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Test strip and kit for helicobacter pylori colloidal gold typing detection

A technology for detecting Helicobacter pylori and test paper, which is applied in the field of medical biology, can solve the problems of low sensitivity, long time consumption, radioactivity in the human body, etc., and achieves the effect of simple operation method and guaranteed reliability.

Inactive Publication Date: 2017-07-07
蔡长春 +2
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AI Technical Summary

Problems solved by technology

[0004] 1) Helicobacter pylori culture was taken from the gastric mucosa by gastroscopy: although the culture method of Helicobacter pylori is the most reliable and regarded as the gold standard for the diagnosis of Helicobacter pylori, the sensitivity is not high
In addition, Helicobacter pylori is a microaerophilic bacterium, and its cultivation requires certain equipment, so it is not suitable for routine use in general hospitals.
[0005] 2) Rapid urease and histological examination: the combined application of the two greatly improves the positive rate of diagnosing Helicobacter pylori, but it can only determine whether it is due to Helicobacter pylori infection, and cannot be further typed
[0007] 1) Bacterial culture: complex, time-consuming, and certain experimental conditions are required. Specimen transfer culture requires a special transfer solution and cryopreservation
[0008] 2) Anti-Helicobacter pylori antibody detection in serum: The detected antibody is IgG, and positive only indicates past or current infection with Helicobacter pylori. After eradication of Helicobacter pylori, serum antibodies, especially cytotoxic Cag / A antibodies, can last for a long time, up to several years , so the result cannot judge the situation after Helicobacter pylori treatment in time
[0009] 3) Carbon-13 / carbon-14 breath test: Although the sensitivity and specificity are high, the carbon-13 breath test uses stable isotopes, and the detection requires a special mass spectrometer for detection, which is time-consuming and expensive, and is not suitable for promotion in primary hospitals Carbon 14 breath test is radioactive to the human body, pregnant women and children should use it with caution
In addition, this breath test is not reliable when the detection value is near the critical value
In addition, none of the above methods can classify
[0010] It can be seen that the existing mainstream detection technology for Helicobacter pylori is still at the level of detecting the presence or absence of Helicobacter pylori infection through the urease test, and there is a lack of typing products

Method used

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  • Test strip and kit for helicobacter pylori colloidal gold typing detection
  • Test strip and kit for helicobacter pylori colloidal gold typing detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1. Core antigen expression of Helicobacter pylori cytotoxicity (CagA), vacuolar toxin (VacA), urease subunit A protein and urease subunit B protein

[0036]The four gene sequences of Helicobacter pylori cytotoxicity (CagA), vacuole toxicity (VacA), urease subunit A protein and urease subunit B protein are the sequences of Helicobacter pylori strain 26695 collected by the applicant according to GenBank, after Preferably, the found core antigen sequence is artificially synthesized by Jinkai Rui Company. Wherein, the gene sequence of HP cytotoxicity (CagA) is Seq ID No.1, and the gene sequence of HP vacuolar toxin (VacA) is Seq ID No.2, and the gene sequence of the HP urease subunit A protein antigen is Seq ID No.2. ID No.3, the gene sequence of HP urease subunit B is Seq ID No.4.

[0037] 1.1 Artificial synthesis of the above four gene sequences

[0038] And add restriction site fragments at both ends of the gene as needed

[0039] 1.2 Construction of expressio...

Embodiment 2

[0048] Embodiment 2 detects the preparation of chromatographic membrane

[0049] 2.1. Preparation of coating buffer

[0050] KC10.2g, Na 2 Helicobacter pylori O 4 12H 2 O 2.9g, KH 2 PO 4 0.2g, methanol 30ml, distilled deionized water to 1000ml, 0.22um membrane filtration

[0051] 2.2 Preparation of nitrocellulose chromatography membrane

[0052] T line (detection line):

[0053] Dilute CagA, VacA, UreA and UreB core antigen proteins with coating buffer to 50-100ng / ml, adjust the machine, draw four detection lines (T lines) from the end of the gold standard pad, respectively CagA, VacA, UreB and UreA.

[0054] C line (quality control line):

[0055] Dilute the rabbit anti-mouse IgG antibody to 50-100 ng / ml with coating buffer, adjust the machine, draw the C line, which is the quality control line, and the C line is close to the absorbent pad, about 3mm away from the absorbent pad. The distance between the two lines is 5-8mm, and the lines should be fine and uniform. ...

Embodiment 3

[0056] Example 3 Preparation of gold-labeled anti-human IgG Fc segment monoclonal antibody

[0057] 3.1. Preparation of required solutions

[0058] (1) prepare chloroauric acid solution

[0059] 10g chloroauric acid; double distilled deionized water to 1000ml. The prepared solution was stored at 4°C.

[0060] Formulated 1% Trisodium Citrate

[0061] Dissolve trisodium citrate in double-distilled deionized water, filter through a 0.22um membrane, and prepare and use immediately.

[0062] Prepare 0.1Mol / L Potassium Carbonate

[0063] Prepare with double-distilled deionized water, 13.8g of potassium carbonate; dilute to 1000ml with double-distilled deionized water, filter with 0.22um membrane, and store at 4°C.

[0064] Formulated 2% PEG-20000

[0065] 20g PEG-20000 double-distilled deionized water to 1000ml, filter with 0.22um membrane, and store at 4°C.

[0066] Prepare labeling wash preservation solution

[0067] 2% bovine serum albumin (BSA), 0.05% sodium azide (NaN3)...

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Abstract

The invention provides a colloidal gold typing detection test strip for Helicobacter pylori. The test strip includes a support plate, and a sample pad, a gold standard pad, and nitrocellulose are arranged in sequence from the sample loading end on the surface of the support plate. These four parts are the plain film and the absorbent pad. The gold standard pad contains colloidal gold-labeled anti-human IgG Fc segment monoclonal antibody; Four detection lines and quality control lines for foamy toxin (VacA) antigen, HP urease subunit A protein antigen and HP urease subunit B protein antigen, and the above four detection lines and quality control lines do not overlap with each other. The test strip of the present invention belongs to the non-invasive detection technology, can detect 4 kinds of Helicobacter pylori antibodies of urease A, urease B, Cag / A and Vac / A at the same time, adopts the internationally recognized standard Helicobacter pylori strain and quality Control serum to ensure the reliability of detection. The operating method of the test strip is simple, fast and popular, and has application prospects and potential social value.

Description

technical field [0001] The invention relates to the field of medical biotechnology, in particular to a detection method for pathogenic bacteria, in particular to a colloidal gold type detection test strip for pathogenic Helicobacter pylori. Background technique [0002] It has been confirmed that Helicobacter pylori is an important pathogenic factor leading to severe gastrointestinal diseases such as gastric ulcer and gastric cancer. At present, the infection rate of Helicobacter pylori has reached about 50%. However, because the pathogenicity of Helicobacter pylori is inconsistent (divided into low-virulence type and high-virulence type), not all Helicobacter pylori infections require treatment. Therefore, it has important guiding significance for clinical treatment to diagnose whether or not to be infected by Helicobacter pylori and what kind of Helicobacter pylori infection. Currently, methods for diagnosing Helicobacter pylori infection can be broadly classified into i...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/569G01N33/558
CPCG01N33/577G01N33/558G01N33/56911
Inventor 孙明宽蔡长春贾乙
Owner 蔡长春
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