CN107858433B - It is a kind of for detecting the kit of seminoma - Google Patents

Abstract

The invention discloses a kind of kits with for detecting seminoma, contain the detection reagent that can detect molecular labeling relevant to seminoma.The kit of seminoma of the invention has many advantages, such as that detection is simple, accurate, quick, high specificity.

Description

It is a kind of for detecting the kit of seminoma

Invention field

The present invention relates to detection technique fields, and in particular to a kind of for detecting the kit of seminoma.

Background technique

Seminoma (seminoma) originates from testis archaeocyte, is the most common tumour of testis, mostly occurs After the middle age, often to be Unilateral, right side is slightly more than left side.The more normal position testis of probability for betiding cryptorchidism is tens times high.It should Tumor is low potential malignancy.Naked eyes are seen, and testis enlargement, sometimes up to 10 times of normal volume, a small number of case Testicular Sizes are normal.Tumour Volume is not of uniform size, and only several millimeters of small person, for big person up to more than ten centimetres, general diameter is 3~5cm.

The I phase: male castration is postoperative to answer conventional line diaphragm paraaortic lymph nodes area radiotherapy (20-30Gy) included below. Preventative mediastinal irradiation is not done, because the region recurrence rate is extremely low.Risk of a part of patient since irradiation complication occurs The patient of larger and a small number of T1, T2 lesions can not do radiotherapy and simple Follow-up After is selected to observe.Two methods are former to I phase essence thin Born of the same parents' tumor patient's cure rate is almost 100%.But 15-20% can recur in the patient of non-row adjuvant radiotherapy, middle position recurrence time It is postoperative 12 months, is recurred after also having postoperative 5 years.It can still be cured after recurrence through chemotherapy.

The II phase: male castration is postoperative should row diaphragm aorta side included below and ipsilateral iliac vessels side lymph interface radiotherapy (35-40Gy).Preventative mediastinal irradiation is not done.If patient suffers from horseshoe kidney, is then not suitable for radiotherapy, is given by the GCT of good prognosis Chemotherapy.

IIC the and III phase: IIC phase patient has huge retroperitoneal lymph node to shift, then (sees below by the GCT row chemotherapy of good prognosis State risk factor classification).Whether Tumoral survival is had according to imageological examination after chemotherapy, take respectively observation follow-up, operation excision/ Biopsy or radiotherapy.Such as select to perform the operation, then it is not recommended that row Retroperitoneal benign tumor because seminoma patient due to Extensive fibrosis and there are technical difficulties, may cause serious complication.The row solution treatment if CT prompts lesion growth It treats.Row chemotherapy is classified according to prognosis if (such as mediastinum) seminoma patient outside III phase or sexual gland.Internal organ turn in addition to there are lung Shifting person is other than poor risk, other patients IIIC are the patient of good prognosis, and about 90% advanced stage can be by controlling containing cisplatin More.

The recurrence after radiotherapy of I phase and IIA, IIB: 3 period BEP or 4 periods can be given by the nonseminoma of good prognosis EP scheme chemotherapy.Cure rate 90% or so.Patient's (having visceral metastases other than lung) of medium prognosis gives 4 period BEP or participation Clinical test.After chemotherapy, if remaining lesion, which is greater than 3cm, is contemplated that operation or radiotherapy or follow-up observation.

Initially the research about GCT combined chemotherapy starts from 1970s, using bleomycin (BLM), vinblastine (VLB) and the BVP Regimen Chemotherapy metastatic GCT of cis-platinum (DDP) can make patient 70-80% up to complete incidence graph (CR).But the program With adverse reaction serious recent and at a specified future date, including neurotoxicity, bone marrow suppression, renal toxicity, ototoxicity, the relevant lung of BLM Toxicity, VP16 may cause leukaemia, Raynaud's phenomenon etc..Since the high-efficiency of chemotherapy and serious toxicity, people are continuous Patient is layered by exploration, corresponding treatment is given according to different Prognostic Characteristics, to avoid over-treatment and insufficient therapy. Research finds that staging and serum markers and prognosis have close relation, therefore, patient was once divided into good prognosis and poor prognosis Two groups.The early symptom of gastric cancer has also been proposed international germinoma meeting classification (international germinoma cooperation recently Group, 1997), and prognosis grouping is added in germinoma AJCC staging scale.The classification by patient be divided into good prognosis, Prognosis is medium and poor prognosis three classes (being shown in Table 1).And etoposide (VP16) is made into neurotoxicity instead of the BEP scheme of VLB Rate is substantially reduced.

