CN101974645A

CN101974645A - Kit for detecting cervical carcinoma and detection method

Info

Publication number: CN101974645A
Application number: CN 201010555993
Authority: CN
Inventors: 张秀茹
Original assignee: Individual
Current assignee: Individual
Priority date: 2010-11-24
Filing date: 2010-11-24
Publication date: 2011-02-16

Abstract

The invention belongs to the field of medicine, and particularly provides a kit for detecting cervical carcinoma and a detection method. The genes of SOX1, PAX1, LMX1A, NKX6-1, WT1 and ONECUT1 are tumor suppressor genes related to the cervical carcinoma, and the methylation of CpG islands of promoter sites of the genes is one of main reasons for the inactivation of the genes. In the kit and the detection method, a patient can be clearly determined whether to suffer from the cervical carcinoma or not by detecting the methylation of the CpG islands of the promoter sites of the genes of SOX1, PAX1, LMX1A, NKX6-1, WT1 and ONECUT1 respectively and detecting whether HPV-L1 exists or not, the methylation of the genes is treated in advance and outcome is detected by using the HPV-L1 proteins, so that the actual conditions of cervical canceration and outcome of the patient can be perfectly evaluated in terms of molecular level, treatment is effectively guided, and misdiagnosis, over-treatment and missed diagnosis in the conventional treatment of the cervical carcinoma can be further avoided.

Description

A kind of test kit and detection method that detects cervical cancer

Technical field

The invention belongs to medical field, particularly relate to a kind of test kit and detection method that detects cervical cancer.

Background technology

Cervical cancer is the present clear and definite cancer of unique a kind of cause of disease, can effectively be prevented early discovery and the cancer of effectively curing.How asymptomatic uterine cervix precancerous lesion and early cancer be, and gynecologial examination is also difficult to be found, when symptom occurring, for invading the profit cancer, is difficult to cure more than 80%.

Infect the time that precancerous lesions of uterine cervix will experience 5-20 from HPV, current cervical cancer inspection: the first step is in the Pap smear mode, has several factors and causes and fail to pinpoint a disease in diagnosis.Second step be human papillomavirus (HPV) detection technique detect the women whether infective virus with suffer from cervical cancer, this gimmick also can cause false-positive result.Moreover, also be difficult to the period whether judgement should perform the operation and perform the operation, it is unfavorable to cause treating, and influences result of treatment.

Summary of the invention

In view of above deficiency, main purpose of the present invention is to provide a kind of test kit and detection method that detects cervical cancer.

In order to reach above purpose, this kind provided by the invention detects the test kit of cervical cancer, comprising:

A. right at the primer of the methylation-specific of SOX1, PAX1, LMX1A, NKX6-1, WT1 and ONECUT1 gene and non-methylation-specific; And/or,

B. right at proteic specificity of HPV-L1 and non-specific primer.

The mentioned reagent box also comprises required reagent such as being used to extract DNA, PCR, hybridization, colour developing, as extracting solution, amplification liquid, hybridization solution, enzyme, contrast liquid etc.

The present invention also provides a kind of method that detects cervical cancer, may further comprise the steps:

A). from the histocyte of cervical smear, extract DNA;

B). gene SOX1, PAX1, LMX1A, NKX6-1, WT1 and ONECUT1 are carried out PCR propagation, adopt abnormal methylation detection method (MSP) to detect and draw the result that whether methylates, if draw the result that methylates, the HPV that this patient is described infects the variation that has related to gene level, prompting the pernicious of the state of an illness lapses to, and needs and early treatment;

C). cervical tissue is carried out PCR propagation, and electrophoresis detects whether there is HPV-L1, if detect the positive findings of HPV-L1, the prompting that this patient has the direction of becoming better to lapse to is described.

SOX1, PAX1, LMX1A, NKX6-1, WT1 and ONECUT1 gene are the tumor suppressor genes relevant with cervical cancer, and it is one of main mechanism of SOX1, PAX1, LMX1A, NKX6-1, WT1 and ONECUT1 gene inactivation that its promoter site CpG island methylates.The present invention uses methylation status of PTEN promoter (Methylation specific PCR, MSP) different SCC clone SOX1, PAX1, LMX1A, NKX6-1, WT1 and ONECUT1 gene promoter CpG island methylation state are detected, on molecular level, understand methylate dependency with cervical cancer of SOX1, PAX1, LMX1A, NKX6-1, WT1 and ONECUT1, and make diagnosis in early days; And the proteic existence of HPV-L1 points out precancerous lesions of uterine cervix to lapse to, and just can self repair.

If under the prerequisite that the both exists, at first to observe its prognosis to uterine neck relevant gene SOX1, PAX1, LMX1A, NKX6-1, WT1 and ONECUT1 demethylation then.

Whether above-mentioned detection SOX1, PAX1, LMX1A, NKX6-1, WT1 and ONECUT1 methylated method, reaches 99% for the judging nicety rate of cervical cancer.

Whether the present invention methylates and detects whether have HPV-L1 by detecting SOX1, PAX1, LMX1A, NKX6-1, WT1 and ONECUT1 respectively, can very clear and definite judgement patient whether suffer from cervical cancer, and the methylating and lapsing to of therapeutic gene ahead of time with the HPV-L1 Protein Detection.The cervical carcinogenesis of very perfect assess patient and the practical situation that lapse on molecular level effectively instruct treatment like this.Thereby avoided the mistaken diagnosis of cervical cancer treatment now and crossed treatment, and failed to pinpoint a disease in diagnosis.

