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A group of probes for detecting HPV high-risk and intermediate-risk nucleic acids, detection kits and applications thereof

A kit, medium-risk technology, applied in the field of molecular detection, can solve problems such as loss of probe function

Active Publication Date: 2022-02-18
GUANGDONG HECIN SCI INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

According to the result of sequence comparison, the overlapping regions of the 18 genotypes are less, which may require the design of dozens or hundreds of probes, which may easily cause the probes to lose their function due to the combination of probes

Method used

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  • A group of probes for detecting HPV high-risk and intermediate-risk nucleic acids, detection kits and applications thereof
  • A group of probes for detecting HPV high-risk and intermediate-risk nucleic acids, detection kits and applications thereof
  • A group of probes for detecting HPV high-risk and intermediate-risk nucleic acids, detection kits and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1 A preferred embodiment of a kit for detecting HPV high-risk and intermediate-risk nucleic acid molecular levels of the present invention

[0061] The kit of this embodiment contains HPV double FAM-labeled probes, streptavidin-labeled microspheres, pre-hybridization solution, hybridization solution, washing solution and probes.

[0062] Wherein, the probe is a probe for detecting high-risk and intermediate-risk HPV nucleic acids, and the probe is at least one of the sequences shown in SEQ ID NO: 1-28. The sequence shown in SEQ ID NO:1-28 is specifically as follows:

[0063] HPV-1:5'-ACGTGTTCTTGATGATCTGC-3'

[0064] HPV-2:5'-TAGAGATCAGTTGTCTCTGG-3'

[0065] HPV-3:5'-TGTAGAGTCACACTTRCAACA-3'

[0066] HPV-4:5'-TGTGTGCTTTGTACGCACAA-3'

[0067] HPV-5:5'-GGTCTTCCAAAGTACGAATG-3'

[0068] HPV-6:5'-ATTCCTAGTGTGCCCATTAA-3'

[0069] HPV-7:5'-TGTCGCTTAATTGCTCRTGAC-3'

[0070] HPV-8:5'-CCTCCCACACCACGGACACACAAAGGACAGTGGGAGG-3'

[0071] HPV-9:5'-GGACACAGCGCCCTGTCCAACG...

Embodiment 2

[0096] Example 2 A preferred embodiment of a kit for detecting HPV high-risk and intermediate-risk nucleic acid cellular levels of the present invention

[0097] The kit of this embodiment contains HPV double FAM-labeled probes, a fixative solution with a volume percent concentration of 4% paraformaldehyde, a volume percent concentration of 1% TritonX-100 permeation solution, a pre-hybridization solution, and a hybridization solution , wash solution and probes.

[0098] Wherein, the probes are probes for detecting high-risk and intermediate-risk HPV nucleic acids, and the probes have the sequences shown in SEQ ID NO: 1-28. The sequence shown in SEQ ID NO:1-28 is specifically as follows:

[0099] HPV-1:5'-ACGTGTTCTTGATGATCTGC-3'

[0100] HPV-2:5'-TAGAGATCAGTTGTCTCTGG-3'

[0101] HPV-3:5'-TGTAGAGTCACACTTRCAACA-3'

[0102] HPV-4:5'-TGTGTGCTTTGTACGCACAA-3'

[0103] HPV-5:5'-GGTCTTCCAAAGTACGAATG-3'

[0104] HPV-6:5'-ATTCCTAGTGTGCCCATTAA-3'

[0105] HPV-7:5'-TGTCGCTTAATTGCTCR...

Embodiment 3

[0131] Embodiment 3: detect one of high-risk HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 58, 59, 68 and intermediate-risk HPV26, 82 genotypes with the kit in Example 1 preferred embodiment

[0132] The probe in the kit in Example 1 is an HPV-27 probe, and the kit in Example 2 is used in this example to detect high-risk HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 58, 59, 68 and intermediate-risk HPV26, 82 genotypes. The specific experimental method is as follows:

[0133] Biotin-labeled HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 58, 59, 68, 26, 82 mRNA were respectively transcribed in vitro. After purification, 100 ng of mRNA was added to the streptavidin-labeled microspheres washed in the pre-hybridization solution. For the negative control, 1 μl of pure water was added to the streptavidin-labeled microspheres washed in the prehybridization solution. Add 5 μl of HPV-27 probe with a concentration of 0.5 μM to each group, make up to 10 μl with pre-hybridization solution, and mix well. Th...

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Abstract

The invention discloses a group of probes for detecting high-risk and intermediate-risk HPV nucleic acids, such as at least one of the sequences shown in SEQ ID NO: 1-28. In order to amplify the fluorescent signal, the probe set of the present invention may also comprise bridging probes. The invention also discloses the application of the probe in the detection of HPV high-risk type and medium-risk type nucleic acid and a detection kit. The prehybridization solution in the kit consists of Tris, Tween‑20, BSA, MgCl 2 , NaCl and formamide; hybridization solution consists of dextran sulfate, Tris, Tween-20, BSA, MgCl 2 , NaCl and formamide; the washing solution consists of Tris, Tween‑20, BSA, MgCl 2 and formamide. The probe of the present invention can reduce the number of probes when detecting multiple genotypes, realize simultaneous detection of any one or more genotypes in high-risk HPV types and intermediate-risk types, and has high sensitivity.

Description

technical field [0001] The invention relates to the technical field of molecular detection, in particular to a group of probes for detecting high-risk and intermediate-risk HPV nucleic acids, a detection kit and applications thereof. Background technique [0002] HPV infection is the main cause of cervical cancer, but not all HPV infections can induce cervical cancer lesions. Only those infected with high-risk HPV and always active can have the opportunity to induce cervical cancer lesions. Therefore, the detection of active and high-risk HPV has great clinical significance. [0003] FISH-Flow is a technology that combines fluorescence in situ hybridization and flow cytometry, which can simultaneously detect the expression of cellular genes and proteins. Compared with real-time fluorescence quantitative PCR, there is no need to extract nucleic acid from cells, reducing errors caused by personnel operations. [0004] The probe is an important tool for FISH-Flow detection. A...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12N15/11C12R1/93
CPCC12Q1/708
Inventor 李小峰李晨阳梁伟伟
Owner GUANGDONG HECIN SCI INC