CN108977576A - A kind of liver cancer earlier evaluations method and its detection primer and detection kit - Google Patents
A kind of liver cancer earlier evaluations method and its detection primer and detection kit Download PDFInfo
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Abstract
The invention discloses a kind of liver cancer earlier evaluations method and its detection primers and detection kit, and described detection method includes the following steps: (1) extracting the DNA of sample to be tested；(2) specific primer is used, using DNA obtained by step (1) as template, carries out PCR amplification detection, wherein the specific primer is upstream detection primer, sequence is as shown in SEQ ID No:1 in sequence table；The specific primer is downstream detector primer, and sequence is as shown in SEQ ID No:7 in sequence table；(3) according to step (2) PCR amplification testing result, liver cancer risk is assessed.The detection method and detection kit have high sensitivity, advantage easy to operate.In each reaction system, as long as can be detected comprising 5 HBV DNA moleculars, the kit index of the existing import of remolding sensitivity is significantly improved, can be with earlier evaluations liver cancer risk.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of liver cancer earlier evaluations method and its detection primer and detection
Kit.
Background technique
Hepatitis B (Hepatitis B Virus, HBV) is a kind of important communicable disease, can be by blood etc.
Various body fluid communications cause oxyhepatitis, chronic hepatitis, cirrhosis, liver cancer etc., seriously threaten the world mankind health and
Life security.Wherein, China is the hotspot of hepatitis B again, it is estimated that, there are about 60% people once to infect hepatitis B,
Up to 10% is carry, at least 15~25% ultimately succumbs to the relevant hepatopathy of HBV in infection population, most of is hepatocellular carcinoma.
Therefore, accurate, rapid diagnosis hepatitis B seems particularly important to the earlier evaluations of liver cancer and treatment and prognosis.
It currently, is mainly enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction to the detection means of HBV
(PCR).Wherein the foundation of ELISA scheme is the blood serum designated object (HBV-M) that hepatitis B is detected according to antigen-antibody reaction
The concentration of HBsAg, HBeAg, HBcAb, HBsAb, HBeAb are detected, however the mainly human body of program reflection is to HBV infection
Immune state, but cannot directly reflect that HBV, can not be as judging whether patient has biography in the duplication situation of patient's body
The positive evidence of metachromia.PCR scheme directly expands and detects the DNA of serum HBV, therefore is able to reflect out HBV in vivo
It is true to expand state and its whether there is situations such as infectiousness.Therefore should be the result is that judging whether internal HBV is replicating, it is multiple
Processing procedure degree or the important evidence that whether HBV is removed etc. after treating, and turn for clinical observation curative effect of medication, the state of an illness
Change etc. provides direct basis.Since PCR scheme is a kind of simple and effective, has very high detection sensitivity and specificity,
The program has become the powerful of HBV molecular diagnosis as ELISA scheme.
PCR scheme has been widely adopted since 1997 are approved for clinical detection, and there has also been many kits
It is used.But the kit sensitivity that HBV detection kit sensitivity at present based on PCR scheme is not high, domestic
In 10E3 copy/ml, and the sensitivity of import reagent box is 300 copies/ml, cause the application of HBV detection kit still have compared with
Big limitation.
Summary of the invention
Therefore, the problem not high for the HBV detection method sensitivity based on current PCR scheme, the present invention provides
A kind of new liver cancer earlier evaluations method and its detection primer and detection kit.
In order to solve the above technical problems, one of the technical solution that the present invention takes are as follows: a kind of liver cancer earlier evaluations method, institute
State that detection method includes the following steps:
(1) DNA of sample to be tested is extracted；
(2) specific primer is used, using DNA obtained by step (1) as template, PCR amplification detection is carried out, wherein the specificity is drawn
Object is upstream detection primer, and sequence is as shown in SEQ ID No:1 in sequence table；The specific primer draws for detected downstream
Object, sequence is as shown in SEQ ID No:7 in sequence table；More preferably, the specific primer further includes closing primer, the envelope
Closing primer is SEQ ID No:2, SEQ ID No:3, SEQ ID No:4, SEQ ID No:5, SEQ ID No:6, SEQ ID
One or more of No:8, SEQ ID No:9, SEQ ID No:10, SEQ ID No:11 and SEQ ID No:12 sequence；
(3) according to step (2) PCR amplification testing result, hepatitis B is detected.
Wherein step (1) is to extract the DNA of sample to be tested.The sample to be tested is the sample to be tested of this field routine, packet
Serum is included, is organized, cell or other samples to be tested.Wherein the extracting method of the DNA of the sample to be tested is that this field is conventional
DNA extraction method, as long as can extract the DNA in sample to be tested, the DNA extraction method includes phenol chloroform
Method, or the DNA using commercially available DNA extraction agent box extracting sample.
