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High-sensitivity HPV (human papillomavirus) detection kit

A kit and reagent technology, applied in the field of high-sensitivity HPV virus detection kits, can solve the problems of difficult elution, nucleic acid shedding, low yield, etc., and achieve high nucleic acid extraction yield, high grafting uniformity, high The effect of sensitivity

Pending Publication Date: 2022-06-07
杭州康大晨星医学科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Positively charged magnetic beads adsorb nucleic acids through electrostatic attraction, and the adsorption of nucleic acids is strong, but the elution is difficult, resulting in low yields
Due to the existence of the above problems, silica magnetic beads and carboxyl magnetic beads are usually used in the prior art (such as patents CN201511007550.7 and CN202110951242.9). Positively charged salt ions form a salt bridge between magnetic beads and negatively charged nucleic acids, thereby realizing the adsorption of magnetic beads to nucleic acids, which is easy to elute nucleic acids from magnetic beads (by removing the salt in the system ions), but the adsorption effect of magnetic beads on nucleic acid is poor. In the process of washing the magnetic beads before elution to remove impurities such as proteins, the nucleic acid is easy to fall off from the magnetic beads, which will also cause low yields.
And high yield is very important for the sensitivity of virus detection, especially in the early stage of the disease, the content of viral nucleic acid in the sample to be tested is usually low, if the nucleic acid cannot be extracted with high yield, false negatives are easy to occur, resulting in missed detection

Method used

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  • High-sensitivity HPV (human papillomavirus) detection kit
  • High-sensitivity HPV (human papillomavirus) detection kit
  • High-sensitivity HPV (human papillomavirus) detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1: Preparation of high yield magnetic beads

[0064] To prepare a high-yield magnetic bead for nucleic acid extraction, the steps are as follows:

[0065] (A) Grafting hyperbranched dextran: Dissolve hyperbranched dextran and tungstophosphoric acid in water according to the ratio of 1g:4g:50mL, add carboxyl magnetic beads (the mass ratio to hyperbranched dextran is 1:2) ), after uniform dispersion, the reaction was stirred at 80 °C for 7 h, followed by magnetic separation, washing with an aqueous ethanol solution, and drying to obtain dextran-magnetic beads;

[0066] (B) Grafted cysteine: Dissolve S-trityl-L-cysteine ​​and tungstophosphoric acid in water at a ratio of 0.4g:1g:10mL, add dextran-magnetic beads (with S -The mass ratio of trityl-L-cysteine ​​is 1:0.8), after uniform dispersion, the reaction was stirred at 65 °C for 4 h, followed by magnetic separation, washing with ethanol aqueous solution, and drying to obtain Cys(Trt) - Magnetic beads;

[0067]...

Embodiment 2

[0069] Example 2: HPV virus detection kit

[0070] The HPV virus detection kit of this embodiment is composed of nucleic acid extraction reagents and PCR amplification reagents, and the compositions are described in Table 1 and Table 2, respectively.

[0071] Table 1 Nucleic acid extraction reagents

[0072]

[0073] In Table 1, the magnetic beads in the magnetic bead suspension are the high-yield magnetic beads prepared in Example 1.

[0074] Table 2 PCR amplification reagents

[0075]

[0076]In Table 2, the HPV amplification primers are universal primers GP5+ / GP6+, that is, the upstream and downstream primer sequences are 5'-TTTGTTACTGTGGTAGATACYAC-3' and 5'-GAAAAATAAACTGTAAATCATATTC-3' respectively; HPV detection Taqman probes include HPV-16 detection The probe for HPV-18 detection and the probe for HPV-18 detection, the sequences are 5'-CATACACCTCCAGCACCTAA-3' and 5'-GGATGCTGCACCGGTCTGA-3'; the internal standard primer is used to amplify β-Globin in HPV, upstream ...

Embodiment 3

[0077] Example 3: HPV virus detection

[0078] Utilize the kit in embodiment 2, carry out nucleic acid extraction to the sample to be tested, and the steps are as follows:

[0079] (1) Add 400 μL sample to be tested, 20 μL magnetic bead suspension, 20 μL proteinase K solution and 400 μL lysate to the centrifuge tube, after mixing, adjust the pH to 8 with hydrochloric acid and ammonia water, and incubate for 10 min; to one side of the centrifuge tube, discard the waste liquid;

[0080] (2) Add 600 μL of washing solution A to the centrifuge tube, mix for 2 minutes; magnetically attract the magnetic beads to one side of the centrifuge tube, and discard the waste liquid;

[0081] (3) Add 600 μL of washing solution B to the centrifuge tube and mix for 2 minutes; magnetically attract the magnetic beads to one side of the centrifuge tube, and discard the waste liquid;

[0082] (4) Add 100 μL of eluate to the centrifuge tube, adjust the pH to 10 with hydrochloric acid and ammonia wa...

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Abstract

The invention relates to the technical field of virus detection, and discloses a high-sensitivity HPV virus detection kit. According to the kit, a high-yield magnetic bead for nucleic acid extraction is adopted, and cysteine-asparagine dipeptide is grafted on the surface of the high-yield magnetic bead; the cysteine-asparagine dipeptide is grafted on the surface of the high-yield magnetic bead through carboxyl in cysteine. According to the magnetic bead, the electrical property of the surface of the magnetic bead can be changed by changing the pH value, so that the magnetic bead can adsorb nucleic acid through electrostatic attraction, and the nucleic acid and the magnetic bead can be separated by utilizing electrostatic repulsion, so that the adsorption and elution effects of the nucleic acid are improved, the nucleic acid extraction has higher yield, and the detection sensitivity of the kit is improved.

Description

technical field [0001] The invention relates to the technical field of virus detection, in particular to a high-sensitivity HPV virus detection kit. Background technique [0002] Human papillomavirus (HPV) is a spherical DNA virus belonging to the genus Papillomavirus A of the family papovaviridae, which can infect host cells through minimally invasive epithelial openings and cause scaly human skin and mucous membranes. Epithelial cells proliferate and change, causing a variety of human benign and malignant tumors. More than 200 HPV genotypes have been found, which can be divided into high-risk types (HR-HPV) that mainly cause malignant lesions and low-risk types (LR-HPV) that generally lead to benign lesions. Among them, HPV-16 and HPV -18 is the most common carcinogenic type and a major factor in cervical cancer. At present, the detection of HPV virus usually adopts the PCR method. After nucleic acid is extracted from the sample to be tested, PCR amplification is perform...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/70C08B37/02C07K5/062C12R1/93
CPCC12N15/1013C12Q1/708C08B37/0006C07K5/0606C12Q2600/166C12Q2531/113C12Q2527/125Y02P20/55
Inventor 沈晓亮余向东张赟
Owner 杭州康大晨星医学科技有限公司