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Composition, reagent kit and method for detecting HPV

A technology of composition and kit, which is applied in the field of molecular biological detection and HPV detection, can solve the problems of sensitivity sacrifice, etc., and achieve the effect of reducing difficulty and saving cost

Active Publication Date: 2019-12-20
SANSURE BIOTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In this regard, Shuoshi Biotechnology Co., Ltd. provided a HPV detection kit called Shuoshi High-risk Human Papillomavirus (HPV16+2) Nucleic Acid Detection Kit, which can detect 18 types, including 16 , 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 82; 16 and 18 can be typed specifically, but this detection reagent is in terms of sensitivity sacrificed, its sensitivity is 5×10 5 copy / ml

Method used

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  • Composition, reagent kit and method for detecting HPV
  • Composition, reagent kit and method for detecting HPV
  • Composition, reagent kit and method for detecting HPV

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Embodiment 1, primer and probe design and screening used in the present invention

[0056] 1. Primer and probe design

[0057] Using the SeqMan and MegAlign software in the DNAStar software package, the genome sequences of 15 high-risk HPV types retrieved from Genbank were compared for homology, and the specific and conserved segments in the E interval were found. With the aid of Primer Express 3.0 and Primer Premier 5.0 software, and through the search and analysis of the Blast tool in the GenBank database, 3 sets of primers and corresponding probes were designed for each type. At the same time, find out the conserved segment of the β-globin gene, design 3 sets of primers and corresponding probes for the β-globin gene in the same way, see Table 1 for the nomenclature, and Table 2-4 for the sequence.

[0058] 3 sets of primer probe numbers designed in Table 1

[0059]

[0060]

[0061] Table 2 The sequence information of the first set of primers and probes

[0...

Embodiment 2

[0086] Embodiment 2, primer and probe dosage optimization

[0087] In general, the amount of primers used in PCR reactions affects the amplification. If the primer concentration is too low, the amount of product will decrease. If the primer concentration is too high, it will not only promote the production of non-specific products, but also increase the formation of primer-dimers. 12 pairs of primer probes were designed for 15 types, and then 15 kinds of pseudoviruses with high, medium and low concentrations of HPV high-risk types were used against 12 pairs of primer probes (concentration 1.00~5.00E+08 copies / ml, concentration 1.00 ~5.00E+06 copy / ml, concentration 1.00~5.00E+04 copy / ml) for detection and comparison.

[0088]Taking HPV16 type as an example, first fix the amount of other components, and then use 0.05μmol / L, 0.1μmol / L, 0.2μmol / L, 0.4μmol / L primers as shown in Table 6 to prepare the PCR reaction solution, and then take 5μl The HPV16 type pseudovirus is carried ou...

Embodiment 3

[0097] Example 3 HPV detection experiment

[0098] 1. Reagent preparation:

[0099] According to the number of samples to be tested, negative control and positive control, take the corresponding amount of reaction solution and enzyme mixture according to the proportion (PCR reaction solution 38-44 μl / person + enzyme mixture 3-5 μl / person), and mix well to form PCR-mix, the primers and probes in the PCR-mix were added as described in Table 8 above, and the PCR-mix was centrifuged briefly for later use.

[0100] 2. Sample processing

[0101] Draw 1mL sample from the sample collection tube to a 1.5mL centrifuge tube as the sample to be tested. It is recommended to use the sample release agent of Hunan Shengxiang Biotechnology Co., Ltd. to operate according to its instructions for nucleic acid extraction. Or use nucleic acid extraction or purification reagents from Hunan Shengxiang Biotechnology Co., Ltd. to perform nucleic acid extraction according to its instructions. 10 μl o...

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Abstract

The invention relates to the field of molecules biological detection, in particular to the field of HPV detection, and provides a composition. HPV16 / 18 can be subtyped, and HPV 16, 18, 31, 33, 35, 39,45, 51, 52, 53, 56, 58, 59, 66 and 68 types can also be specially detected. Besides, the invention further provides an application of the composition for detecting HPV, the reagent kit containing thecomposition, and the method for detecting the HPV.

Description

technical field [0001] The invention belongs to the field of molecular biology detection, more specifically, to the field of HPV detection. Background technique [0002] Human papillomavirus (HPV) is a type of non-enveloped double-stranded circular DNA virus with a small molecular weight and a genome length of about 8000 base pairs (bp), which is divided into three functional regions, namely the early transcription region (E region), late transcribed region (L region) and non-transcribed region (long control region, LCR), obligately infect and parasitize epithelial cells of human reproductive organs and other tissues and organs. After HPV infection, not only genital warts or skin warts may appear, but it is more likely to induce female cervical cancer. According to related reports, more than 90% of cervical cancers are caused by HPV infection. [0003] Clinically, human papillomaviruses can be divided into high-risk types and low-risk types according to the different pathog...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12R1/93
CPCC12Q1/708C12Q1/686C12Q2600/166C12Q2563/107C12Q2537/143
Inventor 李勃刘保生罗琳谢泽群邓中平刘佳戴立忠
Owner SANSURE BIOTECH INC
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