Embodiment

Below in conjunction with specific embodiment technical scheme of the present invention is further specified, but its qualification of not opposing:

This kind provided by the invention detects the test kit of cervical cancer, comprising:

B. right at the proteic Auele Specific Primer of HPV-L1.

The above-mentioned methylation-specific and the primer of non-methylation-specific at SOX1, PAX1, LMX1A, NKX6-1, WT1 and ONECUT1 gene is right, adopt abnormal methylation detection method (MSP), available result is: methylation-specific primer and non-methylation-specific primer all amplify the purpose band, present the heterozygosity state that methylates; Or the methylation-specific primer amplification gone out the purpose band, and non-methylation-specific primer does not amplify the purpose band, shows as the exhaustive methylation state.Dna methylation changes not only can directly or indirectly influence genetic transcription, also can bring out cytosine(Cyt) and change thymus pyrimidine into by 5 methylcystein deaminizatings, cause gene unconventionality expression, also can cause alteration of chromosome structure, cause the genetic expression instability, judge the generation of cervical cancer thus by methylating of above and the closely-related cancer suppressor gene of cervical cancer.

Above-mentioned right at proteic specificity of HPV-L1 and non-specific primer, same form with PCR propagation, detect the existence of HPV-L1, HPV L1 glutelin is the primary structure albumen of HPV virus, L1 glutelin monomer can form immunocomplex with T cell (CD4/CD8) and MHHC I/II (major histocompatibility complex), and only single immunocomplex promptly can the activate immunity chain reaction and formed the immune antibody of high density in the part.The expression of HPV L1 in body often illustrates that body is subjected to the HPV infection and virus is in duplicate stage, and under the normal circumstances, the expression of L1 helps exciting human cellular immunization, removes the cell that infects.But after the HPV viral DNA is incorporated into host gene, HPV L1 glutelin expression deletion, body can't be discerned the removing sick cell, causes the progress of cervical lesions or pathology.Therefore, the existence of HPV L1 glutelin means the pathology of non-progressivity, and its disappearance then is the molecular basis of the relevant vicious transformation of HPV.

A). from the cervical smear cell, extract DNA;

C) detect whether there is HPV-L1,, the prompting that this patient has the direction of becoming better to lapse to is described if detect the positive findings of HPV-L1.

SOX1, PAX1, LMX1A, NKX6-1, WT1 and ONECUT1 gene are the tumor suppressor genes relevant with cervical cancer, its promoter site CpG island methylate be SOX1, PAX1, LMX1A, NKX6-1, WT1 and ONECUT1 gene inactivation main mechanism it.The relevant information of above-mentioned SOX1, PAX1, LMX1A, NKX6-1, WT1 and ONECUT1 gene and cervical cancer is as shown in table 1:

Table 1

The present invention uses methylation status of PTEN promoter (Methylation specific PCR, MSP) different SCC clone SOX1, PAX1, LMX1A, NKX6-1, WT1 and ONECUT1 gene promoter CpG island methylation state are detected, on molecular level, understand methylate dependency with cervical cancer of SOX1, PAX1, LMX1A, NKX6-1, WT1 and ONECUT1, and make diagnosis in early days; And the proteic existence of HPV-L1 points out precancerous lesions of uterine cervix to lapse to, and just can self repair.If under the prerequisite that the both exists, at first to observe its prognosis to uterine neck relevant gene SOX1, PAX1, LMX1A, NKX6-1, WT1 and ONECUT1 demethylation then.

Whether above-mentioned detection SOX1, PAX1, LMX1A, NKX6-1, WT1 and ONECUT1 gene methylated method, reaches 99% for the judging nicety rate of cervical cancer.

Below technology contents of the present invention has been done detailed description.For persons skilled in the art, any conspicuous change of under the prerequisite that does not deviate from the principle of the invention it being done can not exceed the protection domain of the application's claims.

Claims

1. a test kit that detects cervical cancer is characterized in that, comprising:

B. right at proteic specificity of HPV-L1 and non-specific primer.

2. the test kit of detection cervical cancer according to claim 1 is characterized in that, also comprises being used to extract DNA, PCR, hybridization, the required reagent of colour developing.

3. the test kit of detection cervical cancer according to claim 2 is characterized in that, described reagent comprises extracting solution, amplification liquid, hybridization solution, enzyme, contrast liquid.

4. a method that detects cervical cancer is characterized in that, may further comprise the steps:

A). from cervical smear, extract DNA;

B). gene SOX1, PAX1, LMX1A, NKX6-1, WT1 and ONECUT1 are carried out PCR propagation, whether adopt the abnormal methylation detection method to detect to draw the baseization result;

C). cervical tissue is carried out PCR propagation, and electrophoresis detects whether there is HPV-L1.

5. the method for detection cervical cancer according to claim 4, it is characterized in that, obtain a result to methylating in step b), and step c) draws the positive findings of HPV-L1, further may further comprise the steps:, observe its prognosis then uterine neck relevant gene SOX1, PAX1, LMX1A, NKX6-1, WT1 and ONECUT1 demethylation.