Wherein step (2) are as follows: specific primer is used, using DNA obtained by step (1) as template, carries out PCR amplification detection,
Wherein the specific primer is upstream detection primer, and sequence is as shown in SEQ ID No:1 in sequence table；The specificity is drawn
Object is downstream detector primer, and sequence is as shown in SEQ ID No:7 in sequence table；More preferably, the specific primer further includes
Primer is closed, the closing primer is SEQ ID No:2, SEQ ID No:3, SEQ ID No:4, SEQ ID No:5, SEQ ID
One of No:6, SEQ ID No:8, SEQ ID No:9, SEQ ID No:10, SEQ ID No:11 and SEQ ID No:12
Or several sequences.The preparation method of specific primer of the present invention is this field customary preparation methods, preferably complete sequence
It is artificial synthesized.Closing primer of the present invention is preferably meant that primer 3 ' holds base to be closed, and cause the closing primer can not
PCR reaction, wherein the closed method is the closed method of this field routine, the 3 ' of the preferably described closing primer
Terminal bases are closed with amino, phosphoric acid closing or double deoxidation are closed, it is made to lose the ability for carrying out PCR reaction.
Wherein step (2) specific primer is upstream detection primer, and sequence is preferably SEQ ID in sequence table
Shown in No:25；The specific primer is downstream detector primer, and sequence is preferably SEQ ID No:31 institute in sequence table
Show；The specific primer further includes closing primer, and the closing primer is more preferably SEQ ID No:26, SEQ ID No:
27, SEQ ID No:28, SEQ ID No:29, SEQ ID No:30, SEQ ID No:32, SEQ ID No:33, SEQ ID
One or more of No:34, SEQ ID No:35 and SEQ ID No:36 sequence.
Wherein step (2) specific primer is upstream detection primer, and sequence is more preferably SEQ ID in sequence table
Shown in No:13；The specific primer is downstream detector primer, and sequence is more preferably SEQ ID No:19 institute in sequence table
Show；The specific primer further includes closing primer, and the closing primer is more preferably SEQ ID No:14, SEQ ID No:
15, SEQ ID No:16, SEQ ID No:17, SEQ ID No:18, SEQ ID No:20, SEQ ID No:21, SEQ ID
One or more of No:22, SEQ ID No:23 and SEQ ID No:24 sequence.Specific primer sequence of the present invention
In, the meaning of the mononucleotide character in addition to ACGT is preferably: A, T, C=H；G,T,C=B；G,A,T=D；A,C,G=V.
Wherein the condition of step (2) the PCR amplification detection is preferably: (1) 90-96 DEG C initial denaturation 10 seconds -600
Second；(2) it is denaturalized -90 seconds 5 seconds for 90-96 DEG C；(3) it anneals -99 seconds 5 seconds for 45-65 DEG C；(4) extend -300 seconds 3 seconds for 55-73 DEG C；Step
(2)-(4) cycle-index is 28-60；(5) extend -600 seconds 5 seconds for 60-73 DEG C.The condition of the PCR amplification is more preferably (1) 95
DEG C initial denaturation 3 minutes；(2) 95 DEG C are denaturalized 30 seconds, 47-60 DEG C of (3) annealing 30 seconds, 63-68 DEG C of (4) extension 1 minute, step (2)-
(4) cycle-index is 36 times, and (5) 72 DEG C extend 5 minutes.
Wherein the reaction system of step (2) the PCR amplification detection is preferably: 2 × PCR reaction buffer, 25 μ l, spy
1 μ l of specific primer solution middle and upper reaches primer solution, described its close any one or whole in primer, and it is molten that closing primer is added
Liquid 0.1-5 μ l；Then 1 μ l of downstream primer solution, 5 μ l of gained sample DNA extract are added MiliQ water and supply to 50 μ l, described
The concentration of specific primer solution and closing primer is preferably 10mM.Preferably, doing quantitative PCR in PCR reaction system
When, negative control is done with the blank pipe of no positive control sample, while gradient dilution is made according to the titer being had, from every microlitre
1-3 copies 10E8 copy to and does quantitative PCR reaction, regression equation is calculated through Ct value, so as to HBV in absolute assessment sample
Absolute concentration.Preferably, in Standard PCR detection scheme, gradient dilution is made according to the titer being had simultaneously, from every micro-
Liter 1-3 copies 10000 copies to and does PCR reaction, as positive control, while having blank pipe as negative control.Preferably, this hair
The bright liver cancer earlier evaluations method, is the detection method not for the purpose of the diagnosing and treating of disease.