The classification of risks

Seminoma-clinical stages:

The histological type of seminoma is related with prognosis, and the range of degree and transfer that tumour is spread also affects pre- Afterwards.Therefore clinician is not only it is to be understood that the histological type of tumour, but also to be formulated according to the difference of extent of disease and be controlled accordingly Treatment scheme.It is thus determined that each disease variation phase is of practical significance.Commonly used method by stages now are as follows:

I phase: tumour is confined in testis and epididymis, and not yet breaks through coating or intrusion spermatic cord, no lymphatic metastasis.

II phase: having transfer by physical examination, x-ray inspection confirmation, can spread spermatic cord, scrotum, Ilioinguinal approach lymph node, But without departing from lymphatic districts after peritonaeum.Metastatic lymph node clinic fails to lay one's hand on and person was II a phase, and clinical examination is laid one's hand on and lymphonodi coeliaci Person was II b phase.

III phase: the existing above lymphatic metastasis of diaphragm or DISTANT METASTASES IN.Also there is researcher that DISTANT METASTASES IN person was included into for IV phase.

Seminoma is the pernicious enblastoma of the most common mediastinum, accounts for the 2%~4% of mediastinal tumor, it is pernicious to account for mediastinum The 13% of tumour accounts for the 50% of mediastinum malignant germ cell tumors.It is nearly all young man, peak age of onset 20-40 It is located at anterior mediastinum in year, 80% has symptom.The patient of 20%-30% is asymptomatic, Symptomatic patient symptom be pectoralgia, cough, Expiratory dyspnea, hemoptysis etc., can there is drowsiness, weight loss.There is obstruction of superior vena cava syndrome in 10%~20% patient.These Clinical symptoms are often related with compressing, infringement of the tumour to mediastinal structures.A part of seminoma is grown in intratracheally, and local Extend to neighbouring mediastinum and lung.General mediastinum seminoma is sent out through Lymphatic channel transfer, and hematogenous metastasis, bone can also occur Bone and lungs are the positions most often shifted.

The common huge tumor of anterior mediastinum of rabat, sometimes it can be found that tumour is along intratracheal growth.CT is mostly even density Big mass, 50% it is visible it is intrathoracic transfer or extend beyond anterior mediastinum without that can perform the operation.CT and MRI aids in determining whether the model of tumour It encloses, to the infringement situation of mediastinal structures.Resection rate of going to a doctor for the first time is lower than 25%.

It is horizontal to cope with all young men measurement blood α-FP, β-hCG for suffering from tumor of anterior mediastinum.Simple seminoma is several Without the raising of AFP, hCG, 7%-10% has hCG raising, but is often no more than 100ng/ml, and AFP is not increased.CA125 can also It can be biological marker.The chromosome analysis of tumor tissues can find characteristic isochromosome on No. 12 chromosomes, this is to mirror Other germinoma and other kinds of tumour help.

In the diagnosis of seminoma, the direct detection mainly sampled at present by sample, this method has certain Hysteresis quality, until find be disease later period, for early diagnose it is nonsensical, delayed the state of an illness.And from molecular level Diagnosis is the conventional method of current cancer detection, has discovery property earlier.But currently, alpha-fetoprotein (AFP), human chorionic The diagnosing tumour of gonadotrophin beta-subunit (HCG) and lactic dehydrogenase (LDH) as TGCTs marks (9).However, not yet reflecting Make seminoma specific tumor marker.There has been no endoplasmic reticulum nuclear signal transducin 2 (ERN2) and spermatogoniums at present The report of tumor correlation.Therefore, the side of further exploitation detection seminoma people at highest risk and susceptible individual are badly in need of in this field Method and its kit.

Summary of the invention

For the above-mentioned prior art, present inventor is by deeply widely studying, to a large amount of candidate genes It is determined and analyzes, find that ERN2 and seminoma neurological susceptibility are closely related for the first time, it is susceptible to can be used as seminoma Property detection or early diagnosis molecular labeling, therefore, the present invention provides one kind for detecting seminoma neurological susceptibility point Method of the primer and kit and vitro detection sample of son label with the presence or absence of the single nucleotide polymorphism of ERN2 gene.

To achieve the above object, the present invention adopts the following technical solutions:

A kind of molecular labeling relevant to seminoma neurological susceptibility, nucleotide sequence as shown in SEQ ID NO:1, Compared with normal ERN2 gene, difference is: the 61st there are a single nucleotide polymorphism: C > T, i.e. the 61st base It is normal ERN2 gene, when for base T, for mutation when for base C including two kinds of situations of base C and base T ERN2 gene.

