High-risk human papilloma virus kit and detection method

A technology of human papillomavirus and detection method, applied in the direction of microorganism-based method, biochemical equipment and method, microorganism measurement/testing, etc., can solve the problems of increasing HPV subtypes, linear increase of detection cost, etc. Uniformity, extended annealing time, beneficial effects of binding and extension

Inactive Publication Date: 2017-06-13
SUZHOU MUNICIPAL HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to further reduce the cost of fluorescently labeled multiplex PCR, and to solve the problem of the increase of HPV subtypes and the linear increase in detection costs, we have introduced general fluorescently labeled primers into the multiplex PCR system, and only need to design three fluorescently labeled primers to be able to Amplify more than a dozen amplicons of different lengths or different fluorescence

Method used

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  • High-risk human papilloma virus kit and detection method
  • High-risk human papilloma virus kit and detection method
  • High-risk human papilloma virus kit and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] The kit of the present invention comprises multiplex PCR primers and fluorescent labeling primers as shown in Table 1 and Table 2

[0065]

[0066]

[0067]

[0068] Table 1

[0069]

[0070] Table 2

[0071] Table 1 includes the sequence information and concentration of the multiplex PCR primers, and Table 2 is the information table of the fluorescently labeled primers.

Embodiment 2

[0073] 1. Multiplex PCR reaction

[0074] Reaction system 10uL: DDH2O 2.35uL, 10*PCR Buffer 1uL, 25nM Mg2+1uL, 2nM DNTP1.5uL, adapter primer mixture (see Table 1 for primer information) 1uL, fluorescent labeling primer mixture (primer information see Table 2) 1uL, 5U / uL Faststart Taq enzyme 0.15uL, HPV16 positive sample DNA 2uL. PCR cycle program: 95°C 4min; 11cycles x (94°C 30s, 68°C-0.5°C / cycle 120s); 36cycles x (94°C 30s, 58°C 60s); 72°C 10min; 4°C for ever.

[0075] 2. Genetic analyzer on the PCR product

[0076] Take 1uL of the PCR product and dilute it 50 times; take 1uL of the diluted solution, mix it with 0.06μl Liz120SIZE STANDARD and 8.9μl Hi-Di, and perform capillary electrophoresis and genotype analysis on a genetic analyzer ABI3130. HPV subtypes are determined based on peaks of specific color and position on the capillary electrophoresis map.

[0077] 3. Result Analysis

[0078] Test results such as image 3 Shown as HPV16 positive.

[0079] When using the s...

Embodiment 3

[0081] 1. Multiplex PCR reaction

[0082] Reaction system 10uL: DDH2O 2.35uL, 10*PCR Buffer 1uL, 25nM Mg2+1uL, 2nM DNTP1.5uL, adapter primer mixture (see Table 1 for primer information) 1uL, fluorescent labeling primer mixture (primer information see Table 2) 1uL, 5U / uL Faststart Taq enzyme 0.15uL, HPV52 positive sample DNA 2uL. PCR cycle program: 95°C 4min; 11cycles x (94°C 30s, 68°C-0.5°C / cycle 120s); 36cycles x (94°C 30s, 58°C 60s); 72°C 10min; 4°C for ever.

[0083] 2. Genetic analyzer on the PCR product

[0084] Take 1uL of the PCR product and dilute it 50 times; take 1uL of the diluted solution, mix it with 0.06μl Liz120SIZE STANDARD and 8.9μl Hi-Di, and perform capillary electrophoresis and genotype analysis on a genetic analyzer ABI3130. HPV subtypes are determined based on peaks of specific color and position on the capillary electrophoresis pattern.

[0085] 3. Result Analysis

[0086] Test results such as Figure 5 Shown as HPV16 positive.

[0087] When using ...

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Abstract

The invention provides a high-risk human papilloma virus detection kit which comprises a HPV amplification primer. Universal fluorescent mark primers are introduced into a multiplex PCR (polymerase chain reaction) system, and more than ten amplicons different in length or fluorescence can be obtained by amplification by designing three fluorescent mark primers; a specific joint of 20bp is introduced to a 5' terminal of a specific amplification primer, the universal fluorescent mark primers are introduced into the PCR system, length of fluorescent marked amplicons obtained through multiplex PCR amplification is larger than that of a target amplification area by two joints, and a multiplex PCR product is applied to capillary electrophoresis analysis to obtain genotype. By the high-risk human papilloma virus detection kit, expense for fluorescent marking of multiplex PCR is further lowered, and the problems of increase of HPV subtypes and linear increase of detection cost are solved. Taking using a method to detect 14 high-risk HPV subtypes as an example, multiplex PCR cost can be lowered by 50%, and cost reduction amplitude is higher as detected HPV subtypes are increased.

Description

technical field [0001] The invention belongs to the field of life science and technology, in particular to the detection of human papillomavirus HPV. Background technique [0002] Human papillomavirus (HPV), persistent high-risk HPV infection (HR-HPV) is the main cause of cervical cancer, and HR-HPV E6 and E7 genes are the most important oncogenes that promote the occurrence and development of cervical cancer. Current research shows that different high-risk HPV subtypes have different carcinogenic abilities in causing cervical cancer, among which HPV16 and 18 subtypes are recognized as the strongest carcinogenic subtypes. Therefore, the detection of high-risk HPV subtypes in women's cervical epithelial tissue plays an important role in predicting the occurrence of cervical cancer. The latest cervical cancer screening guidelines also recommend high-risk HPV typing as the first-line cervical cancer screening method. At present, more than 125 HPV detection methods have been r...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/6855C12Q1/708C12Q2600/16C12Q2531/113C12Q2537/143C12Q2563/107
Inventor 王本敬戴建荣李文静王挺刘敏娟李海波侯顺玉霍薇薇李红
Owner SUZHOU MUNICIPAL HOSPITAL
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