In order to solve the above technical problems, the two of the technical solution that the present invention takes are as follows: a kind of liver cancer earlier evaluations it is special
Property primer, the specific primer be upstream detection primer, sequence is as shown in SEQ ID No:1 in sequence table；It is described special
Property primer be downstream detector primer, sequence is as shown in SEQ ID No:7 in sequence table；More preferably, the specific primer is also
Including closing primer, the closing primer is SEQ ID No:2, SEQ ID No:3, SEQ ID No:4, SEQ ID No:5,
In SEQ ID No:6, SEQ ID No:8, SEQ ID No:9, SEQ ID No:10, SEQ ID No:11 and SEQ ID No:12
One or more of sequences.
In order to solve the above technical problems, the three of the technical solution that the present invention takes are as follows: a kind of liver cancer earlier evaluations it is special
Property primer, the specific primer be upstream detection primer, sequence is as shown in SEQ ID No:13 in sequence table；It is described special
Property primer be downstream detector primer, sequence is as shown in SEQ ID No:19 in sequence table；More preferably, the specific primer is also
Including closing primer, the closing primer is SEQ ID No:14, SEQ ID No:15, SEQ ID No:16, SEQ ID No:
17, SEQ ID No:18, SEQ ID No:20, SEQ ID No:21, SEQ ID No:22, SEQ ID No:23 and SEQ ID
One or more of No:24 sequence.
In order to solve the above technical problems, the four of the technical solution that the present invention takes are as follows: a kind of liver cancer earlier evaluations it is special
Property primer, the specific primer be upstream detection primer, sequence is as shown in SEQ ID No:25 in sequence table；It is described special
Property primer be downstream detector primer, sequence is as shown in SEQ ID No:31 in sequence table；More preferably, the specific primer is also
Including closing primer, the closing primer is SEQ ID No:26, SEQ ID No:27, SEQ ID No:28, SEQ ID No:
29, SEQ ID No:30, SEQ ID No:32, SEQ ID No:33, SEQ ID No:34, SEQ ID No:35 and SEQ ID
One or more of No:36 sequence.
The preparation method of specific primer of the present invention is this field customary preparation methods, and preferably complete sequence is artificial
Synthesis.Closing primer of the present invention is preferably meant that primer 3 ' holds base to be closed, and makes the closing primer that can not cause PCR anti-
It answers, wherein the closed method is the closed method of this field routine, 3 ' ends of the preferably described closing primer
Base is closed with amino, phosphoric acid closing or double deoxidation are closed, it is made to lose the ability for carrying out PCR reaction.
In order to solve the above technical problems, the five of the technical solution that the present invention takes are as follows: a kind of detection of liver cancer earlier evaluations
Kit, the detection kit include: specific primer, and the specific primer is upstream detection primer, sequence such as sequence
In table shown in SEQ ID No:1；The specific primer is downstream detector primer, SEQ ID No:7 in sequence such as sequence table
It is shown；More preferably, the specific primer further includes closing primer, and the closing primer is SEQ ID No:2, SEQ ID No:
3, SEQ ID No:4, SEQ ID No:5, SEQ ID No:6, SEQ ID No:8, SEQ ID No:9, SEQ ID No:10,
One or more of SEQ ID No:11 and SEQ ID No:12 sequence；It further include 2 × PCR reaction buffer, positive control,
And negative control.
More preferably, the detection kit further includes specific primer, and the sequence of the specific primer is upstream detection
Primer, sequence is as shown in SEQ ID No:13 in sequence table；The specific primer is downstream detector primer, sequence such as sequence
In list shown in SEQ ID No:19；More preferably, the specific primer further includes closing primer, and the closing primer is SEQ
ID No:14, SEQ ID No:15, SEQ ID No:16, SEQ ID No:17, SEQ ID No:18, SEQ ID No:20, SEQ
One or more of ID No:21, SEQ ID No:22, SEQ ID No:23 and SEQ ID No:24 sequence；The specificity
Primer is upstream detection primer, and sequence is as shown in SEQ ID No:25 in sequence table；The specific primer is detected downstream
Primer, sequence is as shown in SEQ ID No:31 in sequence table；More preferably, the specific primer further includes closing primer, institute
Stating closing primer is SEQ ID No:26, SEQ ID No:27, SEQ ID No:28, SEQ ID No:29, SEQ ID No:30,
One of SEQ ID No:32, SEQ ID No:33, SEQ ID No:34, SEQ ID No:35 and SEQ ID No:36 or several
Kind sequence.
