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Fluorescent PCR (Polymerase Chain Reaction) kit for quantitively detecting HPV16/18 type infection

A quantitative detection and kit technology, applied in the determination/inspection of microorganisms, microorganism-based methods, microorganisms, etc., to achieve the effect of accurate quantification, simple steps, and reducing the risk of PCR product contamination

Inactive Publication Date: 2010-07-21
道可名康医学发展(上海)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Currently there is no product on the market that utilizes fluorescent PCR technology for HPV detection

Method used

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  • Fluorescent PCR (Polymerase Chain Reaction) kit for quantitively detecting HPV16/18 type infection
  • Fluorescent PCR (Polymerase Chain Reaction) kit for quantitively detecting HPV16/18 type infection
  • Fluorescent PCR (Polymerase Chain Reaction) kit for quantitively detecting HPV16/18 type infection

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1: the preparation of kit

[0044] 1. Primer and probe design and synthesis

[0045] Use Primer Express 2.0 to target the L1 regions of HPV16 and HPV18 types (research shows that there are certain homologous sequences and polymorphic sequences between the L1 region sequences of HPV genomes, and the sequence differences are the basis for the division of different HPV types ), screen the probes in the conserved region, and design 16 and 18 upstream and downstream primers on the basis of the selected probes. Both primers and probes were synthesized by a professional company. The primers were purified by PAGE, and the probes were purified by HPLC. The 5' end of the probe was labeled with a FAM fluorescent group, and the 3' end was labeled with a TAMRA fluorescent group.

[0046] The amplified sequence is shown in Table 1:

[0047] Table 1. Specific probe and primer sequences

[0048] sequence name

[0049] 2. Preparation of HPV plasmid positive templ...

Embodiment 2

[0059] Embodiment 2: the use of kit

[0060] 1. Sample extraction

[0061] Use the DNA extraction solution to extract the HPV virus nucleic acid in the sample to be tested

[0062] 1) Sample pretreatment: wipe off excess secretions from the cervix, use a cotton swab soaked in normal saline to cling to the cervical mucous membrane and rotate it for 2 weeks to obtain secretions and exfoliated cells, and put the cotton swab after sampling Rinse fully in an EP tube with 1ml of sterile saline, and squeeze dry by sticking to the wall. Alkaline lysis method to extract HPV virus.

[0063] Lysis solution formula: 60mmol / L TrisHCl, pH 8.0; 0.5% SDS; 200mmol / L NaCl

[0064] 2) Operation steps: Take 500 μl of secretion and mix it, centrifuge at 13000 rpm for 10 minutes, discard the supernatant; add 50 μl of lysate, incubate at 100°C for 20 minutes, centrifuge at 13000 rpm for 1 minute, take 2 μl of supernatant for PCR reaction.

[0065] 2. Quantitative reference preparation

[0066] ...

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Abstract

The invention discloses a fluorescent PCR (polymerase chain reaction) kit for quantitatively detecting human papilloma virus (HPV) 16 / 18 type infection, belonging to the field of in vitro nucleic acid diagnosis kits. The kit comprises a quantitive reference substance, a negative reference control substance, a positive reference substance, a critical positive reference substance, a fluorescent polymerase chain reaction solution, PCR serotype specific primers, a specific fluorescent probe and a DNA extraction solution. The kit comprises a multi-PCR system based on a fluorescent PCR technology, which consists of the positive and reverse primers and the fluorescent probe which aim at 16 type HPV and 18 type HPV and can simultaneously detect the DNA of the 16 type HPV and the 18 type HPV in a reaction tube under appropriate PCR conditions. The kit can simply, conveniently and rapidly detect the HPV infection in a clinical sample and has high specificity and great clinical value on early prevention and treatment of cervical carcinoma.

Description

technical field [0001] The invention belongs to the field of in vitro nucleic acid detection, in particular to a fluorescent PCR (polymerase chain reaction) kit for quantitatively detecting high-risk 16 and (or) 18 types of HPV (human papillomavirus) infection simultaneously in clinical samples. Background technique [0002] Cervical lesions are one of the most common diseases in women, and the most serious developmental outcome is cervical cancer. Cervical cancer is now considered a global public health problem. There are more than 130,000 new cases in China each year, accounting for 28% of the world's incidence. In recent years, there has been an increasing trend in the vast rural areas, and the age of onset is showing an earlier phenomenon. With the joint efforts of scholars all over the world dedicated to cervical cancer research, WHO announced in 1996 that persistent HPV infection is the direct cause of cervical cancer. [0003] Human papillomaviruses (HPV) are a clas...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12Q1/06C12R1/93
Inventor 穆海东汪宁梅穆宇豪黎飒
Owner 道可名康医学发展(上海)有限公司
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