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Up-conversion luminescent particle detection card for porcine cysticercosis as well as preparation method and application thereof

A technology of porcine cysticercosis and luminescent particles, which is applied in the fields of botanical equipment and methods, biochemical equipment and methods, applications, etc., and can solve problems such as errors, affecting detection results, and low sensitivity

Pending Publication Date: 2021-09-14
INNER MONGOLIA UNIV FOR THE NATITIES
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

The most valuable imaging techniques for cysticercosis are computed tomography (CT) and magnetic resonance imaging (MRI), but the accuracy and sensitivity of these two techniques are highly dependent on cyst location and infection stage, so they have low sensitivity. , high cost and low usability, it is not feasible to use it as a detection of porcine cysticercosis in a timely manner.
In addition, the two detection methods cannot realize the detection of egg invasion to cyst formation stage and human tapeworm disease
Immunological detection methods have the characteristics of good applicability and are widely used. At present, the antigens used in the immunological detection of Taenia solium / cysticercosis are mostly cystic fluid antigens, parasite antigens, excretion / secretion antigens, etc. These antigens are not only derived from Limited, cumbersome to prepare, and severe cross-reactivity with a variety of parasites
Serological methods based on a single recombinant antigen, such as enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunoelectrotransfer blotting (EITB) detection methods, overcome the above shortcomings, but they still have differences in detection with similar diseases (such as echinococcosis, thin neck echinococcosis) has cross-reactivity, and more important defects are the need for expensive instruments and equipment, professional operators, complex data processing, and unsatisfactory sensitivity, which makes the two methods stay at the research stage In the primary stage, it cannot meet the requirements of timely detection
The emergence of colloidal gold immunochromatographic detection method has solved the problems existing in the above detection methods to a certain extent, but its method is difficult to realize multiple analysis and quantitative analysis of samples, and the error of result judgment cannot be avoided by various immunological detection methods. question
In addition, the expression of recombinant antigens selected for diagnosis nowadays, the epitopes of which are easily changed by modification after translation or intricately folded internally, resulting in significantly reduced or lost activity, which affects the effect of detection. The quality and quantity of recombinant antigens is difficult, and the cost is very expensive

Method used

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  • Up-conversion luminescent particle detection card for porcine cysticercosis as well as preparation method and application thereof
  • Up-conversion luminescent particle detection card for porcine cysticercosis as well as preparation method and application thereof
  • Up-conversion luminescent particle detection card for porcine cysticercosis as well as preparation method and application thereof

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preparation example Construction

[0043] Embodiments of the present invention also relate to the preparation method of the upward light-emitting particle detection card, including the following steps:

[0044] S1, preparation of porcine sac and active protein GP50;

[0045] S2, prepare goat anti-pig IgG in the prepared light-emitting particle labeled;

[0046] S3, fixed the porrobilized active protein GP50 on the detection line, fixing the rabbit anti-goat IgG on the quality control line, and the glowing pork IgG is converted on the binding pad.

[0047] Among them: The concentration of the porcinal saccharge active protein GP50 is 1 to 5 mg / ml, preferably 2 mg / ml; the concentration of the solution of the rabbit anti-mountain IgG is 30 to 80 μg / ml, preferably 50 μg / ml; upper conversion luminous particles The solution of the labeled goat anti-pig IgG is 0.3 to 1 mg / ml, preferably 0.5 mg / ml.

[0048] Among them: Preparation of Pig Ballochi Active Protein GP50 includes:

[0049] S11, extract the total RNA...

Embodiment 1

[0081] Example 1 silicide particles and the modified embodiment amination

[0082] (1) 16.7mL of isopropanol was added to 50mg UCP particles after sonication 2min, magnetic stirring heating mantle 42 ℃ stirred for 30min. Upon the addition of 84μL orthosilicate (TEOS) was stirred for 1h, 4 ℃ 12000r centrifuged 5min, washed twice with isopropanol, resuspended and sonicated solution 30s, stirring was continued for 30min.