2 × PCR reaction buffer of the present invention is the PCR reaction buffer of this field routine, and formula is preferably
Are as follows: 10mM-200mM TrisCl, 50mM-200 mM KCl, the 1mM-15mM MgCl that pH is 8.82, every kind of 10 μM of dNTP-
800 μM, 0.1M-2M glycine betaine, 0.1-3M trehalose, 0.5U-5U Taq enzyme/100 μ l, 0.01 μ g-10 μ g/ml SYBR
Green；Mass percent is that 0.001%-0.5% is phenol red；0.02%-2% bovine serum albumin(BSA), 0.001%-5% gelatin；Volume hundred
Divide than being 5%-30% glycerol, 0.1%-20% deionized formamide, 0.1%-10% dimethyl sulfoxide and 0.01 μ l-10 μ l Taq enzyme
Antibody/ml.
Wherein the positive control is the positive control of this field routine, and the negative control is the yin of this field routine
Property control, the preferably serum of no hepatitis B virus DNA pollution.
On the basis of common knowledge of the art, above-mentioned each optimum condition, can any combination to get each preferable reality of the present invention
Example.
The reagents and materials used in the present invention are commercially available.
The positive effect of the present invention is that:
1, high sensitivity, in each reaction system, as long as both can detect comprising 5 HBV DNA moleculars.It is extracted according to this system
HBV DNA, and PCR reaction is carried out, in this way, when the HBV DNA in serum reaches 100 molecules, so that it may detected, spirit
Sensitivity is significantly increased than the corresponding index of the kit of existing report import.
2, easy to operate, when operating using Standard PCR scheme, inspection can be realized using common domestic PCR instrument
Survey purpose.
3, it due to the raising of detection sensitivity, can be detected within the incubation period of HBV, so as to liver
Cancer risk gives warning in advance.
4, for clinical detection, especially medication effect, stronger support is provided.
5, the detection of HBV is carried out according to the program, it is not necessary to purchase expensive import equipment, and require testing laboratory
It reduces, is not only suitable for the perfect sensing chamber of laboratory equipment, the at county level clinical laboratory relatively simple and crude for experimental assembly condition
Liver cancer earlier evaluations also can be smoothly unfolded in room.
6, newly-designed primer: selected primer itself has and efficiently synthesizes, the extremely low advantage of nonspecific reaction.
The use for closing primer, significantly reduces nonspecific amplification, while PCR reaction can be made in a more relaxed item
It is carried out under part, to annealing and the temperature requirement extended no longer as previous scheme is so stringent, thus the requirement to PCR instrument is non-
Constant width pine, while yet loose many to the temperature requirement of detection environment does not need between constant temperature.
7, due to 2 × PCR reaction buffer using oneself unique design, so that PCR reaction can be reached at 60-66 DEG C
To best, this low temperature extension program, the use limitation of routine PCR reaction is broken through.
Specific embodiment
The present invention is further illustrated below by the mode of embodiment, but does not therefore limit the present invention to the reality
It applies among a range.In the following examples, the experimental methods for specific conditions are not specified, according to conventional methods and conditions, or according to quotient
The selection of product specification.
Embodiment 1
Extract sample DNA.Specific primer: specific upstream detection primer, sequence such as SEQ ID No:1 institute in sequence table
Show；Downstream detector primer, sequence is as shown in SEQ ID No:7 in sequence table；Special closing primer is SEQ ID No:2, SEQ
ID No:3, SEQ ID No:4, SEQ ID No:5, SEQ ID No:6, SEQ ID No:8, SEQ ID No:9, SEQ ID
One or more of No:10, SEQ ID No:11 and SEQ ID No:12 sequence；
The reaction system of PCR amplification: 10mM TrisCl (pH8.8), 50 mM KCl, 1.5 mM MgCl2, every kind of dNTP 200
μM, the SYBR Green of 1M glycine betaine, 1M trehalose, 0.5U Taq enzyme/100 μ l, 0.5 μ g/ml；Mass percent is 0.02%
Bovine serum albumin(BSA) (BSA), 0.015% gelatin (Gelatin)；Percent by volume is 10% glycerol, 10% deionized formamide, 10%
Dimethyl sulfoxide (DMSO) and 10 μ l Taq enzyme antibody/ml.
The reaction condition of PCR amplification: the condition of the PCR amplification be more preferably (1) 95 DEG C initial denaturation 3 minutes；(2) 95 DEG C
Denaturation 30 seconds, (3) 55 DEG C are annealed 30 seconds, and (4) 65 DEG C extend 1 minute, and step (2)-(4) cycle-index is 36 times, and (5) 72 DEG C are prolonged
It stretches 5 minutes.Acquired results are analyzed, it can be determined that test object whether Hepatitis B carrier, and to liver cancer risk carry out
Earlier evaluations.
