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Method for distinguishing early and late infection of syphilis

A technology for syphilis and Treponema pallidum, applied in chemical instruments and methods, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problem of affecting diagnosis and treatment, unable to correctly diagnose patients with early or late infection, unable to distinguish syphilis Infection time and other issues, to achieve the effect of simple detection method and avoid excessive treatment

Inactive Publication Date: 2019-02-12
柯吴坚
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above methods cannot distinguish the time of syphilis infection, and cannot correctly diagnose whether the patient is early or late infection, which affects the clinical diagnosis and treatment.

Method used

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  • Method for distinguishing early and late infection of syphilis
  • Method for distinguishing early and late infection of syphilis
  • Method for distinguishing early and late infection of syphilis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Preparation of Treponema pallidum TP0136 protein

[0033] Passaging and isolation of Treponema pallidum strains: Treponema pallidum Nichols strain was propagated and extracted from the testes of New Zealand white rabbits by intratesticular inoculation. Before the infection experiment, all New Zealand white rabbits were tested by Venereal Disease Research Laboratory (VDRL) and fluorescent treponemal antibody-absorbed (FTA-ABS) to rule out T.paraluiscuniculi infection. Only VDRL and FTA-ABS negative animals were used for this experiment.

[0034] Treponema pallidum DNA Extraction from Rabbit Testis Tissue: Treponema pallidum DNA was isolated from rabbit tissue fragments by low-speed centrifugation from infected rabbit testis, and then the Treponema pallidum was pelleted by centrifugation in a microcentrifuge. Treponema pallidum was resuspended in 1X cell lysis buffer (containing 10 mM Tris, 0.1 M EDTA, 0.5% SDS). Resuspend Treponema pallidum and freeze at -20°...

Embodiment 2

[0039] Embodiment 2 The preparation of the method for detecting TP0136 antibody

[0040] 1) Antibody detection strip: each kit contains 2 ELISA plates, each plate contains a detachable ELISA strip coated with the detection antigen, and the specification is 8 wells x 12 strips.

[0041] Preparation of ELISA strips (antigen plates): Dilute the purified TP0136 recombinant protein obtained in Example 1 with 0.05M carbonate buffer (pH9.6), and determine the optimal coating concentration of 0.236 μM / L, take a detachable 96-well ELISA plate, add 100 μl to each well, coat at 4°C for 24 hours, wash twice with PBS (pH7.4) washing solution (PBST) containing 0.05% Tween-20 (volume ratio) , blocked with 5% skimmed milk powder (mass volume ratio) at 37° C. for 1 hour, washed thoroughly with PBST, and air-dried at room temperature.

[0042] 2) Negative and positive control serum (1.5 mL each): the negative control serum is the serum of a healthy person not infected with syphilis, and the p...

Embodiment 3

[0047] Example 3 The use of the ELISA detection kit for distinguishing early and late syphilis infections

[0048] After diluting the serum sample to be tested 100 times with the serum diluent, add 100 μl to the antibody detection plate, directly add 100 μl to the positive and negative control serum without dilution, and repeat the two wells, incubate at 37°C for 30 minutes, then discard the wells Liquid, each well was washed 4 times with 250 μl of washing solution.

[0049] Add 100 μl enzyme-labeled secondary antibody to each well, incubate the enzyme-labeled plate at 37°C for 30 minutes, discard the liquid in the well, and wash 4 times with 250 μl washing solution per well.

[0050] Add substrate chromogenic solution, 50 μl per well, and develop color at 37°C in the dark for 15 minutes.

[0051] Add stop solution, 50 μl per well, to stop color development.

[0052] Read the OD value with a microplate reader at a wavelength of 450 nm.

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Abstract

The invention relates to a method for distinguishing early and late infection of syphilis. A molecular biology gene cloning and expression technique is utilized, a syphilis TP0136 recombinant proteinis obtained, the purified syphilis TP0136 recombinant protein serves as an antigen, an ELISA method for detecting a TP0136 antibody is established, and a kit is assembled; and early and late infectionis distinguished based on the TP0136 antibody level of a syphilis infected patient, thus clinical rational drug use is advantageously guided, overtreatment is avoided, and a fast, simple and convenient detection method is provided for diagnosis and treatment of syphilis. The syphilis infection stages are distinguished by detecting the TP0136 antibody level, and the blank of a syphilis staging diagnosis and detection method is filled; and the method has the advantages of large detection flux, accurate results, high sensitivity and easy operation, and is easy to popularize at the base level.

Description

technical field [0001] The invention relates to the field of syphilis antibody detection, in particular to a method for distinguishing early and late syphilis infection. Background technique [0002] Syphilis is a sexually transmitted disease caused by Treponema pallidum subsp.pallidum infection. Epidemiological data show that a total of 25 million people worldwide are infected with syphilis, and there are more than 11 million new cases of syphilis every year. In addition, evidence suggests that congenital transmission of syphilis is the leading cause of fetal and perinatal mortality in developing countries; and syphilis infection increases human immunodeficiency virus transmission and acquisition, making syphilis an important global health problem. [0003] According to the time of syphilis infection, it is divided into early syphilis (within 2 years) and late syphilis (more than 2 years). According to the clinical manifestations of syphilis, it can be divided into primar...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569C12N15/31C12N15/70C07K14/20
CPCG01N33/56911C07K14/20C12N15/70G01N2333/20G01N2469/20
Inventor 柯吴坚刘雅慧杨斌
Owner 柯吴坚
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