A kind of for detecting the kit of seminoma, including specific primer, the primer is a pair, has SEQID The sequence of NO:2 and SEQ ID NO:3 is capable of the primer of the amplification ERN2 gene of specificity, can specifically amplify length 241bp。

Further, kit further includes PCR reaction solution, and PCR reaction solution is by dNTP, Mg2+, Taq enzyme and Buffer group At.The dosage relation of each component is conventional in PCR reaction solution, is common knowledge for one of ordinary skill in the art.

A kind of vitro detection sample is with the presence or absence of the method for the single nucleotide polymorphism of ERN2 gene, and steps are as follows:

(1) with the ERN2 gene of ERN2 gene-specific primer amplification sample, amplified production is obtained；The primer is one It is right, the sequence with SEQ ID NO:2 and SEQ ID NO:3；

(2) sequencing is carried out to amplified production, detects and whether there is following single nucleotide polymorphism in amplified production: 61C>T；The mutated site numbers the sequence based on SEQ ID NO:1.

The method that a kind of pair of individual seminoma neurological susceptibility or risk are detected or diagnosed, steps are as follows:

Detect ERN2 gene, transcript and/or the albumen of the individual, and with normal ERN2 gene, transcript and/or egg It is white to be compared, a possibility that indicating that the individual seminoma is had differences higher than normal population.The difference refers to No there are single nucleotide polymorphism: 61C > T；The mutated site numbers the sequence based on SEQ ID NO:1.

Of the invention examines for detecting the molecular labeling, kit and its method of seminoma neurological susceptibility, and in vitro Sample is with the presence or absence of the method for the single nucleotide polymorphism of ERN2 gene, and detection is simple, accurate, quick, high specificity, for Seminoma patient implements effective early diagnosis, early control is of great significance with individuation prevention and treatment.

Specific embodiment

Below by specific example, the present invention will be further elaborated, it should explanation, following the description be only for It explains the present invention, its content is not defined.

The detection of 1 ERN2 gene mutation of embodiment and association analysis with seminoma

1.1 research object

Seminoma Patients with 300 Cases is chosen, in January, 2009~2016 year are true in First Affiliated Hospital of Soochow University,Suzhou October Diagnosis and treatment are treated, and clinical data is obtained from case history.Age similar control 300, is medical center physical examination healthy population.Take the experimenter Member peripheral blood 1.5ml, it is stored refrigerated in -80 DEG C.All subjects are according to First Affiliated Hospital of Soochow University,Suzhou ethics committee member It may require that informed consent.

1.2 DNA are extracted

Subject's peripheral blood DNA is extracted with conventional phenol chloroform method, specific as follows:

1) take 1ml peripheral blood, 3-6 DEG C of 1600g is centrifuged 8-12min, by supernatant in: after 4 DEG C of 16000g centrifugation 8-12min Supernatant is taken again；

2) it takes the supernatant being centrifuged in step 1) through 16000g in centrifuge tube, adds isometric digestive juice and protease Compound, 55-62 DEG C of incubation 30min；

3) sample that step 2) obtains is cooled down on ice, NH is then added₄AC protein precipitation, and mix well, 5000g It is centrifuged 4-6min；

4) transfer step 3) supernatant mixes with 0.8 times of volume magnetic bead suspension, it is incubated at room temperature 1min；

5) step 4) is obtained sample to be placed on magnetic frame after standing adsorption, draws supernatant；

6) supernatant obtained step 5) and 1.5 times of volume magnetic bead suspensions are incubated at room temperature lOmin, are placed on magnetic frame After standing adsorption, supernatant is discarded, with 75% ethanol wash 2 times to get magnetic bead-target DNA complex.

7) DNA eluent is used, magnetic bead-target DNA complex that step 6) is obtained vortex in DNA eluent shakes It swings, is placed on magnetic frame and stands, Aspirate supernatant obtains target DNA.

1.3 primers, PCR amplification and sequencing

ERN2 genome sequence (GeneLocation:XM_011545712.2) is downloaded from GenBank, uses primer Premier5.0 design primer.Specific primer details are shown in Table 2.

2 primer sequence table of table

Primer	Sequence	SEQIDNO:
			Upstream primer	5'-CCGAACACAGTATACGGTCA-3'	2
Downstream primer	5'-CAGGCCGTCCTGGTGCCAGG-3'	3

PCR amplification condition: 94 DEG C 3 minutes, (94 DEG C 30 seconds, 62 DEG C 25 seconds, 72 DEG C 40 seconds) × 32,72 DEG C 10 minutes, 10 DEG C heat preservation.Pcr amplification product is 241bp.

Pcr amplification product delivers the sequencing of Shanghai Sheng Gong biotech company, and sequencing result application software is tested and divided Analysis, this implementation are analyzed, and there are following SNP:61C > T for discovery.

