CN105256075B - The fluorescent PCR kit and detection method of eight kinds of Genotypings of hepatitis type B virus - Google Patents
The fluorescent PCR kit and detection method of eight kinds of Genotypings of hepatitis type B virus Download PDFInfo
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Abstract
The present invention provides a kind of fluorescent PCR kit and detection method of eight kinds of Genotypings of hepatitis type B virus, belongs to bio-assay technology field.The technical solution adopted is that：Design synthesis specificity is directed to the primer and probe of eight kinds of gene hypotypes of hepatitis type B virus, and carries out fluorescent marker to probe respectively, and specific detection is carried out with fluorescence PCR method.Beneficial effects of the present invention are：（1）This product combines fluorescent probe technique using PCR (PCR), and it is good to detect A H-type hepatitis type B virus partings, designed probe and primer specificity type and high sensitivity, accuracy in sample.（2）Cost economy, operating method is fast and convenient, suitable for wide popularization and application.
Description
Technical field
The invention belongs to bio-assay technology fields, and in particular to a kind of fluorescence of eight kinds of Genotypings of hepatitis type B virus
PCR kit and detection method.
Background technology
Virus B hepatitis is the problem of long-standing problem whole world medical field, is acute, chronic hepatitis, hepatic sclerosis, the weight of liver cancer
Want one of virulence factor and one of the public health problem of whole world most serious.It is reported according to WTO, global more than 60 hundred million populations
In, there are about 3,000,000,000 population lives in hepatitis type B virus（HBV）HBV was infected in high Prevalent district more than 2,000,000,000 people, wherein
Chronic infection is more than 3.5 hundred million.The annual hepatitis B incidence in China is a B virus at 6 000 ten thousand person-times or more
Property incidence of hepatitis big country.In the countries and regions such as China, Southeast Asia and Africa, there are about 50% hepatic sclerosis and 70-90%
Liver cancer is caused by Chronic HBV infection, and the HBV carrier for having infected HBV variants has 7-30%.
It is at present 8 kinds of gene types of A~H by HBV points according to the nucleotide difference of gene order, according to complete genome sequence
The diversity factor of nucleotide difference >=8% or its S gene order nucleotide difference >=4% is determined as different types, and same gene type expires
Sufficient sequence difference≤5.6%.HBV gene type is there are obvious regionality distribution feature, and A types are European more with Central Africa
See；Type B, c-type are distributed mainly on Asia；D types distributed areas are most wide, are popular in the areas such as Mediterranean, the Middle East；E types using Africa as
It is main；G types are detected in US and European；F, H-type is distributed in the U.S..China is advantage type, and is existed based on Type B and c-type distribution
Differences between the south and the north, the north is using c-type as advantage type, and south is using Type B as advantage type；It is few that D types are distributed in such as Xinjiang, Tibet, Ningxia more
Occasionally there are A, E, F type report in number ethnic mimority area, there is not yet G, H-type.Some researches show that there are the other mixing senses of different genotype by HBV
Dye.
The detection method of hepatitis B virus gene typing includes gene sequencing, polymerase chain reaction-limitation at present
Property fragment length polymorphism analysis（PCR-RFLP）, it is Serotype-dependent multiplex-PCR parting (i.e. labelled by nested-PCR method), special
Property linear probe detection method (INNO-LiPA) INNO-LiPA, PCR microplate making nucleic acid molecular hybridization ELISA method, monoclonal antibody
ELISA methods and genetic chip.But more or less there are some defects for these methods：As gene sequencing classifying method result is accurate
It is really reliable, but process it is cumbersome, it is time-consuming, of high cost, be difficult to promote, be not suitable for the detection of large sample, and lack sensibility, it is impossible to send out
The mixed infection of two or more HBV gene type in existing same individual.PCR-RFLP is for single nucleotide acid site
Endonuclease digestion is hindered in restriction enzyme digestion sites change caused by polymorphism, and digestion is not exclusively present with complicated item
Band, restriction enzyme mapping identification is complicated, thus influences the confidence level of Genotyping, and the method cannot equally find the mixed of several genotype
Close infection.Labelled by nested-PCR method method needs to synthesize a plurality of primer, need to last length, opportunities for contamination compared with RFLP methods through two-wheeled PCR processes
It is larger, and non-specific amplification band can not be avoided the occurrence of completely and interfere observation genotyping result.There are prices to hold high by INNO-LiPA
Deficiency expensive, specificity is slightly lower.Monoclonal antibody ELISA methods cannot carry out gene point to the HBV infection of serum HBsAg feminine gender
Type and minority HBsAg can not partings.There are cost is higher, false positive rate is high, the integrated level of different chips deficiency for genetic chip
The shortcomings of, PCR microplate making nucleic acid molecular hybridizations ELISA method is suitable for the higher laboratory of high degree of automation.
