CN105177163B - A kind of kit for cervical carcinoma early screening - Google Patents
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Abstract
The invention discloses a kind of kit for cervical carcinoma early screening, including QlAamp DNA Mini Kit kits, QIAamp EpiTect Bisulfite Kits kits, the forward primer sequence of JAM3 M4 is as shown in SEQ ID No.6, reverse primer sequences are as shown in SEQ ID No.7, the forward primer sequence of β actin is as shown in SEQ ID No.16, and reverse primer sequences are as shown in SEQ ID No.17, PCR reaction solution and ddH2O, the kit, the further verification of the preliminary and substantial amounts of cervical exfoliated cell of detection sensitivity, specificity, positive predictive value and negative predictive value through clinical tissue sample, reaches the requirement of clinical diagnosis.
Description
Technical field
The present invention relates to a kind of kit for cervical carcinoma early screening, belong to molecular biology and medical domain.
Background technology
Cervical carcinoma is one of common gynecologic malignant tumor, and the annual new cases in the whole world are about 528,000, death
266,000 is there are about, wherein about 85% occurs in developing country.The incidence of China remains high, and is 7.5/100,000,
The death rate is 3.4/100,000.In the backward areas of some reasonable examinations of shortage, incidence is even as high as 81/100,000.Just
Step estimation, to the year two thousand thirty, the patient for dying of cervical carcinoma every year there are about 474,000, wherein may betide backwardness more than 95%
And developing country.Therefore, the molecular labeling of cervical carcinoma early screening is found, examination goes out precancerous lesions of uterine cervix and tumour is suffered from
Person, it is significant to implement effective early diagnosis, early gauge.
At present, China is using the cervical carcinoma screening strategy using cervical liquid-based cells as main contents, while local
Using HPV-DNA+ cytology United screenings.The national examination system of China's not yet Erecting and improving, and due to the injustice of examination
Weighing apparatus property (influence factor such as regional economy, medical resource, public cognitive), causes current examination shortage excessive with examination and deposits, no
Canonical management and over-treatment are simultaneously deposited.Cervical liquid-based cells are not high (about 50%-85%) there are 1, single application sensitiveness；
2nd, the specificity of ASCUS and LSIL is not high；3rd, horizontal uneven of materials, film-making, diagosis, lacks the quality control of standardization
The defects of.Therefore single dependent cells inspection be easy to cause false negative, and omission factor is high.HPV-DNA detections can significantly improve
Sensitivity, but be often possible to detect transient HPV infection, rather than there is pathogenic high-risk HPV persistent infection.Therefore it is single
By HPV examinations, excessively diagnosis and over-treatment can be caused.
DNA methylation is an important epigenetics mechanism.DNA methylation, i.e., non-methylated cytosine-phosphoric acid-
Guanine (Cytosine-phosphate-Guanosine, CpG) dinucleotides methylates, and is tumor suppressor gene inactivation
One of key mechanism.Tumor suppressor gene (Tumor Suppressor Genes, TSG) occurs lose, be mutated, methylating
When change causes the normal function to lose, the proliferation out of control of cell forms malignant tumour.Clear and definite DNA methylation is TSG inactivations
One of three kinds of mechanism, and be probably unique mechanism in some cases.Thus DNA methylation is sent out in cancer occurrence and development
Wave important function.As cancer occur earliest events, DNA abnormal methylations detection can patient occur clinical manifestation or
Accomplish that molecule diagnoses before radiological evidence, new approach is provided for early diagnosis of cancer.
Domestic and international researcher is detected using Relationship Between Methylation of Tumor Suppressor Gene, is taken in the research of cervical carcinoma early screening
Obtained exciting progress, but same gene between different researchs there are larger difference, analyzing its reason may be：1、
The specificity in gene methylation site；2nd, the otherness of gene genetic background；3rd, operating method and the heterogeneity of experiment reagent.Cause
This, this area is badly in need of being further discovered that the more effective more stable cervical carcinoma specific early stage that methylates for being more easy to be applied to clinic sieves
The biomarker looked into.