[0083] (2) Add 167μL of 3-aminopropyl triethoxysilane (the APTES) magnetic stirring heating mantle 42 ℃ stirred for 30min, after centrifugation 12000r 4 ℃ 5min, washed twice with deionized water, then 500μl of deionized water UCP resuspended particles.

[0084] (3) Add 500μL 20mM 2- morpholino ethanesulfonic acid (MES) buffer, 0.5mg modified after the ultrasound UCP particles added 30μL N- hydroxysuccinimide (NHS) and 30μL 1- (3- dimethyl amino) -3-ethyl carbodiimide (EDC) mixing, ultrasound again.

[0085] (4) Take 200μL activate UCP particles added 20mM MES buffe...

Embodiment 2

[0087] Example 2 Cysticercus recombinant GP50 (RTGP50) Preparation of

[0088] (1) induce expression of recombinant protein

[0089] a. Total RNA extraction parasites

[0090] Each sample was added 50 ~ 100mg 1mL TRIzoL reagent. Glass homogenizer homogenizer 20min, and grind tissue. Standing at room temperature 5min. Add 0.2mL of chloroform / mL, with vigorous shaking 15s, allowed to stand at room temperature 3min. 12000g, 4 ℃ centrifuged 15min, after centrifugation, the mixture is divided into upper, middle and lower three. Draw the upper colorless aqueous phase to a sterile centrifuge tube, was added 0.5mL of isopropanol / mL, mix by inversion at room temperature was allowed to stand 10min. 12000g, 4 ℃ centrifuged 10min, the supernatant was discarded. Add 1mL 75% ethanol (DEPC water formulation) RNA precipitate was washed. 7500g, 4 ℃ centrifuge 5min, discarded the supernatant. The net absorption liquid, an RNA dried, 30μL with RNase-free DEPC water to dissolve RNA, 55 ℃ water ba...

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Abstract

The invention relates to the technical field of biological detection, in particular to an up-conversion luminescent particle detection card for porcine cysticercosis as well as a preparation method and application thereof. The up-conversion luminescent particle detection card comprises a sample pad, a combination pad, an analysis membrane and a water absorption pad. A detection line and a quality control line are arranged on the analysis membrane. The combination pad is coated with goat anti-pig IgG marked by up-conversion luminescent particles, and the detection line is coated with porcine cysticercosis active protein GP50. A nucleotide sequence for coding the cysticercus cellulosae active protein GP50 is as shown in SEQ ID NO: 1. The detection card has the advantages of being simple in antigen preparation, simple, convenient and rapid, easy in result interpretation and the like, does not generate cross reaction with other parasite positive serum, is high in specificity, good in stability, high in practicability and easy to store, can be used for large-scale detection, and has great market prospects.

Description

Technical field [0001] The present invention relates to the field of biological detection, and in particular, to the upper transmissive particle detection card for porcine bladder, the preparation method and application thereof. Background technique [0002] Pig vesicular disease is a human beast who caused by larvae in pig straps in pigs or human parasitic diseases. The disease not only causes huge losses to the pig industry, but also endangered human health, and even cause death. At present, the World Health Organization (WHO) has been listed as "one of the 17 ignored tropical diseases" and determines it as a focus research and control. The "Ten Crison Human Food Severe Parasites", which is published by the United Nations Food and Agriculture Organization (FAO) and the World Health Organization, and the first place in the capsule, and is also one of the key parasitic diseases of the "National Health Department's planning prevention". Diagnosis of porch tape tapkoda depends on i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/558G01N33/58G01N33/531G01N33/533C12N15/70C12N15/12
CPCG01N33/6893G01N33/558G01N33/582G01N33/531G01N33/533C12N15/70C07K14/43554G01N2333/43543G01N2800/26
Inventor 孙树民齐昱刘明远刘晓雷吴秀萍赵权骆学农
Owner INNER MONGOLIA UNIV FOR THE NATITIES
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