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Method and kit for detecting treponema pallidum

A Treponema pallidum and kit technology, applied in the field of medical immunoassay, can solve the problems of poor specificity, poor detection ability of early syphilis infection, false positive results, etc.

Inactive Publication Date: 2007-02-21
ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this type of reagent has a high detection rate for the second phase, its specificity is not good, and false positive results often occur in pregnant women, patients with autoimmune diseases, and chronic persistent hepatitis
At the same time, the ability to detect early syphilis infection is poor (the sensitivity to primary syphilis is only 80%), the window period is long, and the missed blood often leads to serious consequences when it becomes the source of infection

Method used

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  • Method and kit for detecting treponema pallidum
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  • Method and kit for detecting treponema pallidum

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Example 1: Construction of protein engineering bacteria with high expression of Treponema pallidum-specific outer membrane proteins TP0684 and TP0453

[0033] Preparation of TP genome DNA: Treponema pallidum Nichols strain was provided by the Academy of Military Medical Sciences, and the extraction of TP genome was carried out according to the operating instructions of the Tianwei Times Bacterial Genome Extraction Kit.

[0034] According to the nucleotide coding sequences of the TP0684 and TP0453 genes published by Genbank, the segments rich in antigenic determinants (SEQ ID NO: 1; SEQ ID NO: 2) were selected respectively, and the primers were designed with the biological software Primer Premier V5.0 and synthesis (underlined enzyme cleavage sites), and then carry out polymerase chain reaction (PCR) to TP0684 and TP0453 genes to amplify the desired amplified gene fragments:

[0035] P0684(1): 5′TAT CATATG AAGGAGAATTCTTGCACGGCG-3', (SEQ ID NO: 3)

[0036] (wherein the...

Embodiment 2

[0056] Example 2: Fermentation of recombinant bacteria and isolation and purification of expression products

[0057] A B.Bron 10L fermenter was used to inoculate the seed bacteria at a ratio of 10%, maintaining 70% dissolved oxygen, temperature 37°C, and pH 7.0. A 600 No feed was added when it did not reach 2, and feed was added every 0.5 hours to make the final concentrations of glucose, tryptone, and 8% yeast extract 0.5%, 0.2%, and 0.2%, respectively. After the fourth feed, when the glucose concentration dropped to 0.1%, IPTG 500 μmol / L was added to induce the harvest for 4 hours. The fermentation process is based on the batch culture controlled by cascading dissolved oxygen, and feeding is added.

[0058] The medium used in the fermentation process is an improved M9-CAA medium, that is, on the basis of M9-CAA, 0.6% yeast extract and 2mg / L ZnCl are added 2 4H 2 O, 2mg / LCoCl 2 4H 2 O, 4mg / L FeSO 4 16H 2 O, 5mg / L H 3 BO 3 , 1.6mg / L MnCl 2 4H 2 O, 4mg / L CuSO 4 ma...

Embodiment 3

[0065] Example 3. Analysis of immunological activity of antigenic protein

[0066] Basically according to "Molecular Cloning" (Sam Brook J., Felici EE Mani Artis T., Molecular Cloning Experiment Guide 2nd Edition, Beijing: Science Press, 1999, 888-897) Western blot analysis method, TP0684 and TP0453 recombinant proteins were electrotransferred to NC membrane, the positive sera of syphilis patients and healthy human sera confirmed by the TPPA method collected clinically were used as the primary antibody, and HRP-labeled goat anti-human IgG was used as the secondary antibody , Western blot detection of recombinant proteins.

[0067] Immunoblotting results show that recombinant syphilis antigenic protein TP0684 of the present invention, TP0453 have good immunoreactivity with positive sera of early syphilis patients diagnosed by TPPA method (results such as Figure 8 with 9 shown), but did not react with syphilis-negative sera.

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Abstract

A method for detecting luetin uses TP 0684 and TP 0453 syphilis specific antigens unitedly to make full use of characteristics and advantages of two said antigens for greatly raising sensitivity and specificity of detection. The kit for realizing said method to detect syphilis in early stage is also disclosed.

Description

technical field [0001] The invention relates to a medical immunological detection method, in particular to a method and a kit for detecting early syphilis infection with an immunological method using a new syphilis-specific antigen. Background technique [0002] Syphilis is an ancient sexually transmitted disease. The pathogen of human syphilis is Treponema pallidum, also known as Treponema pallidum (TP). The pathogen is an obligate human parasite, and it is not easy to cultivate artificially. It is well known that syphilis has a long course and serious harm. In the early stage, it invades the genitals and skin, and in the late stage, it invades various organs of the body, especially the cardiovascular and central nervous system. Syphilis can be divided into acquired syphilis (acquired) and congenital syphilis according to the route of infection. Penicillin is very effective against syphilis but does not prevent reinfection of TP. [0003] In recent years, the incidence...

Claims

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Application Information

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IPC IPC(8): G01N33/571G01N33/543G01N33/53
Inventor 邹全明肖洁郭刚曾明杨珺钟情宁亚蕾张卫军
Owner ARMY MEDICAL UNIV
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