The content of the invention
For the studies above progress and the prior art, present inventor is by retrieving and consulting lot of documents and right
The data analysis of The Cancer Genome Atlas (TCGA) platform, preferably goes out multiple sites of several candidate genes, wherein
There are multiple sites never to report.By measuring, verifying and analyze, find the M4 sites of JAM3 genes with cervix disease first
The degree that the exacerbation of change degree methylates gradually rises, can be as the molecular labeling of cervical carcinoma early screening, and therefore, the present invention carries
A kind of kit for cervical carcinoma early screening is supplied.
To achieve these goals, the present invention adopts the following technical scheme that：
A kind of kit for cervical carcinoma early screening, including QlAamp DNA Mini Kit kits, QIAamp
EpiTect Bisulfite Kits kits, the forward primer sequence of JAM3-M4 is as shown in SEQ ID No.6, reverse primer
Sequence is as shown in SEQ ID No.7, and the forward primer sequence of β-actin is as shown in SEQ ID No.16, and reverse primer sequences are such as
Shown in SEQ ID No.17, PCR reaction solution and ddH2O。
The application method of mentioned reagent box, step are as follows：
(1) the DNA extractions of cervical exfoliated cell：The cervical exfoliated cell of patient is taken, using QlAamp DNA Mini Kit
Kit extracts, and obtains DNA solution；
(2) free serum DNA methylates modification：The cast-off cells DNA for taking step (1) to extract, using QIAamp
EpiTect Bisulfite Kit kits are modified, DNA after must modifying；
(3) DNA carries out PCR amplification after step (2) is modified；
(4) formula is utilized：2[Ct(β-actin)-Ct(target)]× 10,000 calculate the methylation of sample gene.
The PCR reaction conditions：
JAM3-M4, CADM1-M2, CADM1-M8, DAPK1-M2 or DAPK1-M3, nucleotide sequence are respectively：
JAM3-M4:CGCAGCCAGGGCTGGGACTCGGGCCTGGCTAGGGCGGGGGCCCTGGGACGCCCGGCAGTTG
GACCGGGGCGGGGAGCTAATTTGGGATGGGGGGCCCGTCCCAGGCAGTGAGTCGGTCCCGGACAGCCGTCGGACCCC
(SEQ ID NO:1),
CADM1-M2:GTCCTCCGCCACTTGTTGCTCCCGGGTCCTGCAGCTCTGGAGCTGCAAGGAGGGGCTTTG
CAGGCTCAGAGCCCTGCTGCATCCCCTCCTGCATCGCCGCTCGCACCCCGCGGCCCC(SEQ ID NO:2),
CADM1-M8:GCCGCCGCACACTGGGATCCGCTCGGCAGCACTACACTCGCCATGTCGGGCACCTGCCTC
AGACTGGCGGCGTTGGCTGCCTCCGGAGCCCGAGCGGACAGCTAATGAGA(SEQ ID NO:3),
DAPK1-M2:CGCTTGCAGGGTCCCCATTGGCCGCCTGCCGGCCGCCCTCCGCCCAAAAGGCGGCAAGGA
GCCGAGAGGCTGCTTCGGAGTGTGAGGAGGACAGCCGGACCGAGCCAACGCCGGGGACTTTGTTCCCTCCGCGGAGG
GGACTCGGCAACTCGCAGCGGCAG
(SEQ ID NO:4),
DAPK1-M3:GTCCCCATTGGCCGCCTGCCGGCCGCCCTCCGCCCAAAAGGCGGCAAGGAGCCGAGAGGC
TGCTTCGGAGTGTGAGGAGGACAGCCGGACCGAGCCAACGCC(SEQ ID NO:5)。
The principle of DNA methylation and methylation status of PTEN promoter (Methylation specific PCR, MSP)：DNA first
Baseization is non-methylated cytosine-phosphoric acid-guanine (Cytosine-phosphate-Guanosine, CpG) dinucleotides hair
Life methylates, and is the modification of the most common type epigenetics, and the main site that occurs is CpG islands.CpG sites go out in genome
Existing frequency is far below other dinucleotide sequences in genome, but very high in some regions CpG site densities, up to average
More than 5 times, the CpG islands rich in guanine and cytimidine are formed, CpG islands are frequently located in promoter region and the 1st exon 1 of gene,
It is about 1kb.In normal human genome, the CpG sites outside CpG islands are typically what is methylated, and the CpG sites in CpG islands lead to
Non- methylation state is often in, this form to methylate is in the heredity stablized with cell division.When tumour occurs, press down cancer base
Because of the non-methylation increase in CpG sites beyond CpG islands, and the CpG sites in CpG islands are in hyper-methylation state, cause to contaminate
The increase of colour solid helix degree, Transcription inhibition, gene expression missing.Methylation status of PTEN promoter PCR (Methylation
Specific PCR, MSP) it is the current first studied one of the most sensitive experimental technique that methylates, can find minimum about 50pgDNA
Base, its basic principle are:Single stranded DNA after bisulphite modified, all unmethylated cytimidines (cytosine,
C) deamination is changed into uracil (uracil, U), and the cytimidine to methylate in CpG sites remains unchanged, therefore separately designs two
To for the primer to methylate with non-methylated DNA fragments, will can be methylated (Methylation, M) and non-first by PCR amplification
Base (Unmethylation, U) DNA sequence dna distinguishes.