For the conventional detection of great amount of samples in Molecular Laboratory, it is badly in need of that accuracy is high, specificity is good, price economy, fast
The detection method of speed.
The content of the invention
In order to solve the above technical problems, the present invention provides a kind of fluorescent PCR examination of eight kinds of Genotypings of hepatitis type B virus
Agent box and detection method are directed to the primer and probe of eight kinds of gene hypotypes of hepatitis type B virus by designing synthesis specificity, and
With reference to the technical solution of PCR detection method, quick, Accurate Determining hepatitis type B virus parting is realized, cost of determination reduces.
The technical solution adopted by the present invention is：The fluorescent PCR kit of eight kinds of Genotypings of hepatitis type B virus, feature
It is, the kit includes as followsTo at least one PCR reaction solution system in 8.：
1. for the PCR reaction solution system of hepatitis type B virus A type genotype, including following primer and probe
HBV-A-F ACCTGGGTGGGTAATAATTTGGA
HBV-A-R GAAACCACAATAGTTGCCTGATCTT
HBV-A-P AATTGACTACTAGATCCCTG
2. for the PCR reaction solution system of hepatitis type B virus Type B genotype, including following primer and probe
HBV-B-F CTCGTGGTGGACTTCTCTCAATT
HBV-B-R ATCCAGCGATAACCAGGACAAAT
HBV-B-P CAAATCTCCAGTCACTCAC
3. for the PCR reaction solution system of hepatitis B virus C type genotype, including following primer and probe
HBV-C-F GAGCCAACTCAAACAATCCAGAT
HBV-C-R GAATGCTCCCGCTCCTACCT
HBV-C-P CCCCAACAAGGATC
4. for the PCR reaction solution system of hepatitis type B virus D type genotype, including following primer and probe
HBV-D-F GGGAACTTTACTGGGCTTTATTCTT
HBV-D-R GGGCCTACAAACTGTTCACATTT
HBV-D-P CTCATTGGAAAACACC
5. for the PCR reaction solution system of hepatitis B virus E type genotype, including following primer and probe
HBV-E-F AGTGAACCCTGTTCCGACTACTG
HBV-E-R GGTCCCCAATCCTCGAGAA
HBV-E-P CTCACTCATCTCGTCAAT
6. for the PCR reaction solution system of hepatitis type B virus F type genotype, including following primer and probe
HBV-F-F GAGTCCCTTTATACCGCTGTTACC
HBV-F-R GTAATGATCCCCAACTGCCAAT
HBV-F-P ATGGATACCCACAGATAA
7. for the PCR reaction solution system of hepatitis type B virus G type genotype, including following primer and probe
HBV-G-F TGGAAAGTCTGTCAACGAATAACTG
HBV-G-R AAGGCAGGGTAACCACATTGG
HBV-G-P TTTCGCTGCTCCTTTT
8. for the PCR reaction solution system of hepatitis type B virus H-type genotype, including following primer and probe
HBV-H-F CCCGTGTGTCCTCTACTTCCA
HBV-H-R AAGAGTGGTGCAGGTTTTGCA
HBV-H-P ATCTACAACCACCAGCACG。
Preferably, each described probe one end be marked with fluorescent reporter group FAM, TET, HEX, ROX, JOE, CY3, CY5,
TAMRA or Texas Red, the other end be marked with fluorescent quenching group MGB, BHQ-1, BHQ-2, Lowa Black RQ,
LowaBlackTMFQ or Dabcyl.