1.4 SNP partings and association analysis

Application card side (X²) examine it is for statistical analysis to the distribution in subject site；When the expectation of a cell It accurately examines and is analyzed using Fisher when number is less than 5.All P values are bilateral probability, think to have as P < 0.05 aobvious Write statistical significance.Using unconditional logistic regression analysis to genotype frequency, gene frequency and seminoma illness Between correlation assessed, calculate its odds ratio (Odds ratio, OR) and 95% credibility interval (confidence Interval, CI), and application SPSS17.0 software (SPSS Inc.Chicago, Illinois, USA) is for statistical analysis.

Significant related, the wherein additive inheritance as a result, it has been found that the site 61C > T in SEQ ID NO:1 and seminoma are fallen ill The P value of model (C vs T) is 1.31x10^-8(OR=2.94,95%CI (2.00,4.31)), dominant inheritance model (C/C vs C/T+T/T P value) is 2.33x10^-13(OR=2.96,95%CI (2.92,3.98)), recessive inheritance model (C/C+C/T vs T/T P value 1.31x10)^-4(OR=6.21,95%CI (2.14,18.00)).Detailed results are shown in Table 3.

3 61C of the table > distribution of T loci gene type and allele in seminoma group and healthy control group

Embodiment 2

Seminoma neurological susceptibility detection kit

Due to the mutation of 61C > T in SEQ ID NO:1 deliver with seminoma it is highly relevant, can be prominent based on this Become design NER2 gene-specific primer and is extended detection by template of the DNA of patient again.

Prepare a kit (100 person-times), the composition of kit are as follows: upstream and downstream primer, dNTP, Mg2+, Taq enzyme, PCR Buffer, reaction system are 20 microlitres.

Extracting subject's peripheral blood 2ml, (wherein subject is 10 newly identified seminomas, 2 control Healthy Peoples Group), conventional method extracts DNA.PCR reaction, PCR amplification condition: 94 are carried out using seminoma neurological susceptibility detection kit DEG C 3 minutes, (94 DEG C 30 seconds, 62 DEG C 25 seconds, 72 DEG C 40 seconds) × 32,72 DEG C 10 minutes, 10 DEG C of heat preservations.Pcr amplification product is 241bp.Reaction product is sequenced, sequencing result is tested using Meglign 7.0 and 2.33 software of Chromas And analysis.The subject that testing result contains 61C > T mutation is seminoma patient, and 2 controls do not detect described Mutation.

It should be understood that those skilled in the art can be the present invention respectively after having read above content of the invention Kind modification or change, but equivalent forms of these changes or modification are also fallen in limited range of the present invention.

Sequence table

<110>Suzhou Li Hao Biotechnology Co., Ltd

<120>a kind of for detecting the kit of seminoma

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 241

<212> DNA

<213>people (Homo sapiens)

<400> 1

ccgaacacag tatacggtca ccatgcatga cccaagagcc ccagccctgc gctggaacac 60

tacctaccgc cgctactcag cgccccccat ggatggctca cctgggaaat acatgagcca 120

cctggcgtcc tgcgggatgg gcctgctgct cactgtggac ccaggaagcg ggacggtgct 180

gtggacacag gacctgggcg tgcctgtgat gggcgtctac acctggcacc aggacggcct 240

g 241

<210> 2

<211> 20

<212> DNA

<213>artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 2

ccgaacacag tatacggtca 20

<210> 3

<211> 20

<212> DNA

<213>artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 3

caggccgtcc tggtgccagg 20

Claims (5)

1. a kind of for detecting the kit of seminoma, it is characterised in that: it, which contains, is able to detect and seminoma phase The detection reagent of the molecular labeling of pass, molecular labeling its nucleotide sequence such as SEQ ID NO relevant to seminoma: Shown in 1, compared with normal ERN2 gene, difference is: in SEQ ID NO:1 the 61st, there are a mononucleotide polymorphics Property: C > T.

2. kit as described in claim 1, which is characterized in that the detection reagent is following primer, sequence such as sequence table Shown in middle SEQ ID NO:2 and SEQ ID NO:3.

3. such as the described in any item kits of claim 1-2, which is characterized in that the kit further includes having PCR reaction solution, PCR reaction solution is by dNTP, Mg²⁺, Taq enzyme and Buffer composition.

Primer pair shown in 4.SEQ ID NO:2 and SEQ ID NO:3 is in preparation for detecting the relevant reagent of seminoma Application in box.

5. a kind of molecular labeling, nucleotide sequence is as shown in SEQ ID NO:1, compared with normal ERN2 gene, difference Be: in SEQ ID NO:1 the 61st, there are a single nucleotide polymorphism: C > T.