Beneficial effects of the present invention：
Present inventor is by retrieving and consulting lot of documents and to The Cancer Genome Atlas
(TCGA) data analysis of platform, preferably goes out multiple sites of several candidate genes, wherein there is multiple positions never to report
Point.By measuring, verifying and analyze, find that the methylation in the M4 sites of JAM3 genes is related with cervical lesions first, can
Molecular labeling as cervical carcinoma early screening.
The present invention be used for cervical carcinoma early screening molecular labeling primer and kit, detection sensitivity, specificity,
The preliminary and substantial amounts of cervical exfoliated cell of positive predictive value and negative predictive value through clinical tissue sample is further tested
Card, reaches the requirement of clinical diagnosis.
JAM3-M4 is (particularly small for ASCUS for patient and the cytology screening results for shunting the high-risk HPV positive
In the patient of 30 years old) and LSIL patient clinicals efficiency height, also there is certain carry in terms of shunting clinically remains the CIN2 of dispute
Signal justice.
The present invention be used for cervical carcinoma early screening molecular labeling primer specificity it is strong, primer using the present invention into
During row amplification, amplification condition is easy to grope, convenient to carry out specific amplification, better than other primers.
Brief description of the drawings
Fig. 1 is 27 sites of methylation status of PTEN promoter (MSP) 4 genes of Preliminary detection in cervical cancer tissues and normal
Compare methylation differential expression in cervical tissue；
Fig. 2 is-CADM1-M2, CADM1-M8, DAPK1-M2, DAPK1-M3 and JAM3-M4 are in early diagnosis and examination
Application value；
Fig. 3 is JAM3-M4 in methylation level of the uterine neck without knurl change-precancerous lesion-cancer patient's cast-off cells；
Fig. 4 is that ROC curve analyzes the clinical diagnosis efficiency that JAM3-M4 methylates；
Fig. 5 is the immunohistochemical staining and pyrosequencing of CIN2 cases.
Embodiment
The invention will be further described with embodiment below in conjunction with the accompanying drawings.
