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In-situ hybridization detection kit and method thereof for human papilloma virus

A technology of human papillomavirus and detection kit, which is applied in the field of biotechnology detection, can solve problems such as high missed diagnosis rate, low sensitivity, and influence on detection accuracy, and achieve the effects of easy interpretation, pain relief, and improved signal-to-noise ratio

Active Publication Date: 2012-04-11
福州泰普生物科学有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the lower limit of detection of HC is about 5000 genomes of HPV-DNA, and can only be identified in groups but not individual genotyping. The cross-reaction between the two groups of mixed probes also affects the accuracy of its detection.
Since the status of HPV infection requires the identification of a specific genotype of HPV, this method cannot be used for research and efficacy observations such as specific typing, type-specific vaccines or type-specific drugs
[0014] Although the hybrid capture method is mature, its sensitivity is not high and it has a high rate of missed diagnosis
Since HPV load is a valuable indicator for disease prediction and observation of curative effect, it is difficult to establish a sensitive enough total HPV-DNA quantitative analysis method

Method used

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  • In-situ hybridization detection kit and method thereof for human papilloma virus
  • In-situ hybridization detection kit and method thereof for human papilloma virus
  • In-situ hybridization detection kit and method thereof for human papilloma virus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1 kit of the present invention forms

[0058] The in situ hybridization (human papillomavirus type 16, 18) detection kit for liquid-based thin-layer cells provided by the invention consists of three parts: glass slides, hybridization solution, and in situ hybridization reagents. The hybridization solution includes Dig-probes (digoxigenin-labeled specific hybridization probes).

[0059] HPV16, 18 type-specific Dig-probes were prepared by PCR with 4 pairs of primers, which were HPV16, 18 L1 region and E6 region respectively. Primer sequences are listed in Table 1.

[0060] Table 1

[0061]

[0062] Preferably, the enzyme used to prepare the probe by PCR may be hot start enzyme or Taq enzyme. Hot-start enzymes and other routine components of the PCR reaction can be commercially available products, such as products produced by NEB Company, and prepared according to the instructions of the commercially available products. Taq enzymes and other conventional ...

Embodiment 2

[0078] (1) Preparation of the sample used to prepare the target Dig-probe:

[0079] The HPV-positive samples collected from 6 clinical cases were confirmed by sequencing to be HPV16 and 18 respectively. Transfer the sample solution in the test tube to a 1.5ml centrifuge tube, and mark the tube cap with a Mark pen. Centrifuge at 10,000rpm for 5 minutes, discard the supernatant and keep the precipitate;

[0080] Sample precipitation 50μl DNA extraction solution (composition: 25mM NaOH, 10mM Tris-HCl (pH8.0), 1% TritonX-100, 1% NP-40, 2% Chelex-100, 0.1mM EDTA (pH8.0)) and mix well , boiled for 10 minutes, centrifuged at 10,000rpm for 5 minutes, and the supernatant was used for later use.

[0081] (2) Preparation of Dig-probe:

[0082] The PCR reagents were prepared using hot-start enzyme or Taq enzyme respectively, wherein Taq enzyme was produced by NEB Company, hot-start enzyme was produced by Fermentas, and 10× hot-start PCR buffer was commercially available. The preparatio...

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Abstract

The invention relates to the field of detection by biotechnology, and aims at providing a visualized in-situ hybridization detection kit with high sensitivity and reliability and a method thereof for human papilloma virus. The invention has one technical scheme of providing an in-situ hybridization detection kit for human papilloma virus, comprising a slide, a hybridization solution and an in-situ hybridization reagent, wherein the hybridization solution comprises a labeled hybridization probe, and the sequences of the hybridization probe are as shown in SEQ ID NO:9 to SEQ ID NO:12. The invention has the beneficial effects of overcoming the limitation that an in-situ hybridization detection sample has to be a paraffin section or frozen section by special treatment of the slide, introducing a hybridization enhancing solution and a development synergic solution in the in-situ hybridization detection procedure, increasing the signal / noise ratio of the detection result, and facilitating reading.

Description

technical field [0001] The invention relates to the field of biotechnology detection, in particular to a human papillomavirus in situ hybridization detection kit and a method thereof. Background technique [0002] Cervical cancer is the second largest female malignant tumor. There are about 500,000 new cases every year in the world. The prevalence and fatality rate in my country account for about 1 / 3 of the world, and its incidence rate is 2% to 3% per year. Speed ​​growth With the research and development of cervical cancer etiology, its prevention and treatment methods are also constantly improving and developing. The practice of various countries has proved that the census can reduce the incidence and death of invasive cervical cancer. The reason is that cervical cancer has a series of precursor lesions. Its occurrence and development change from quantitative change to qualitative change, and gradually change to mutation. →moderate→severe)→carcinoma in situ→early invasive...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
Inventor 彭春梅胡守旺谢佐福周晓强姚铭锋
Owner 福州泰普生物科学有限公司
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