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A highly sensitive human papillomavirus 6,11 nucleic acid detection kit

A technology of human papillomavirus and kit, which is applied in the biological field, can solve the problems of low detection sensitivity, missed detection and misdiagnosis, etc., and achieve the effect of high nucleic acid purity, labor cost saving, and full removal

Active Publication Date: 2021-02-19
SHANGHAI XINGYAO MED TECH DEV CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The nucleic acid extraction reagent in the kit is based on the principle of magnetic bead nucleic acid extraction, which can quickly extract HPV DNA from samples and remove PCR inhibitors. The obtained DNA has high purity and is suitable for automatic operation of instruments. The highly sensitive HPV6,11 provided by the invention The new nucleic acid detection kit can overcome the problems of low detection sensitivity and misdiagnosis caused by the existing technology, and is suitable for clinical promotion and use

Method used

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  • A highly sensitive human papillomavirus 6,11 nucleic acid detection kit
  • A highly sensitive human papillomavirus 6,11 nucleic acid detection kit
  • A highly sensitive human papillomavirus 6,11 nucleic acid detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1: Design of primer probe of HPV 6,11 type nucleic acid detection kit

[0030] According to the HPV 6 and 11 gene sequences queried in the NCBI GenBank database, using Vector NTI, Oligo and other primer design software, the optimally obtained primer probe sequences are shown in Table 1, and the target sequences amplified by the HPV 6 and 11 primers are HPV The viral L1 gene, the optimally designed primers and fluorescent probes are the conserved homologous sequences of these two subtypes of viruses.

[0031] Table 1 Primer probes designed for HPV 6,11 kits

[0032]

[0033] The above primers and probes were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.

Embodiment 2

[0034] Embodiment 2: Preparation of HPV 6,11 type nucleic acid fluorescent PCR detection kit

[0035] The amplification reagent of the kit, that is, the PCR reaction buffer is self-prepared. According to the concentration and volume of the components in Table 2, the PCR reaction buffer of the kit is prepared for 32 people. The prepared reaction buffer is divided into 20 μl for each reaction. Add the template After 10 μl the total reaction volume was 30 μl.

[0036] Table 2 The volume of each component prepared by the reaction buffer of the kit

[0037]

[0038] The negative control of the kit uses physiological saline, and the positive control is Escherichia coli containing HPV6, 11 type SEQ ID NO: 1 and 2 primer amplified fragment plasmids, the concentration is 4.0x10 3 copy / ml, the negative control and positive control are distributed according to 400μl / tube.

[0039] Kit nucleic acid extraction reagents include lysis buffer, magnetic beads, washing buffer W1 and W2, e...

Embodiment 3

[0042] Embodiment 3: the determination of kit detection sensitivity, specificity

[0043] (1) Sample template preparation

[0044] Take the HPV6 and 11 plasmid Escherichia coli constructed by the kit (concentration 4x10 3 copy / ml), using normal saline 10-fold serial dilution to 4x10 2 copy / ml, 4x10 1 copy / ml, take 5 copies of the above-mentioned dilution gradient samples, and 1 copy of the negative control and positive control of the kit, each sample volume is 400 μl, and use the magnetic bead method nucleic acid extraction reagent in Example 2 together on the nucleic acid extraction instrument. For nucleic acid extraction, 50 μl of nucleic acid was obtained from each sample.

[0045] At the same time, the specificity of the kit was evaluated by using the national reference material for human papillomavirus L1 genotyping (batch number 360002-201001) of the China National Institutes for Food and Drug Control, which is composed of 30 sequenced HPV genotype reference produc...

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Abstract

The invention relates to a kit for highly sensitive detection of low-risk human papillomavirus (HPV) 6 and 11 nucleic acids by using fluorescent PCR technology. It discloses specific amplification primers and fluorescent probes for detection, PCR reaction buffer, reference substances and nucleic acid extraction reagents. The invention also provides preparation and application methods of the kit for detecting HPV6 and 11 types. The invention is suitable for the qualitative detection of HPV6 and 11 viruses in the genitourinary tract secretion samples of human condyloma acuminatum. The kit is easy and fast to operate, and is convenient for automatic operation of instruments, and overcomes the problems of low detection sensitivity and easy misdiagnosis in the prior art. .

Description

technical field [0001] The invention relates to a high-sensitivity human papillomavirus 6 and 11 type nucleic acid detection kit, which belongs to the field of biotechnology. Background technique [0002] Human papillomavirus (Human Papillomavirus, HPV) belongs to the papillomaviridae family, is a small molecule, non-encapsulated, circular double-stranded DNA virus, the genome length is about 8000 base pairs (bp), divided into 3 There are three functional regions, namely early transcribed region (E region), late transcribed region (L region) and non-transcribed region (long control region, LCR). HPV infects humans through direct or indirect contact with contaminated objects or through sexual transmission. The virus is not only host-specific but also tissue-specific, and can only infect human skin and mucosal epithelial cells, causing various papillomas or warts on human skin and hyperplastic lesions of the genital tract epithelium. [0003] According to the different nucle...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6848C12Q1/6806
CPCC12Q1/6806C12Q1/6848C12Q1/708C12Q2563/143C12Q2563/149C12Q2527/125C12Q2521/531C12Q2561/00C12Q2563/107C12Q2545/113
Inventor 吴大治夏懿吴梅
Owner SHANGHAI XINGYAO MED TECH DEV CO LTD
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