1st, the DNA extractions of cervical exfoliated cell：
The cervical exfoliated cell of patient is taken, using QlAamp DNA Mini Kit (Qiagen GmbH, Germany) reagent
Box extracts, 80 μ L of final gained DNA solution；
2nd, free serum DNA methylates modification：
Take 1 μ g extract cast-off cells DNA, using QIAamp EpiTect Bis μ Lfite Kits (Qiagen GmbH,
Germany) kit is modified, 30 μ L of DNA solution after final gained modification；
3rd, the structure of real-time quantitative methylation status of PTEN promoter (QMSP) reaction system and optimization：
1) the real-time primer sequences of：
The primer of JAM3-M4：MF:CGTAGTTAGGGTTGGGATTC(SEQ ID NO:6)
MR:GAAATCCGACGACTATCCGA(SEQ ID NO:7),
The primer of β-actin：MF:TGGTGATGGAGGAGGTTTAGTAAGT(SEQ ID NO:16)
MR:AACCAATAAAACCTACTCCTCCCTTAA(SEQ ID NO:17)
2) optimization of real-time PCR reactions system：
Real-time PCR reactions suit comes from 1 × PowerGreen PCR Master Mix(ABI,USA)
Real-time PCR reactions system：
Template：DNA after 1 μ L------ modifications；
Primer：The 1 final concentration of 50nM of μ L-------；
MIX：10μL
ddH2O：8μL
Detection sensitivity, specificity, positive predictive value and negative predictive value and clinical mark to the detection method of the present invention
The verification and assessment of this detection application
1st, used kit is：
(1) DNA of cervical tissue and cast-off cells is extracted：QlAamp DNA Mini Kit(Qiagen GmbH,
Germany) kit；
(2) DNA methylation is modified：QIAamp EpiTect Bis μ Lfite Kits Qiagen GmbH, Germany) examination
Agent box；
(3) methylate and non-methylate DNA standard items：EpiTect PCR Control DNA Set(QIAGEN,
59695)；
(4) real-time PCR reactions are set with：1×PowerGreen PCR Master Mix(ABI,USA)；
(5) PCR reactions suit：AmpliTaq360Master Mix (ABI, USA)；
2nd, real-time PCR conditions：
Real-time PCR reactions condition：
3rd, Real time PCR results judge
(1) calculation formula of JAM3-M4 methylations is 2[Ct(β-actin)-Ct(target)]×10,000；
(2) the CT values of internal reference β-actin are more than 32, it is believed that sample does not meet Quality Control, and sample is unqualified；
(3) solubility curve of SYBR Green PCR systems is monitored, if the solution temperature of experiment sample and standard items
Solution temperature deviation ± 2 DEG C, then the sample be considered as invalid, need to detect again.
First, 27 sites of methylation status of PTEN promoter (MSP) 4 genes of Preliminary detection are in cervical cancer tissues and normal right
According to methylation differential expression in cervical tissue
The modification that methylate of 2 μ g clinical tissues sample DNAs is taken to obtain reaction template；As seen from Figure 1, in representativeness
Cancer and normal cervical tissues in, the visible obvious positive band of cancer, normally has no obvious band.Standard items：M standard items and U marks
Quasi- product and unmodified template demonstrate the specificity of primer.It is 5 most obvious that P value of the table 1 based on two groups lists difference
The positive number of cases and statistics P values in site.Explanation：By preliminary screening, the most obvious MSP primers of 5 pairs of differences are have selected into one
Step carries out the research of real-time PCR.
Table 1
Primer sequence is for example shown below：
The primer of JAM3-M4：MF:CGTAGTTAGGGTTGGGATTC(SEQ ID NO:6)
MR:GAAATCCGACGACTATCCGA(SEQ ID NO:7),
The primer of JAM3-U4：UF:TGTAGTTAGGGTTGGGATTT(SEQ ID NO:18)
UR:CAAAATCCAACAACTATCCA(SEQ ID NO:19),
The primer of CADM1-M2：MF:GTTTTTCGTTATTTGTTGTTTTC(SEQ ID NO:8)
MR:GAAACCGCGAAATACGAACG(SEQ ID NO:9),
The primer of CADM1-U2：UF:GTTTTTTGTTATTTGTTGTTTTTG(SEQ ID NO:20)
UR:AAAACCACAAAATACAAACA(SEQ ID NO:21),
The primer of CADM1-M8：MF:GTCGTCGTATATTGGGATTC(SEQ ID NO:10)
MR:TCTCATTAACTATCCGCTCG(SEQ ID NO:11),
The primer of CADM1-U8：UF:GTTGTTGTATATTGGGATTT(SEQ ID NO:22)
UR:TCTCATTAACTATCCACTCA(SEQ ID NO:23),
The primer of DAPK1-M2：MF:CGTTTGTAGGGTTTTTATTGGTC(SEQ ID NO:12)
MR:CTACCGCTACGAATTACCGA(SEQ ID NO:13),
The primer of DAPK1-U2：UF:TGTTTGTAGGGTTTTTATTGGTTG(SEQ ID NO:24)
UR:CCTACCACTACAAATTACCA(SEQ ID NO:25),
The primer of DAPK1-M3：MF:GTTTTTATTGGTCGTTTGTC(SEQ ID NO:14)
MR:GACGTTAACTCGATCCGACT(SEQ ID NO:15),
The primer of DAPK1-U3：UF:TTTTATTGGTTGTTTGTTGG(SEQ ID NO:26)
