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DNA chip for genotyping of human papilloma virus, kit having same, and method for genotyping

A technology of human papillomavirus and DNA chip, applied in the field of DNA chip, can solve problems such as lack of HPV DNA chip, error, and unstandardized conditions

Active Publication Date: 2013-07-17
GOODGENE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because there are many variations in the DNA base sequence of the HPV genome, if primers or probes are not designed taking them into account, PCR or hybridization will not proceed as expected and errors will occur
[0019] Third, because no internal reference gene (control gene) is used, it is not easy to know whether a negative result is a true negative or a false negative
[0020] Fourth, so-called universal probes capable of testing for the presence of all genotypes of HPV are not considered
[0021] Fifth, PCR is the most important step before HPV DNA analysis, but conditions are not standardized
[0023] Seventh, while many HPV DNA diagnostic kits are available, the scale of the tests and how accurate they are compared to standard tests and how useful they are in screening for cervical cancer and precancerous lesions is highly dependent on research. comparisons are not enough
Existing HPV DNA chips lack such data

Method used

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  • DNA chip for genotyping of human papilloma virus, kit having same, and method for genotyping
  • DNA chip for genotyping of human papilloma virus, kit having same, and method for genotyping
  • DNA chip for genotyping of human papilloma virus, kit having same, and method for genotyping

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0130] Example 1: Preparation of control samples and extraction of DNA

[0131] Prepare samples to be used as standard materials and extract DNA from them.

[0132] As the first sample, human cervical cancer cells infected with HPV and whose type has been identified and have been widely used in HPV genotyping studies were purchased from ATCC (Manassas, VA20108, USA) and Korean cell line bank (KCLB; Seoul National University Cancer Research Institute, South Korea), and used after monolayer culture. Isolate genomic DNA from it.

[0133] The second sample was obtained from CIN cervical tissues of 100 Korean women who had been diagnosed with cervical cancer or carcinoma in situ. The formalin-fixed paraffin-embedded tissue was cut into 5-10 pieces (10-μm thick) and placed on a microscope slide. Then, only the cancer cells were microdissected. Among 100 cases of cervical cancer injury, 98 cases had cervical epithelioma (CIN).

[0134] As the third sample, cervical samples were taken fr...

Embodiment 2

[0184] Example 2: Preparation of standard samples and control samples

[0185] Prepare a plasmid DNA clone of HPV L1 gene to be used as a standard material in the following genotyping and analysis.

[0186] First, extract DNA from human cervical cancer cells and obtain the PCR product of HPV L1 gene. Secondly, the PCR products of 42 types of HPV L1 gene were obtained from the Korea Food & Drug Administration (KFDA). Once again, HPV PCR products were obtained from cervical cancer tissues from 100 Korean women and cervical swab samples from 15,708 women. After the HPV L1 gene was genotyped by post-PCR sequencing, the PCR product was cloned into the pGEM-T Easy vector to obtain L1 clones of each HPV genotype. In the process of establishing the reaction conditions of the DNA chip of the present disclosure, these clones are used as standard samples and control samples. Clone as follows:

[0187] 1) Separate the PCR product of the amplified L1 gene on an agarose gel using a gel recove...

Embodiment 3

[0200] Example 3: PCR amplification

[0201] The HPV L1 gene and human β-actin gene as internal control genes were amplified to investigate the genotype of HPV.

[0202] For PCR amplification, first select and design oligonucleotide primers. The primers include MY11 primers, GP6-1 primers and GP6+ primers (SEQ ID NO1-SEQ ID NO3) for detecting HPV L1 gene, and ACTBF of human β-actin gene for confirming DNA extraction and PCR efficiency ( Forward) primer and ACTBR (reverse) primer. The GP6-1 primer, ACTBF primer and ACTBR primer were designed by the inventors, and the other primers were selected from previously known primers. The length of the PCR product of the HPVL1 gene is 185 bp, and the length of the PCR product of the β-actin gene is 102 bp. Table 2 shows the base sequences of PCR primers for each gene.

[0203] Table 2. PCR primers

[0204]

[0205]

[0206] (In the base sequence, M is A or C, W is A or T, and Y is C or T.)

[0207] The optimal conditions for double PCR were ...

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Abstract

The present invention relates to a DNA chip, or a DNA microarray, having a conglomeration of probes thereon, wherein the probes complementarily bind with 44 types of HPV nucleic acids, which are the main cause of cervical cancer and the most common cause of sexually transmitted diseases, a genotyping kit having same, and a method for genotyiping using same. The present invention enables recognition of all 44 types of HPV that invade the genitals, accurate diagnosis of multiple infections from more than one HPV types, high sensitivity and specificity for the HPV genotype diagnosis at near 100%, and quick testing of a plurality of specimens, and is very useful in prognosis of cervical cancer and precancerous lesions.

Description

Technical field [0001] The present disclosure relates to a DNA chip for detecting the genotype of human papillomavirus (HPV), a kit containing the chip, and a method for HPV genotyping. More specifically, the present disclosure relates to a DNA chip (or DNA microarray), a genotyping kit containing the DNA chip, and a genotyping method using the DNA chip. There are 44 kinds of spots on the DNA chip. HPV (which is the main cause of cervical cancer and the most common cause of sexually transmitted diseases) nucleic acid complementary probe. Background technique [0002] Human papillomavirus (HPV) is a virus that is transmitted to humans through sexual contact and is very important in two aspects. [0003] First, HPV infection is the most common sexually transmitted infection in humans, with the highest prevalence rate. In the United States, 26.8% of women between the ages of 14 and 59 have HPV infection and it is believed that 80% of women have been infected at least once. This inf...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/37
CPCC12Q1/6837C12Q1/6886C12Q1/708B01L3/50855
Inventor 文宇哲吴明烈
Owner GOODGENE
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