It is furthermore preferred that the PCR reaction solution system for hepatitis type B virus A-D type genotype forms the first reactant
System, the 5' ends flag F AM of wherein HBV-A-P, the 5' ends mark HEX of 3' ends mark MGB, HBV-B-P, 3' ends mark MGB, HBV-
The 5' ends mark ROX of C-P, the 5' ends mark CY5 of 3' ends mark MGB, HBV-D-P, 3' ends mark MGB；
The second reaction system, wherein HBV-E- are formed for the PCR reaction solution system of hepatitis B virus E-H-type genotype
The 5' ends mark HEX of the 5' ends flag F AM of P, 3' ends mark MGB, HBV-F-P, the 5' ends mark of 3' ends mark MGB, HBV-G-P
ROX, 3' end mark MGB, the 5' ends mark CY5 of HBV-H-P, 3' ends mark MGB.
The standard items of hepatitis type B virus A-H type genotype are further included in the kit.
The present invention also provides the nondiagnostic detection methods of eight kinds of Genotypings of hepatitis type B virus, which is characterized in that institute
The method of stating comprises the following steps：
（1）Synthesize primer and probe described in claim 1；
（2）According to instrument each probe both ends are marked with fluorescent reporter group described in claim 2 and corresponding respectively
Fluorescent quenching group；
（3）Prepare positive criteria product；
（4）Extract sample to be tested DNA；
（5）Prepare PCR reaction systems；
（6）Run PCR；
（7）Data analysis.
Preferably, the step（3）The preparation method of middle positive criteria product includes hepatitis type B virus A-H respectively for structure
The plasmid of eight kinds of gene hypotype detection target gene standard sequences：It is respectively synthesized eight kinds of gene hypotype inspections of hepatitis type B virus A-H
It surveys target gene standard sequence and is cloned into P18-T carriers, be named as PMT-HBV-A, PMT-HBV-B, PMT-HBV-C,
PMT-HBV-D, PMT-HBV-E, PMT-HBV-F, PMT-HBV-G, PMT-HBV-H.
It is furthermore preferred that the step（2）The mark of middle probe is respectively：The 5' ends flag F AM of HBV-A-P, 3' ends mark
The 5' ends mark HEX of MGB, HBV-B-P, the 5' ends mark ROX of 3' ends mark MGB, HBV-C-P, 3' ends mark MGB, HBV-D-P
5' ends mark CY5,3' ends mark MGB；The 5' ends flag F AM of HBV-E-P, the 5' ends mark of 3' ends mark MGB, HBV-F-P
HEX, 3' end mark MGB, the 5' ends mark ROX of HBV-G-P, the 5' ends mark CY5 of 3' ends mark MGB, HBV-H-P, 3' ends mark
Remember MGB；
Step（5）Middle PCR reaction systems are divided into two, for the primer and probe of hepatitis type B virus ABCD type genotype
It adds in the first reaction system, is added in for the primer and probe of hepatitis B virus E FGH type genotype to the second reactant
System.
Preferably, the step（6）Fluorescence channel detects and selects when running PCR：FAM, HEX, ROX and CY5 are selected in detection
Passage.
The step（6）The response procedures that operation PCR is are 95 DEG C of 2min；Again by 95 DEG C of 15s, 60 DEG C of 1min, Xun Huan
40 times.