Embodiment 2
Extract sample DNA.The upstream detection primer of specificity, sequence is as shown in SEQ ID No:13 in sequence table；Downstream inspection
Primer is surveyed, sequence is as shown in SEQ ID No:19 in sequence table；Specificity closing primer is SEQ ID No:14, SEQ ID
No:15, SEQ ID No:16, SEQ ID No:17, SEQ ID No:18, SEQ ID No:20, SEQ ID No:21, SEQ ID
One or more of No:22, SEQ ID No:23 and SEQ ID No:24 sequence.
The reaction system of PCR amplification: 10 mM TrisCl (pH8.8), 50 mM KCl, 1.5 mM MgCl2, every kind of dNTP
200 μM, 1.5M glycine betaine, 2M trehalose, 5U Taq enzyme/100 μ l, 0.5 μ g/ml SYBR Green；Mass percent is
0.003% is phenol red；0.5% bovine serum albumin(BSA) (BSA), 0.05% gelatin (Gelatin)；Percent by volume is 10% glycerol, 8% goes
Formamide, 10% dimethyl sulfoxide (DMSO) and 5 μ l Taq enzyme antibody/ml
The reaction condition of PCR amplification: (1) 94 DEG C initial denaturation 3 minutes；(2) 92 DEG C are denaturalized 60 seconds, and (3) 58 DEG C are annealed 30 seconds, (4)
72 DEG C extend 1 minute, and step (2)-(4) cycle-index is 36 times, and (5) 72 DEG C extend 5 minutes.Acquired results are analyzed,
Judge test object whether Hepatitis B carrier, and to liver cancer risk carry out earlier evaluations.
Embodiment 3
Extract sample DNA.Specific primer: upstream detection primer is shown in SEQ ID No:25 in sequence table；Detected downstream is drawn
Object is shown in SEQ ID No:31 in sequence table；The closing primer of specificity is SEQ ID No:26, SEQ ID No:27, SEQ
ID No:28, SEQ ID No:29, SEQ ID No:30, SEQ ID No:32, SEQ ID No:33, SEQ ID No:34, SEQ
One or more of ID No:35 and SEQ ID No:36 sequence.
The reaction system of PCR amplification: 10mM TrisCl (pH8.8), 50 mM KCl, 1.5mM MgCl2, every kind of dNTP
200 μM, 0.1M glycine betaine, 2M trehalose, 1.5UTaq enzyme/100 μ l, 2.5 μ g/ml SYBR Green；Mass percent is
0.02% bovine serum albumin(BSA) (BSA), 0.1% gelatin (Gelatin)；Percent by volume is 5% glycerol, 6% deionized formamide, 1%
Dimethyl sulfoxide (DMSO) and 0 μ l-10 μ l Taq enzyme antibody/ml.Template DNA/reaction of 5-10000 copy.
The reaction condition of PCR amplification: (1) 93 DEG C initial denaturation 3 minutes；(2) 93 DEG C are denaturalized 30 seconds, and (3) 48 DEG C are annealed 60 seconds,
(4) 68 DEG C extend 2 minutes, and step (2)-(4) cycle-index is 36 times, and (5) 68 DEG C extend 2 minutes.Acquired results are divided
Analysis, judge test object whether Hepatitis B carrier, and to liver cancer risk carry out earlier evaluations.
Embodiment 4
Extract sample DNA.Specific primer: upstream detection primer is shown in SEQ ID No:37 in sequence table；Detected downstream is drawn
Object is shown in SEQ ID No:43 in sequence table；The closing primer of specificity is SEQ ID No:38, SEQ ID No:39, SEQ
ID No:40, SEQ ID No:41, SEQ ID No:42, SEQ ID No:44, SEQ ID No:45, SEQ ID No:46, SEQ
One or more of ID No:47 and SEQ ID No:48 sequence.
The reaction system of PCR amplification: 10mM TrisCl (pH8.8), 50 mM KCl, 2mM MgCl2, every kind of dNTP 200
μM, the SYBR Green of 2.5M glycine betaine, 0.3M trehalose, 5U Taq enzyme/100 μ l, 1 μ g/ml；The % phenol that mass percent is 3
It is red；0.08% bovine serum albumin(BSA) (BSA), 0.1% gelatin (Gelatin)；Percent by volume is 18% glycerol, 8% deionization formyl
Amine, 5% dimethyl sulfoxide (DMSO) and 1.8 μ l Taq enzyme antibody/ml.
