also fast, and it has considerable advantages in shortening the detection window period and improving the detection rate of pathogens. It is one of the main methods for CT pathogen detection, but the amplification product contamination of the PCR method leads to false positives, and has adverse effects on hardware facilities. Certain requirements, there must be a dedicated PCR diagnostic laboratory and expensive experimental equipment, which is not conducive to the popularization and application in some communities and remote hospitals

Method used

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  • Chlamydia trachomatis detection colloidal gold chromatography kit and application thereof
  • Chlamydia trachomatis detection colloidal gold chromatography kit and application thereof
  • Chlamydia trachomatis detection colloidal gold chromatography kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] [Example 1] Preparation of nucleic acid detection test strips

[0086] The main raw materials required for the preparation of nucleic acid detection test strips: nitrocellulose membrane (NC membrane), sample pad, absorbent paper, PVC bottom plate, etc.

[0087] 1. Spray film:

[0088] Detection line CT-T: Capable of capturing and binding CT-specific probe CES sequence, 10μM CT-coated probe, spray film volume: 2-3μL / cm;

[0089] Detection line internal reference-T: capable of capturing and binding internal reference-specific probe CES sequence, 10 μM internal reference coated probe, spray film volume: 2-3 μL / cm;

[0090] Quality control line C-line: capable of capturing and combining C-line chromogenic probe sequence, 10μM C-line coated probe, spray film volume: 2-3μL / cm;

[0091] After spraying the film, it will be automatically cross-linked once in the ultraviolet cross-linking instrument, put it in a clean incubator at 37°C for 2 hours, and store it in a dry environme...

Embodiment 2

[0094] [Example 2] Sensitivity Test

[0095] The minimum detection limit of Chlamydia trachomatis derived from ATCC VR-346 is serially diluted, and the dilution of each gradient is repeated 3 to 5 times, and each copy is repeated 20 times, and 90% to 95% of positive detections will be obtained. The level of the rate is used as the minimum detection limit, and the detection results are as follows:

[0096] CT minimum detection limit detection

[0097] Table 1.1 Detection experimental data of CT with different titers

[0098]

[0099] Table 1.2 Experimental data of the lowest detection limit of CT

[0100]

[0101]

[0102] In summary, the sensitivity of the kit of the present invention for detecting CT is 5.0 IFU / mL.

Embodiment 3

[0103] [Example 3] Specificity Verification

[0104] 1. Test Strains

[0105] After extracting nucleic acids from different microorganisms, they are tested to verify the specificity of the design of the primers and probes of the kit of the present invention. Relevant pathogens and titer information are as follows:

[0106] Table 2 Specificity verification test strain information

[0107]

[0108] 2. Specificity Test

[0109] After extracting nucleic acids from different microorganisms, they are tested to verify the specificity of the design of the primers and probes of the kit of the present invention. Relevant pathogens and titers are as follows:

[0110] Table 3 Specificity verification test results

[0111]

[0112]

[0113] 3. Conclusion

[0114] As can be seen from the above data, the detection results of the kit of the present invention to these microorganisms are all negative, proving that there is no cross-reaction between the kit of the present inventio...

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Abstract

The invention discloses a chlamydia trachomatis detection colloidal gold chromatography kit and application thereof. According to the kit, a collected sample is subjected to pyrolysis by using a celllysis buffer to release pathogen nucleic acid, subsequently, under the actions of reverse transcriptase and T7RNA (T7 ribonucleic acid) polymerase, reverse transcription and transcription process areimplemented, and thus amplification of a pathogen nucleic acid fragment is achieved. An RNA product after amplification is recognized and captured by a specific probe in a detection liquid, then an RNA amplification product---amplification product---specific probe---golden probe composition is formed, the composition is fixed on an NC (nitrocellulose filter) membrane through lateral flow chromatography to form visible strips, and thus detection on nucleic acid in pathogen can be achieved. The kit is free of NRA extraction process, needs no special instrument, is based on RNA constant-temperature amplification, is not easy to contaminate in actual detection, has the advantages of being high in sensitivity, good in specificity and simple to operate, and makes wide application of chlamydia trachomatis nucleic acid detection possible.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a kit for detecting chlamydia trachomatis nucleic acid based on RNA constant temperature amplification-gold probe chromatography technology and an application thereof. Background technique [0002] Chlamydia trachomatis (CT) is a type of parasitic microorganisms in cells, with a size of about 250-450nm, Gram-negative, round or oval; it contains DNA, RNA and ribosomes; it has a cell wall, and its structure and composition are similar in Gram-negative bacteria. Chlamydia trachomatis is serotyped based on monoclonal or polyclonal antibodies against the outer membrane protein, which divides Chlamydia trachomatis into 12 serotypes: A-K. Among them, A, B / Ba and C types mainly cause trachoma; D-K types mainly cause urogenital tract infection. [0003] Chlamydia trachomatis (CT) can cause nongonococcal urethritis and pelvic inflammatory disease. Chlamydia trachomatis infe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6813C12Q1/04C12N15/11C12R1/01
CPCC12Q1/689C12Q1/6813C12Q2600/166C12Q2521/107C12Q2521/119C12Q2521/327C12Q2527/101C12Q2563/137C12Q2565/625Y02A50/30
Inventor 李先强姜昕陈巨王琳琳薛金辉
Owner 武汉中帜生物科技股份有限公司
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