In above-mentioned technical proposal, the primer and probe for eight kinds of Genotypings of hepatitis type B virus has good respectively
Specificity, for Fluorescence PCR, there is no interfering with each other, each probe one end is marked with fluorescent reporter group, other end mark
Note has fluorescent quenching group, and the fluorescent reporter group of probe not of the same race is different, when detected sample is in the primer of certain parting
After the lower amplification of guiding, combined with the probe of corresponding parting, by the fluorescent reporter group of its probe, then can detect that sample be by
Which kind of parting it is hepatitis b virus infected, in practical application, in view of the factors such as instrument, fluorescein range of choice, can will be directed to
The PCR reaction solution system of hepatitis type B virus A-D type genotype forms the first reaction system, for hepatitis B virus E-H-type
The PCR reaction solution system of genotype forms the second reaction system.
To improve the accuracy of detection, kit kind is configured with the positive criteria product of eight kinds of hypotypes, which can be
The complete sequence of various hypotypes,（Its sequence can be looked into gene pool）Or include one section of detection target gene site
Gene order, above-mentioned sequence authorized company synthesis, the detection target gene site is each hypotype that the present invention screens
Highly conserved sequence site, in application, being spliced by the method for PCR, synthetic detection target gene sequence is cloned into
In P18-T carrier pMD18-T vector (Takara), structure is respectively comprising eight kinds of hypotype detection targets of hepatitis type B virus A-H
The plasmid of gene standard sequence, is named as：PMT-HBV-A, PMT-HBV-B, PMT-HBV-C, PMT-HBV-D, PMT-HBV-E,
PMT-HBV-F, PMT-HBV-G, PMT-HBV-H.
Beneficial effects of the present invention are：（1）This product combines fluorescent probe technique, inspection using PCR (PCR)
A-H types hepatitis type B virus parting in sample, designed probe and primer specificity type and high sensitivity, accuracy are good.
（2）Cost economy, operating method is fast and convenient, suitable for wide popularization and application.
Specific embodiment
Here is the PCR kit of above-mentioned eight kinds of Genotypings of pin hepatitis type B virus and carries out quantitative detection using it
Method illustrates, but the invention is not limited in any way.Experimental method used in embodiment unless otherwise specified,
It is conventional method.Material, reagent and synthesized primer and probe sequence used etc., unless otherwise specified, can be from business
Industry approach obtains.
Embodiment 1：Primer in the PCR kit of eight kinds of Genotypings of pin hepatitis type B virus, probe sequence design and
Synthesis
From GenBank（The geneseq database that National Institutes of Health is safeguarded）Middle search HBV A-H type bases
Because type complete sequence and be compared using BLAST sequence analyses, sift out highly conserved region, for conserved sequence use primer
Design software Primer Premier 5 separately design primer and corresponding specific probe, and have detected whether hairpin structure
Or dimer is formed, the results showed that the probe of design meets specific requirements, and will not form hairpin structure or dimer.
Designed primer, probe sequence are respectively：
1. for the PCR reaction solution system of hepatitis type B virus A type genotype, including following primer and probe
HBV-A-F ACCTGGGTGGGTAATAATTTGGA
HBV-A-R GAAACCACAATAGTTGCCTGATCTT
HBV-A-P AATTGACTACTAGATCCCTG
2. for the PCR reaction solution system of hepatitis type B virus Type B genotype, including following primer and probe
HBV-B-F CTCGTGGTGGACTTCTCTCAATT
HBV-B-R ATCCAGCGATAACCAGGACAAAT
HBV-B-P CAAATCTCCAGTCACTCAC
3. for the PCR reaction solution system of hepatitis B virus C type genotype, including following primer and probe