UR:CCCAACATTAACTCAATCCA(SEQ ID NO:27)。
2nd, in uterine neck without the verification in knurl change-precancerous lesion-cancer patient
1) 5 preliminary screening go out the verification-QMSP in site
It has collected uterine neck coming off carefully without knurl change-precancerous lesion-cancer patient in Shandong obstetrics and gynecology hospital outpatient service Vaginoscopy Room
Born of the same parents, including chronic cervicitis 53, CIN159, CIN271, CIN363 and cervical squamous cell carcinoma 20.From the above
Sample in extract DNA after, the bisulphite modified template as QMSP is carried out to it.MSP has filtered out cancer and normal group
5 sites -- CADM1-M2, CADM1-M8, DAPK1-M2, DAPK1-M3 and the JAM3- for the frequency difference degree maximum that methylates in knitting
M4, further verifies that it is being early diagnosed and the application value in examination by QMSP.Aggravated with lesion degree, gene loci
Methylation gradually rises, particularly evident with JAM3-M4, and the difference between diagnosis is grouped is obvious, as shown in Figure 2.
2) pyrosequencing verifies JAM3-M4 in methylate water of the uterine neck without knurl change-precancerous lesion-cancer patient's cast-off cells
It is flat
Select NILM (n=8) at random from each packet of sample of real-time PCR, CIN1 (n=10), CIN2QMSP are negative
(QM-) (n=9), the CIN2QMSP positives (QM+) (n=8), CIN3 (n=10), cancer (n=9) sample carry out pyrophosphoric acid survey
Sequence, further quantifies the methylation level in JAM3-M4 sites and verifies (Fig. 3 A and B).In order to real-time PCR before
Interpretation of result it is consistent, with clinic need gynecatoptron handle histodiagnosis be consistent, CIN1-, CIN2, CIN3+ classification Jiao
Phosphoric acid sequencing statistical analysis such as Fig. 3 C.
3rd, applied analyses of the JAM3-M4 in clinic
1) JAM3-M4 is applied individually to any clinical early screening
The method of inspection for being clinically widely used in early screening at present is cytology and HPV, and the inspection of JAM3-M4 is imitated
Energy (sensitiveness, specificity, positive predictive value and negative predictive value), such as table 2, is prompted and HPV and cytology compared with both
Compare, although sensitiveness and negative predictive value slightly decrease, specificity and positive predictive value be significantly increased, with
CIN3 shows particularly evident when being dividing value.
Table 2
2) application-United screening combined with cytology and shunting examination
1. United screening is i.e. in early screening, while application cell and DNA methylation assay, its power of test and and mesh
The contrast of preceding common cytology and HPV United screenings is as shown in table 3, although sensitiveness and negative predictive value slightly decrease,
But specificity and positive predictive value are significantly increased, and are showed when using CIN3 as dividing value particularly evident.
Table 3
2. shunting examination i.e. in early screening, application cell checks first, then to the trouble of cytodiagnosis exception
Person, carries out DNA methylation assay, it is shunted, and decides whether colposcopy and handle, the shunting efficiency of JAM3-M4 and
It is as shown in table 4 with the contrast of common HPV shuntings；In view of the specificity that cytodiagnosis is HSIL is higher, the meaning of shunting is not
Greatly, therefore the patient that JAM3-M4 shunting cytodiagnosises are LSIL and ASCUS is subjected to independent analysis, as shown in table 5, for dividing
The effectiveness analysis that cytodiagnosis is LSIL is flowed, specificity and positive predictive value significantly raise, and sensitiveness and negative predictive value
Do not have an impact；The effectiveness analysis for being ASCUS for shunting cytodiagnosis, specificity and positive predictive value significantly improve, sensitive
Property and negative predictive value have to be declined to a certain degree.But it is less than the cell of 30 years old (age bracket of HPV high infection rates) for the age
Be diagnosed as ASCUS patient, and 7 are the HPV positives, and 5 are CIN1-, and JAM3-M4 is feminine gender, prompt shunting meaning bigger.