The condition of the PCR amplification be (1) 92 DEG C initial denaturation 5 minutes；(2) 92 DEG C are denaturalized 75 seconds, (3) 55 DEG C of annealing 45
Second, (4) 66 DEG C extend 3 minutes, and step (2)-(4) cycle-index is 58 times, and (5) 68 DEG C extend 8 minutes.Acquired results are carried out
Analysis, judge test object whether Hepatitis B carrier, and to liver cancer risk carry out earlier evaluations.
It should be understood that those skilled in the art can make the present invention various after having read above content of the invention
Change or modification, these equivalent forms also fall within the scope of the appended claims of the present application.
Sequence table
<110>lucky grace biotechnology (Kunshan) Co., Ltd
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<400> 10
tttccggaag tgttgataag avbhh 25
<210> 11
<211> 25
<212> DNA
<213>artificial sequence (Homo sapiens)
<400> 11
tttccggaag tgttgataag bvbhh 25
<210> 12
<211> 25
<212> DNA
<213>artificial sequence (Homo sapiens)
<400> 12
tttccggaag tgttgataah bvbhh 25
<210> 13
<211> 22
<212> DNA
<213>artificial sequence (Homo sapiens)
<400> 13
ctcctctgcc gatccatact gc 22
<210> 14
<211> 22
<212> DNA
<213>artificial sequence (Homo sapiens)
<400> 14
ctcctctgcc gatccatact hd 22
<210> 15
<211> 22
<212> DNA
<213>artificial sequence (Homo sapiens)
<400> 15
ctcctctgcc gatccatacv hd 22
<210> 16
<211> 22
<212> DNA
<213>artificial sequence (Homo sapiens)
<400> 16
ctcctctgcc gatccatadv hd 22
<210> 17
<211> 22
<212> DNA
<213>artificial sequence (Homo sapiens)
<400> 17
ctcctctgcc gatccatbdv hd 22
<210> 18
<211> 22
<212> DNA
<213>artificial sequence (Homo sapiens)
<400> 18
ctcctctgcc gatccavbdv hd 22
<210> 19
<211> 26
<212> DNA
<213>artificial sequence (Homo sapiens)
<400> 19
ctaacattga gattcccgag attgag 26
<210> 20
<211> 26
<212> DNA
<213>artificial sequence (Homo sapiens)
<400> 20
ctaacattga gattcccgag attgbh 26
<210> 21
<211> 26
<212> DNA
<213>artificial sequence (Homo sapiens)
<400> 21
ctaacattga gattcccgag atthbh 26
<210> 22
<211> 26
<212> DNA
<213>artificial sequence (Homo sapiens)
<400> 22
ctaacattga gattcccgag atvhbh 26
<210> 23
<211> 26
<212> DNA
<213>artificial sequence (Homo sapiens)
<400> 23
ctaacattga gattcccgag avvhbh 26
<210> 24
<211> 26
<212> DNA
<213>artificial sequence (Homo sapiens)
<400> 24
ctaacattga gattcccgag bvvhbh 26
<210> 25
<211> 22
<212> DNA
<213>artificial sequence (Homo sapiens)
<400> 25
tcgcttcacc tctgcacgta gc 22
<210> 26
<211> 22
<212> DNA
<213>artificial sequence (Homo sapiens)
<400> 26
tcgcttcacc tctgcacgta hd 22
<210> 27
<211> 22
<212> DNA
<213>artificial sequence (Homo sapiens)
<400> 27
tcgcttcacc tctgcacgtb hd 22
<210> 28
<211> 22
<212> DNA
<213>artificial sequence (Homo sapiens)
<400> 28
tcgcttcacc tctgcacgvb hd 22
<210> 29
<211> 22
<212> DNA
<213>artificial sequence (Homo sapiens)
<400> 29
tcgcttcacc tctgcachvb hd 22
<210> 30
<211> 22
<212> DNA
<213>artificial sequence (Homo sapiens)
<400> 30
tcgcttcacc tctgcadhvb hd 22
<210> 31
<211> 25
<212> DNA
<213>artificial sequence (Homo sapiens)
<400> 31
acattgagat tcccgagatt gagat 25
<210> 32
<211> 25
<212> DNA
<213>artificial sequence (Homo sapiens)
<400> 32
acattgagat tcccgagatt gagbv 25
<210> 33
<211> 25
<212> DNA
<213>artificial sequence (Homo sapiens)
<400> 33
acattgagat tcccgagatt gahbv 25
<210> 34
<211> 25
<212> DNA
<213>artificial sequence (Homo sapiens)
<400> 34
acattgagat tcccgagatt gbhbv 25
<210> 35
<211> 25
<212> DNA
<213>artificial sequence (Homo sapiens)
<400> 35
acattgagat tcccgagatt hbhbv 25
<210> 36
<211> 25
<212> DNA
<213>artificial sequence (Homo sapiens)
<400> 36
acattgagat tcccgagatv hbhbv 25
Claims (10)
1. a kind of liver cancer earlier evaluations method, which is characterized in that described detection method includes the following steps:
(1) DNA of sample to be tested is extracted；
(2) specific primer is used, using DNA obtained by step (1) as template, PCR amplification detection is carried out, wherein the specificity is drawn
Object is upstream detection primer, and sequence is as shown in SEQ ID No:1 in sequence table；The specific primer draws for detected downstream
Object, sequence is as shown in SEQ ID No:7 in sequence table；More preferably, the specific primer further includes closing primer, the envelope
Closing primer is SEQ ID No:2, SEQ ID No:3, SEQ ID No:4, SEQ ID No:5, SEQ ID No:6, SEQ ID
One or more of No:8, SEQ ID No:9, SEQ ID No:10, SEQ ID No:11 and SEQ ID No:12 sequence；
(3) according to step (2) PCR amplification testing result, liver cancer risk is assessed.