HBV-C-F GAGCCAACTCAAACAATCCAGAT
HBV-C-R GAATGCTCCCGCTCCTACCT
HBV-C-P CCCCAACAAGGATC
4. for the PCR reaction solution system of hepatitis type B virus D type genotype, including following primer and probe
HBV-D-F GGGAACTTTACTGGGCTTTATTCTT
HBV-D-R GGGCCTACAAACTGTTCACATTT
HBV-D-P CTCATTGGAAAACACC
5. for the PCR reaction solution system of hepatitis B virus E type genotype, including following primer and probe
HBV-E-F AGTGAACCCTGTTCCGACTACTG
HBV-E-R GGTCCCCAATCCTCGAGAA
HBV-E-P CTCACTCATCTCGTCAAT
6. for the PCR reaction solution system of hepatitis type B virus F type genotype, including following primer and probe
HBV-F-F GAGTCCCTTTATACCGCTGTTACC
HBV-F-R GTAATGATCCCCAACTGCCAAT
HBV-F-P ATGGATACCCACAGATAA
7. for the PCR reaction solution system of hepatitis type B virus G type genotype, including following primer and probe
HBV-G-F TGGAAAGTCTGTCAACGAATAACTG
HBV-G-R AAGGCAGGGTAACCACATTGG
HBV-G-P TTTCGCTGCTCCTTTT
8. for the PCR reaction solution system of hepatitis type B virus H-type genotype, including following primer and probe
HBV-H-F CCCGTGTGTCCTCTACTTCCA
HBV-H-R AAGAGTGGTGCAGGTTTTGCA
HBV-H-P ATCTACAACCACCAGCACG。
In above-mentioned probe, the detection target gene site of HBV-A-P is located at hepatitis type B virus A type genotype complete sequences
2143-2162 bases, the detection target gene site of HBV-B-P is located at hepatitis type B virus Type B genotype complete sequence
320-338 bases, the detection target gene site of HBV-C-P are located at the 2985- of hepatitis B virus C type genotype complete sequence
3998 bases, the detection target gene site of HBV-D-P are located at the 2521-2536 of hepatitis type B virus D type genotype complete sequences
Base, the detection target gene site of HBV-E-P are located at the 106-123 bases of hepatitis B virus E type genotype complete sequence,
The detection target gene site of HBV-F-P is located at the 931-814 bases of hepatitis type B virus F type genotype complete sequences, HBV-G-P
Detection target gene site be located at the 1014-1029 bases of hepatitis type B virus G type genotype complete sequences, the inspection of HBV-H-P
Survey the 490-508 bases that target gene site is located at hepatitis type B virus H-type genotype complete sequence, corresponding standard items sequence
It can be looked into gene pool.
Specifically to the fluorescent marker of each probe, it then follows following principle according to instrument to the resolution condition of fluorescence, selects glimmering
Light reporter group and corresponding fluorescent quenching group.For ease of using and improving the specificity of reaction, accuracy, second can will be directed to
The PCR reaction solution system of Hepatitis virus A-D type genotype forms the first reaction system, described to be directed to hepatitis type B virus A-D
The PCR reaction solution system of type genotype forms the first reaction system, is reacted for the PCR of hepatitis B virus E-H-type genotype
Liquid system forms the second reaction system, and the design reduces reaction samples, simplify operation, improve conventional efficient.
Embodiment 2：Positive criteria product is detected using mentioned reagent box
（1）According to gene pool query result, specialized company is entrusted（As work, Invitrogen are given birth in Shanghai（Shanghai）Trade is limited
Company）Synthesis is directed to standard sequence, primer and the probe sequence of eight kinds of gene hypotypes of hepatitis type B virus, and makees such as subscript
Note：The 5' ends mark HEX of the 5' ends flag F AM of HBV-A-P, 3' ends mark MGB, HBV-B-P, 3' ends mark MGB, HBV-C-P
5' ends mark ROX, 3' ends mark MGB, HBV-D-P 5' ends mark CY5,3' ends mark MGB, HBV-E-P 5' ends mark
FAM, 3' end mark MGB, the 5' ends mark HEX of HBV-F-P, the 5' ends mark ROX of 3' ends mark MGB, HBV-G-P, 3' ends mark
Remember MGB, the 5' ends mark CY5 of HBV-H-P, 3' ends mark MGB.
