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Recombinant enzyme amplification detection method for HPV16 and HPV18 genotypes, test strip and application thereof

A genotype and test strip technology, which is applied in the field of recombinase amplification detection for HPV 16 and HPV 18 genotypes, test strips and its application, can solve the problems that limit the wide application of PCR technology and the unavailability of equipment, etc. Achieve the effects of shortening the detection time, avoiding the extension of the amplification time, and getting rid of the shackles of the instrument

Pending Publication Date: 2019-06-28
安徽深蓝医疗科技股份有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, relying on PCR instruments with temperature control functions, electrophoresis, hybridization after hydrolysis of amplified products, or special instrument detection characterization methods greatly limit the wide application of PCR technology.
Other techniques, such as microarray technology and real-time polymerase chain reaction (real-time PCR), also require specialized and expensive equipment that may not be available in resource-constrained laboratories, and require specialized operators to perform the associated assays Work

Method used

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  • Recombinant enzyme amplification detection method for HPV16 and HPV18 genotypes, test strip and application thereof
  • Recombinant enzyme amplification detection method for HPV16 and HPV18 genotypes, test strip and application thereof
  • Recombinant enzyme amplification detection method for HPV16 and HPV18 genotypes, test strip and application thereof

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preparation example Construction

[0052] Another aspect of the embodiments of the present invention also provides a method for preparing a dual lateral flow chromatography test strip for simultaneous detection of HPV 16 and HPV 18 genotypes, comprising:

[0053] Coupling streptavidin with carboxyl-modified latex microspheres to prepare streptavidin-coupled latex microspheres;

[0054] Streptavidin-coupled latex microspheres are applied to the marking area, and FITC antibody, digoxin antibody and secondary antibody are respectively made into detection lines T1, T2 and quality control line C in the marking area; and,

[0055] The sample loading area, the marking area, the marking area and the water absorption area are sequentially arranged on the support plate along the set direction to obtain the double lateral flow chromatography test strip.

[0056] In some preferred embodiments, the preparation method specifically includes: making latex microspheres, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochlorid...

Embodiment 1

[0085] The dual lateral flow chromatography detection method for simultaneous detection of HPV 16 and HPV 18 genotypes provided in this example comprises the following steps:

[0086] (1) Preparation of streptavidin-coupled latex microspheres

[0087] Activation: Add an aqueous solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to an appropriate concentration of latex microsphere solution , so that the molar ratio of carboxyl groups on the surface of latex microspheres to EDC and NHS is 1:5:5, and activate at room temperature for 60 minutes in the dark;

[0088] Coupling: Centrifuge (8000g, 4°C, 15min), remove the supernatant, add PB (10mM, pH 7.0) buffer to resuspend, add an appropriate amount of streptavidin, and incubate at room temperature for 2h;

[0089] Blocking: add BSA solution with a mass volume ratio of 10%, and incubate at room temperature for 1 h;

[0090] Resuspension: Centrifuge (8000g, 4°C, 15min), rem...

Embodiment 2

[0101] The dual lateral flow chromatography detection method for simultaneous detection of HPV 16 and HPV 18 genotypes provided in this example comprises the following steps:

[0102] (1) Preparation of streptavidin-coupled latex microspheres

[0103] Activation: Add an aqueous solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to an appropriate concentration of latex microsphere solution , so that the molar ratio of carboxyl groups on the surface of latex microspheres to EDC and NHS is 1:10:10, and activate at room temperature for 40 minutes in the dark;

[0104] Coupling: Centrifuge (10000g, 4°C, 10min), remove supernatant, add PB (10mM, pH 7.0) buffer to resuspend, add appropriate amount of streptavidin, incubate at room temperature for 2h;

[0105] Blocking: add BSA solution with a mass volume ratio of 15%, and incubate at room temperature for 1 h;

[0106] Resuspension: Centrifuge (10000g, 4°C, 10min), remove the...

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Abstract

The invention discloses a recombinant enzyme amplification detection method for HPV16 and HPV18 genotypes, a test strip and application thereof. The sequences of a primer pair for the HPV16 genotype are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively. The sequences of a primer pair for the HPV18 genotype are shown as SEQ ID NO.3 and SEQ ID NO.4 respectively. The double lateral flow chromatography test strip comprises a supporting plate, wherein the upper end face of the supporting plate comprises a sample loading zone, a labeling zone, a marking-off zone and a water absorption zone which arearranged in sequence; the labeling zone is bound with latex microspheres coupled with streptavidin, and detection lines T1 and T2 and a quality control line C which are bound with an FITC antibody, adigoxin and a second antibody respectively. The detection method is good in specificity, high in sensitivity, good in specificity, good in repeatability, easy to operate and high in applicability.

Description

technical field [0001] The present invention relates to a detection method of HPV 16 and HPV 18 genotypes, in particular to primer pairs for HPV 16 and HPV 18 genotypes, a simultaneous detection of HPV 16 and HPV based on the principle of latex microsphere labeling and RPA amplification technology The invention discloses a dual lateral flow chromatography test strip for 18 genotypes and a corresponding recombinant enzyme amplification detection method, belonging to the field of biomedicine. Background technique [0002] Early diagnosis and prevention of cancer has always been an urgent problem in the field of clinical detection. As the fourth most common cancer among women in the world, the detection and prevention of cervical cancer has always attracted the attention of people all over the world. In 2018, there were an estimated 570,000 new cases and 311,000 deaths from cervical cancer worldwide. Existing studies have shown that human papillomavirus (HPV) infection is act...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6804C12Q1/6844C12N15/11C12R1/93
Inventor 张超陈伟陈奉玲秦盼柱
Owner 安徽深蓝医疗科技股份有限公司
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