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Composition for detecting and typing high-risk HPV, kit and method

A technology of composition and kit, which is applied in the field of HPV detection and molecular biological detection, can solve the problems of performance difference, clinical sensitivity gap, unsuitability, etc., and achieve the effect of accurate results, simple method and good specificity

Active Publication Date: 2019-10-11
SANSURE BIOTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this technology needs to be equipped with special equipment QuantivirusTM cold light instrument, which brings certain restrictions on the promotion of detection products
[0008] Since the above detection methods are based on different technologies, there are also differences in performance, and the coverage of detection types is different, so they are not suitable for the needs of domestic cervical cancer screening, and research shows that there is a big gap between its clinical sensitivity and other aspects compared with DNA. Meet domestic cervical cancer screening and clinical requirements

Method used

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  • Composition for detecting and typing high-risk HPV, kit and method
  • Composition for detecting and typing high-risk HPV, kit and method
  • Composition for detecting and typing high-risk HPV, kit and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Embodiment 1, primers and probes used in the present invention

[0083] Using the SeqMan and MegAlign software in the DNAStar software package, the homology analysis of the 15 high-risk types and β-Actin genome sequences retrieved from Genbank on NCBI was carried out, and multiple pairs of specific primer pairs were designed. Afterwards, the multiple pairs of primers obtained were subjected to primer5 analysis, comprehensively considering primer pairing, neck loop structure, amplification efficiency, and mismatching conditions, and through the search and analysis of the Blast tool in the GenBank database, 3 sets of primers and The corresponding probe is used as an example (where HPmR366 can detect HPV 53, 56, and 66 at the same time), and the sequences are shown in Table 1-Table 3 in turn

[0084] Table 1

[0085]

[0086]

[0087] Table 2

[0088]

[0089]

[0090] table 3

[0091]

[0092]

[0093] Wherein, F and R are forward and reverse primer pai...

Embodiment 2

[0095] Embodiment 2, composition screening

[0096] The detection kit of the present invention independently has the following components:

[0097] RNA extraction solution I: 0.2% to 1.0% (mass / volume) of sodium lauryl sulfate, 1.0% to 4.0% (volume / volume) of triton, 0.2mol / L to 1.0mol / L guanidine isothiocyanate L. Composition of 100-400μg / ml magnetic beads;

[0098] RNA extraction solution II: 4-hydroxyethylpiperazineethanesulfonic acid 100-300mmol / L, pH6.5±0.2, sodium chloride 100-300mmol / L;

[0099] RNA extraction solution III: Triton 0.1%~1.0% (volume / volume), sodium chloride 100~300mmol / L;

[0100] RNA extraction solution IV: mineral oil;

[0101] RNA eluent: Tris-HCl 0.8~1.2mol / L, EDTA0.1~1.0mol / L;

[0102] Reverse transcription PCR-reaction solution: 10×HFM buffer reaction buffer 8~16μl, 0.05mmol / L~0.2mmol / L deoxyribonucleoside triphosphate, Depc water 22~32μl, Mgcl 2 0.05-0.4μl, 0.05μmol / L-0.2μmol / L primers for target polynucleotide amplification upstream and dow...

Embodiment 3

[0129] The content regulation of embodiment 3 different reverse transcriptases

[0130] In this example, the contents of different MMLV reverse transcriptases are discussed, the effect of reverse transcriptase on amplification is verified, and the optimal amount of MMLV reverse transcriptase is determined.

[0131] Taking HPV 16 type as an example, for the E6 / E7 region pseudovirus of known HPV 16 type, the concentration is 1.00-5.00E+06, dilute 3 gradients 10 times with Depc water, and set aside.

[0132] Then get 5 μ l nucleic acid respectively, add the reverse transcription PCR-reaction solution (45 μ l) of the table 6 of 10 μ mol / L or 30 μ mol / L or 50 μ mol / L or 70 μ mol / L reverse transcriptase amount, this reaction solution comprises embodiment 1 The compositions shown in Table 1 were tested on the machine.

[0133] Table 5 reverse transcription PCR-reaction solution system

[0134]

[0135] Perform the fluorescent PCR reaction according to the steps in the above-ment...

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PUM

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Abstract

The invention discloses a composition for detecting and typing high-risk HPV, a kit and a method and relates to the field of molecule biology detection, in particular to the field of HPV detection. The composition can be used for specifically detecting E6 / E7 mRNA of HPV types 16, 18, 31, 33, 35, 39, 45 ,51, 52, 53, 56, 58, 59, 66 and 68. At the same time, the invention also provides application ofthe composition in the detection of HPV E6 / E7 mRNA, the kit comprising the composition and the method for detecting the HPV E6 / E7 mRNA.

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 kind of non-enveloped double-stranded circular DNA virus with small molecular weight, which obligately infects and parasitizes the 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] At present, the US FDA has approved four high-risk HPV detection products for cervical cancer, which are Hybrid Capture 2 based on hybrid capture technology, Cervista HPV based on enzyme digestion signal amplification technology, Cobas HPV based on real-time fluorescent PCR technology, and transcription-mediated Aptima HPV with isothermal amplificat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11
CPCC12Q1/708C12Q1/6851C12Q2531/113C12Q2545/101C12Q2563/107C12Q2521/107
Inventor 邓中平李勃刘保生罗琳谢泽群戴立忠
Owner SANSURE BIOTECH INC
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