PCR reaction systems are prepared using commercially available fluorescent PCR reagent or kit or Premix Ex Taq kits.Point
For the first reaction system and the second reaction system, the first reaction system is included for hepatitis type B virus ABCD type genotype
Primer and probe（With above-mentioned fluorescent marker）, what the second reaction system was included for hepatitis B virus E FGH type genotype
Primer and probe, specifically, the working solution and reaction condition of each PCR reaction systems are as follows：
1）First reaction system：Pcr amplification reaction system is 20 μ L, including 10 × PCR Buffer, 2 μ L, dNTP
（2.5mmoL/L）2 μ L, Taq enzyme（5U/μL）0.07μL、HBV-A-F/R（5μM）0.3μL、HBV-A-P（5μM）1.5μL、HBV-
B-F/R（5μM）0.3μL、HBV-B-P（5μM）1.5μL、HBV-C-F/R（5μM）0.3μL、HBV-C-P（5μM）1.5μL、HBV-
D-F/R（5μM）0.3μL、HBV-D-P（5μM）Each 2 μ L of 1.5 μ L, the DNA of A-D standard sequences, ddH is mended20.73 μ L of O are to whole body
Product is 20 μ L.
2）Second reaction system：Pcr amplification reaction system is 20 μ L, including 10 × PCR Buffer, 2 μ L, dNTP
（2.5mmoL/L）2 μ L, Taq enzyme（5U/μL）0.07μL、HBV-E-F/R（5μM）0.3μL、HBV-E-P（5μM）1.5μL、HBV-
F-F/R（5μM）0.3μL、HBV-F-P（5μM）1.5μL、HBV-G-F/R（5μM）0.3μL、HBV-G-P（5μM）1.5μL、HBV-
H-F/R（5μM）0.3μL、HBV-H-P（5μM）Each 2 μ L of DNA of 1.5 μ L, the DNA of A-D standard sequences, ddH is mended20.73 μ L of O are extremely
Final volume is 20 μ L；
（4）Above-mentioned each 20 μ L systems are added separately in PCR pipes, PCR pipes are put into fluorescent quantitation by lid upper tube cap
In PCR detectors（ABI7500 detectors of ABI companies etc.）, operation PCR reactions, reaction condition：95℃ 2min；95 are pressed again
DEG C 15s, 60 DEG C of 1min are cycled 40 times；Reaction system is 20 μ L.Fluorescence channel detects and selects：FAM, HEX, ROX are selected in detection
With CY5 passages.
（5）Data analysis,
Eight kinds of Genotypings of primer pair B Hepatitis virus provided by the invention have good specific amplification, utilize mark
The result that the fluorescent reporter group of note can obtain is accurate, stable, collimation is good.
Embodiment 3：The method that pattern detection is carried out using mentioned reagent box
（1）According to the sequence of the primer of above-mentioned design and probe, specialized company is entrusted（As work, Invitrogen are given birth in Shanghai（On
Sea）Trade Co., Ltd）Synthetic primer and probe, and mark spare；
（2）Extraction test serum sample simultaneously extracts its hepatitis B virus DNA
Person under inspection venous blood 1ml is extracted, injects in the centrifuge tube of sterile 1.5ml, after being stored at room temperature 2h, supernatant is taken to go to separately
It is serum sample in one sterile centrifugation tube；Hepatitis B virus DNA is conventionally extracted by DNA extracting solutions；
（3）Prepare PCR reaction systems
In the present embodiment eight kinds of gene hypotypes of hepatitis type B virus are carried out at the same time with parting detection, using commercially available common
PCR reagent or kit or Premix Ex Taq kits.Wherein it is divided to two for the PCR reaction systems of hepatitis type B virus,
Respectively the first reaction system and the second reaction system, the first reaction system are included for hepatitis type B virus ABCD type genes
The primer and probe of type, the second reaction system include the primer and probe for hepatitis B virus E FGH type genotype；Institute
Probe in each reaction system is set simultaneously all with the fluorescent reporter group and fluorescent quenching group that can be distinguished by instrument
There is no the positive control PCR system of the blank control of hepatitis B virus DNA and A-H positive criteria products.Specifically, each PCR reactions
The working solution and reaction condition of system are as follows：
1）First reaction system：Pcr amplification reaction system is 20 μ L, including 10 × PCR Buffer, 2 μ L, dNTP
（2.5mmoL/L）2 μ L, Taq enzyme（5U/μL）0.07μL、HBV-A-F/R（5μM）0.3μL、HBV-A-P（5μM）1.5μL、HBV-
B-F/R（5μM）0.3μL、HBV-B-P（5μM）1.5μL、HBV-C-F/R（5μM）0.3μL、HBV-C-P（5μM）1.5μL、HBV-
D-F/R（5μM）0.3μL、HBV-D-P（5μM）1.5 μ L, 2 μ L of sample DNA, ddH is mended26.73 μ L of O to final volume be 20 μ L.