Table 4
Table 5
3) application-United screening combined with HPV and shunting examination
1. United screening i.e. in early screening, while apply HPV and DNA methylation assay, its power of test and with present often
The contrast of cytology and HPV United screenings is as shown in table 6, although sensitiveness slightly decreases, specificity, the positive are pre-
Measured value significantly improves, and negative predictive value is grouped slight rise in CIN3+/CIN1- and CIN3+/CIN2- diagnostics, CIN2+/
CN1- slightly declines.
Table 6
2. shunting examination i.e. in early screening, checked first using HPV, then to the patient of the HPV positives, carry out methyl
Change detection, it shunted, decide whether colposcopy and handle, the shunting efficiency of JAM3-M4 and with it is common thin
The contrast of born of the same parents' credit stream is as shown in table 7；Specificity and positive predictive value significantly raise, and sensitiveness slightly decreases, negative predictive value
Slightly rise.
Table 7
4) the clinical diagnosis efficiency that ROC curve analysis JAM3-M4 methylates
ROC curve is a kind of common statistical method of clinical examination, can more different diagnostic tests to disease identification energy
Power.For different crowds --- the case of Fig. 4 (A) all collections, the case of (B) cytodiagnosis exception, (C) cytology are examined
Break be LSIL for the case of ASCUS, (D) cytodiagnosis case, the case of (E) HPV positives, by ROC curve (under curve
Area is measurement index) the clinical diagnosis efficiency that methylates of the JAM3-4 that analyzed and researched；Area under the curve prompting divides clinic
Stream is more significant.
5) indicative significance of CIN2 is shunted
In view of analyzing above, the clinical diagnosis efficiency using CIN3 as dividing value is above CIN2+/CIN1-.Assemble document point
Analysis, CIN lesions can be divided into science (CIN1 and CIN2) and transformant (CIN2 and CIN3) lesion.Science and transformant
CIN2 cannot be distinguished by by morphological observation, and the guide of 2014 editions WHO proposes to carry out the dye of immunohistochemistry P16 with Histological section
Color, splits into HSIL and LSIL, the immunohistochemical staining and pyrophosphoric acid of representational CIN2 cases are surveyed according to positive feminine gender by CIN
Sequence such as Fig. 5 shows.In all CIN2 cases, the positive rate of P16 is 69.78%, and the positive rate of JAM3-M4 is 48.84%. two
The match rate of person is 60.47% (p=0.119), although statistical significance is not notable, still there is certain shunting instruction meaning
Justice.
Although above-mentioned be described the embodiment of the present invention with reference to attached drawing, model not is protected to the present invention
The limitation enclosed, those skilled in the art should understand that, on the basis of technical scheme, those skilled in the art are not
Need to make the creative labor the various modifications that can be made or deformation still within protection scope of the present invention.
Claims (1)
1. a kind of kit for cervical carcinoma early screening, it is characterized in that：Including QlAamp DNA Mini Kit kits,
QIAamp EpiTect Bisulfite Kits kits, the forward primer sequence of JAM3-M4 is as shown in SEQ ID No.6, instead
To primer sequence as shown in SEQ ID No.7, the forward primer sequence of β-actin is as shown in SEQ ID No.16, reverse primer
Sequence is as shown in SEQ ID No.17, PCR reaction solution and ddH2O.
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| CN110564857B (en) * | 2019-10-21 | 2020-06-09 | 北京鑫诺美迪基因检测技术有限公司 | Composition and kit for early cervical cancer detection |
| CN113256627B (en) * | 2021-07-05 | 2022-06-07 | 深圳科亚医疗科技有限公司 | Apparatus and method for analysis management of cervical images, apparatus and storage medium |
| CN113667749A (en) * | 2021-08-03 | 2021-11-19 | 广东省人民医院 | Diagnostic kit for evaluating breast cancer risk by combining four key genes |
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| DNA methylation analysis in self-sampled brush material as a triage test in hrHPV-positive women;A Boers et al;《British Journal of Cancer》;20141231;第111卷;第1095-1101页 * |
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