2. liver cancer earlier evaluations method as described in claim 1, which is characterized in that wherein step (2) described specific primer is
Upstream detection primer, sequence is as shown in SEQ ID No:13 in sequence table；The specific primer is downstream detector primer,
Sequence is as shown in SEQ ID No:19 in sequence table；More preferably, the specific primer further includes closing primer, and the closing is drawn
Object is SEQ ID No:14, SEQ ID No:15, SEQ ID No:16, SEQ ID No:17, SEQ ID No:18, SEQ ID
One or more of No:20, SEQ ID No:21, SEQ ID No:22, SEQ ID No:23 and SEQ ID No:24 sequence.
3. liver cancer earlier evaluations method as described in claim 1, which is characterized in that wherein step (2) described specific primer is
Upstream detection primer, sequence is as shown in SEQ ID No:25 in sequence table；The specific primer is downstream detector primer,
Sequence is as shown in SEQ ID No:31 in sequence table；More preferably, the specific primer further includes closing primer, and the closing is drawn
Object is SEQ ID No:26, SEQ ID No:27, SEQ ID No:28, SEQ ID No:29, SEQ ID No:30, SEQ ID
One or more of No:32, SEQ ID No:33, SEQ ID No:34, SEQ ID No:35 and SEQ ID No:36 sequence.
4. liver cancer earlier evaluations method as described in claim 1, which is characterized in that wherein step (2) PCR amplification detects
Condition are as follows: (1) 90-96 DEG C initial denaturation -10 minutes 10 seconds；(2) it is denaturalized -90 seconds 5 seconds for 90-96 DEG C；(3) 45-65 DEG C anneal 5 seconds-
99 seconds；(4) extend -300 seconds 3 seconds for 55-73 DEG C；Step (2)-(4) cycle-index is 28-60；(5) extend 5 seconds -600 for 60-73 DEG C
Second.
5. the liver cancer earlier evaluations method as described in claim any one of 1-4, which is characterized in that the liver cancer earlier evaluations method
Not for the purpose of the diagnosing and treating of disease.
6. a kind of specific primer of liver cancer earlier evaluations, which is characterized in that the specific primer is upstream detection primer,
Sequence is as shown in SEQ ID No:1 in sequence table；The specific primer is downstream detector primer, in sequence such as sequence table
Shown in SEQ ID No:7；More preferably, the specific primer further includes closing primer, and the closing primer is SEQ ID No:
2, SEQ ID No:3, SEQ ID No:4, SEQ ID No:5, SEQ ID No:6, SEQ ID No:8, SEQ ID No:9, SEQ
One or more of ID No:10, SEQ ID No:11 and SEQ ID No:12 sequence.
7. a kind of specific primer of liver cancer earlier evaluations, which is characterized in that the specific primer is upstream detection primer,
Sequence is as shown in SEQ ID No:13 in sequence table；The specific primer is downstream detector primer, in sequence such as sequence table
Shown in SEQ ID No:19；More preferably, the specific primer further includes closing primer, and the closing primer is SEQ ID No:
14, SEQ ID No:15, SEQ ID No:16, SEQ ID No:17, SEQ ID No:18, SEQ ID No:20, SEQ ID
One or more of No:21, SEQ ID No:22, SEQ ID No:23 and SEQ ID No:24 sequence.