2）Second reaction system：Pcr amplification reaction system is 20 μ L, including 10 × PCR Buffer, 2 μ L, dNTP
（2.5mmoL/L）2 μ L, Taq enzyme（5U/μL）0.07μL、HBV-E-F/R（5μM）0.3μL、HBV-E-P（5μM）1.5μL、HBV-
F-F/R（5μM）0.3μL、HBV-F-P（5μM）1.5μL、HBV-G-F/R（5μM）0.3μL、HBV-G-P（5μM）1.5μL、HBV-
H-F/R（5μM）0.3μL、HBV-H-P（5μM）1.5 μ L, 2 μ L of sample DNA, ddH is mended26.73 μ L of O to final volume be 20 μ L；
3）Blank control PCR system I：Pcr amplification reaction system be 20 μ L, including 10 × PCR Buffer, 2 μ L,
dNTP（2.5mmoL/L）2 μ L, Taq enzyme（5U/μL）0.07μL、HBV-A-F/R（5μM）0.3μL、HBV-A-P（5μM）1.5μL、
HBV-B-F/R（5μM）0.3μL、HBV-B-P（5μM）1.5μL、HBV-C-F/R（5μM）0.3μL、HBV-C-P（5μM）1.5μL、
HBV-D-F/R（5μM）0.3μL、HBV-D-P（5μM）1.5 μ L, 2 μ L of distilled water, ddH is mended26.73 μ L of O to final volume be 20 μ L；
Blank control PCR system II：Pcr amplification reaction system be 20 μ L, including 10 × PCR Buffer, 2 μ L,
dNTP（2.5mmoL/L）2 μ L, Taq enzyme（5U/μL）0.07μL、HBV-E-F/R（5μM）0.3μL、HBV-E-P（5μM）1.5μL、
HBV-F-F/R（5μM）0.3μL、HBV-F-P（5μM）1.5μL、HBV-G-F/R（5μM）0.3μL、HBV-G-P（5μM）1.5μL、
HBV-H-F/R（5μM）0.3μL、HBV-H-P（5μM）1.5 μ L, 2 μ L of distilled water, ddH is mended26.73 μ L of O to final volume be 20 μ L；
4）Positive control PCR system is referring to the first reaction system and the second reaction system in embodiment 2；
（4）Above-mentioned each 20 μ L systems are added separately in PCR pipes, PCR pipes are put into fluorescence and determined by lid upper tube cap
It measures in PCR detectors（The ABI7500 detectors of such as ABI companies）, operation PCR reactions, reaction condition：95℃ 2min；
Again by 95 DEG C of 15s, 60 DEG C of 1min, cycle 40 times；Fluorescence channel detects and selects：FAM, HEX, ROX and CY5 passage are selected in detection.
（5）Data analysis,
Each item data that composite analyser provides sets rational threshold value and baseline, and blank control group committee is stealthy, various
Positive controls are the positive, and sample to be tested confirms whether corresponding hypotype has expansion according to affiliated system and the fluorescence signal detected
Which kind of increase, so as to be derived as the infection of the hepatitis b virus infected or mixed type of hypotype.
