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Rapid fluorescence PCR detection kit for Chlamydia trachomatis

A technology of Chlamydia trachomatis and detection kit is applied in the field of fluorescent PCR kits and early clinical diagnosis of Chlamydia trachomatis, which can solve the problems of unsuitable basis for clinical diagnosis and diagnosis, unable to meet the needs of rapid diagnosis and treatment, poor detection accuracy, etc. Risk of contamination, fast detection and easy operation

Inactive Publication Date: 2015-11-04
兰州安康伯乐生物技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The culture method is accurate and reliable, but it usually takes 2 to 3 days, the culture period is long, and the operation is cumbersome. Due to the influence of sample collection methods and transportation methods, it often leads to shortcomings such as low positive rate, which can no longer meet the pace of rapid diagnosis and treatment of modern medicine; direct The smear method is simple to operate, but the detection accuracy is poor and depends on subjective judgment; although immunological methods such as colloidal gold technology and enzyme-linked immunosorbent assay have the advantages of simplicity and speed, they have poor specificity, low sensitivity, and high false positive rate. Disadvantages: It is not suitable for clinical diagnosis and confirmation, and can only be used as a method for initial diagnosis

Method used

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  • Rapid fluorescence PCR detection kit for Chlamydia trachomatis
  • Rapid fluorescence PCR detection kit for Chlamydia trachomatis
  • Rapid fluorescence PCR detection kit for Chlamydia trachomatis

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Experimental program
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Effect test

Embodiment 1

[0030] The preparation of embodiment 1 kit

[0031] 1. Design and synthesis of kit-specific primers

[0032] According to the CT sequence published online (GenBank: CP006677.1), the Chlamydia trachomatis PCR primers were designed for the conserved Chlamydia trachomatis gene trpB fragment using the biology software Primer Premier 5 software and Oligo 7 software, and the software Mega4 and NCBI online Blast were used for the design Sequence alignment ensures the specificity and rationality of each pair of primers. The primers were synthesized by PAGE Bioengineering (Dalian) Co., Ltd. and purified by PAGE. The nucleotide sequences of the kit-specific primers are as follows:

[0033] Forward primer: 5'-CTATACAGAATCTAATGGCGGAATG-3',

[0034] Reverse primer: 5'-TAGCAAGCAAACACTGACCTTG-3',

[0035] 2. Design and preparation of CT plasmid positive quality control products

[0036]A pair of amplification primers are newly designed on the periphery of the sequence amplified by the ...

Embodiment 2

[0050] The usage method of embodiment 2 kit

[0051] 1. Sample requirements

[0052] a. Sample collection: A special swab sterilized by autoclaving is inserted into the male urethra or female cervix 1-2 cm, rotated once, take it out after staying for about 10 seconds, put the cotton swab head into 1ml sterile physiological The saline sample is stored in a cryopreservation tube, and the swab is cut off from the neck, and the swab head is immersed in the saline, and rinsed repeatedly to obtain the specimen rinsing solution.

[0053] b. Storage: The obtained specimen rinse solution should be stored at -20°C, and samples that cannot be detected within 24 hours should be stored below -70°C. Specimens should be stored separately in special counters or warehouses.

[0054] c. Transportation: Use curlers with ice packs or foam boxes with ice packs for transportation.

[0055] 2. Sample preparation

[0056] Take 500 μl of specimen washing solution, centrifuge at 10,000 rpm for 2 mi...

Embodiment 3

[0073] The performance evaluation of embodiment 3 kits

[0074] 1. Sensitivity experiment

[0075] CT sensitivity reference product (5.0×10 5 copies / μl), consisting of a plasmid containing the CT target fragment, using TE buffer, the above-mentioned sensitivity reference product was diluted 10 times to obtain the following gradient solution: S1 is 5.0×10 5 copies / μl, S2 is 5.0×10 4 copies / μl, S3 is 5.0×10 3 copies / μl, S4 is 5.0×10 2 copies / μl, S5 is 5.0×10copies / μl, S6 is 5.0copies / μl, S7 is 5.0×10 -1 Copies / μl is used as a sensitivity reference product, and the sensitivity reference product is used for detection. The minimum detection amount is 5.0copies / μl, that is, 10copies / reaction. For the experimental results, see figure 1 . figure 1 It shows that the CT sensitivity reference product of the kit is at 1.0×10 6 There is a good linear relationship between copies / μl and 1.0×10copies / μl, and the minimum detection amount of the kit is 5.0copies / μl, that is, 10 copies / ...

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Abstract

The invention belongs to the field of in-vitro nucleic acid detection and aims to provide a rapid detection kit applicable to Chlamydia trachomatis in a clinical sample and used for assisted diagnosis of Chlamydia trachomatis. A rapid fluorescence PCR detection kit for Chlamydia trachomatis comprises a PCR liquid, a negative quality control material, a positive quality control material, a weak positive quality control material, lysate and proteinase K, wherein the PCR liquid contains TaqDNA polymase for hot starting, forward and reverse primers and SYBRGreenI pigment, and the forward and reverse primers are specific primers designed for specific sequence of Chlamydia trachomatis and are capable of specifically amplifying a target DNA sequence, so that the clinical diagnosis purpose is achieved. The kit disclosed by the invention can be used for simply and rapidly detecting the infection of Chlamydia trachomatis in the clinical sample and has the characteristics of high specificity and high sensitivity.

Description

technical field [0001] The invention belongs to the field of in vitro nucleic acid detection, and the invention provides a method for detecting Chlamydia trachomatis (Chlamydia trachomatis) in clinical samples Chlamydia trachomatis , referred to as CT) fluorescent PCR kit for early clinical diagnosis of Chlamydia trachomatis. Background technique [0002] Chlamydia trachomatis ( Chlamydia trachomatis ) is a type of Chlamydia, which is between viruses and Rickettsia, can pass through bacteria filters, and is a prokaryotic microorganism with obligate intracellular parasitism and a unique life cycle. Chlamydia has affinity for epithelial cells on the mucosa, especially columnar epithelial cells, so it is easy to cause infection of the ocular conjunctiva and genitourinary system mucosa. [0003] Chlamydia trachomatis infection ranks third among sexually transmitted diseases (STDs) in my country, and its incidence is increasing year by year. At present, Chlamydia trachomatis i...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 杨金波王黎明刘志新杜宇平刘音希
Owner 兰州安康伯乐生物技术有限公司
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