8. a kind of specific primer of liver cancer earlier evaluations, which is characterized in that the specific primer is upstream detection primer,
Sequence is as shown in SEQ ID No:25 in sequence table；The specific primer is downstream detector primer, in sequence such as sequence table
Shown in SEQ ID No:31；More preferably, the specific primer further includes closing primer, and the closing primer is SEQ ID No:
26, SEQ ID No:27, SEQ ID No:28, SEQ ID No:29, SEQ ID No:30, SEQ ID No:32, SEQ ID
One or more of No:33, SEQ ID No:34, SEQ ID No:35 and SEQ ID No:36 sequence.
9. a kind of detection kit of liver cancer earlier evaluations, which is characterized in that the detection kit includes: that specificity is drawn
Object, the specific primer are upstream detection primer, and sequence is as shown in SEQ ID No:1 in sequence table；The specificity is drawn
Object is downstream detector primer, and sequence is as shown in SEQ ID No:7 in sequence table；More preferably, the specific primer further includes
Primer is closed, the closing primer is SEQ ID No:2, SEQ ID No:3, SEQ ID No:4, SEQ ID No:5, SEQ ID
One of No:6, SEQ ID No:8, SEQ ID No:9, SEQ ID No:10, SEQ ID No:11 and SEQ ID No:12
Or several sequences；It further include 2 × PCR reaction buffer, positive control and negative control.
10. detection kit as claimed in claim 9, which is characterized in that the detection kit further includes that specificity is drawn
The sequence of object, the specific primer is upstream detection primer, and sequence is as shown in SEQ ID No:13 in sequence table；The spy
Specific primer is downstream detector primer, and sequence is as shown in SEQ ID No:19 in sequence table；More preferably, the specific primer
It further include closing primer, the closing primer is SEQ ID No:14, SEQ ID No:15, SEQ ID No:16, SEQ ID
No:17, SEQ ID No:18, SEQ ID No:20, SEQ ID No:21, SEQ ID No:22, SEQ ID No:23 and SEQ
One or more of ID No:24 sequence；The specific primer is upstream detection primer, SEQ in sequence such as sequence table
Shown in ID No:25；The specific primer is downstream detector primer, and sequence is as shown in SEQ ID No:31 in sequence table；More
Goodly, the specific primer further includes closing primer, and the closing primer is SEQ ID No:26, SEQ ID No:27, SEQ
ID No:28, SEQ ID No:29, SEQ ID No:30, SEQ ID No:32, SEQ ID No:33, SEQ ID No:34, SEQ
One or more of ID No:35 and SEQ ID No:36 sequence, the formula of 2 × PCR reaction buffer are preferably:
10mM-200mM TrisCl, 50mM-200 mM KCl, the 1mM-15mM MgCl that pH is 8.82, 10 μM of -800 μ of every kind of dNTP
M, the SYBR Green of 0.1M-2M glycine betaine, 0.1-3M trehalose, 0.5U-5U Taq enzyme/100 μ l, 0.01 μ g-10 μ g/ml；
Mass percent is that 0.001%-0.5% is phenol red；0.02%-2% bovine serum albumin(BSA), 0.001%-5% gelatin；Percent by volume is
5%-30% glycerol, 0.1%-20% deionized formamide, 0.1%-10% dimethyl sulfoxide and 0.01 μ l-10 μ l Taq enzyme antibody/
ml。
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|---|---|---|---|---|
| CN102433388A (en) * | 2012-01-04 | 2012-05-02 | 河南科技大学 | Kit for early diagnosis of liver cancer |
| CN106636455A (en) * | 2015-10-30 | 2017-05-10 | 上海复阳生物科技有限公司 | A PRINS detecting method for hepatitis B viruses, primers thereof and a detection kit for the method |
| CN107475461A (en) * | 2017-10-11 | 2017-12-15 | 广州立菲达安诊断产品技术有限公司 | A kind of primer, kit and method for detecting hepatitis B virus drug resistance gene |
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| CN102433388A (en) * | 2012-01-04 | 2012-05-02 | 河南科技大学 | Kit for early diagnosis of liver cancer |
| CN106636455A (en) * | 2015-10-30 | 2017-05-10 | 上海复阳生物科技有限公司 | A PRINS detecting method for hepatitis B viruses, primers thereof and a detection kit for the method |
| CN107475461A (en) * | 2017-10-11 | 2017-12-15 | 广州立菲达安诊断产品技术有限公司 | A kind of primer, kit and method for detecting hepatitis B virus drug resistance gene |
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Application publication date: 20181211 |