SEQUENCE LISTING
<110>Hebei international travel health care center
<120>The fluorescent PCR kit and detection method of eight kinds of Genotypings of hepatitis type B virus
<130> 1
<160> 24
<170> PatentIn version 3.3
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Claims (9)
1. the fluorescent PCR kit of eight kinds of Genotypings of hepatitis type B virus, which is characterized in that the kit includes as follows
1. to the PCR reaction solution system in 8.：
1. for the PCR reaction solution system of hepatitis type B virus A type genotype, including following primer and probe
HBV-A-F ACCTGGGTGGGTAATAATTTGGA
HBV-A-R GAAACCACAATAGTTGCCTGATCTT
HBV-A-P AATTGACTACTAGATCCCTG
2. for the PCR reaction solution system of hepatitis type B virus Type B genotype, including following primer and probe
HBV-B-F CTCGTGGTGGACTTCTCTCAATT
HBV-B-R ATCCAGCGATAACCAGGACAAAT
HBV-B-P CAAATCTCCAGTCACTCAC
3. for the PCR reaction solution system of hepatitis B virus C type genotype, including following primer and probe
HBV-C-F GAGCCAACTCAAACAATCCAGAT
HBV-C-R GAATGCTCCCGCTCCTACCT
HBV-C-P CCCCAACAAGGATC
4. for the PCR reaction solution system of hepatitis type B virus D type genotype, including following primer and probe
HBV-D-F GGGAACTTTACTGGGCTTTATTCTT
HBV-D-R GGGCCTACAAACTGTTCACATTT
HBV-D-P CTCATTGGAAAACACC
5. for the PCR reaction solution system of hepatitis B virus E type genotype, including following primer and probe
HBV-E-F AGTGAACCCTGTTCCGACTACTG
HBV-E-R GGTCCCCAATCCTCGAGAA
HBV-E-P CTCACTCATCTCGTCAAT
6. for the PCR reaction solution system of hepatitis type B virus F type genotype, including following primer and probe
HBV-F-F GAGTCCCTTTATACCGCTGTTACC
HBV-F-R GTAATGATCCCCAACTGCCAAT
HBV-F-P ATGGATACCCACAGATAA
7. for the PCR reaction solution system of hepatitis type B virus G type genotype, including following primer and probe
HBV-G-F TGGAAAGTCTGTCAACGAATAACTG
HBV-G-R AAGGCAGGGTAACCACATTGG
HBV-G-P TTTCGCTGCTCCTTTT
8. for the PCR reaction solution system of hepatitis type B virus H-type genotype, including following primer and probe
HBV-H-F CCCGTGTGTCCTCTACTTCCA
HBV-H-R AAGAGTGGTGCAGGTTTTGCA
HBV-H-P ATCTACAACCACCAGCACG,
PCR reaction systems are divided into two, are added in for the primer and probe of hepatitis type B virus ABCD type genotype to first instead
System is answered, is added in for the primer and probe of hepatitis B virus E FGH type genotype to the second reaction system.
2. the fluorescent PCR kit of eight kinds of Genotypings of hepatitis type B virus according to claim 1, which is characterized in that
Each described probe one end is marked with fluorescent reporter group FAM, TET, HEX, ROX, JOE, CY3, CY5, TAMRA or Texas
Red, the other end are marked with fluorescent quenching group MGB, BHQ-1, BHQ-2, Lowa Black RQ, LowaBlackTMFQ or
Dabcyl。
3. the fluorescent PCR kit of eight kinds of Genotypings of hepatitis type B virus according to claim 2, which is characterized in that
The PCR reaction solution system for hepatitis type B virus A-D type genotype forms the first reaction system, wherein HBV-A-P's
The 5' ends mark HEX of 5' ends flag F AM, 3' ends mark MGB, HBV-B-P, the 5' ends mark of 3' ends mark MGB, HBV-C-P
ROX, 3' end mark MGB, the 5' ends mark CY5 of HBV-D-P, 3' ends mark MGB；
The second reaction system is formed for the PCR reaction solution system of hepatitis B virus E-H-type genotype, wherein HBV-E-P's
The 5' ends mark HEX of 5' ends flag F AM, 3' ends mark MGB, HBV-F-P, the 5' ends mark of 3' ends mark MGB, HBV-G-P
ROX, 3' end mark MGB, the 5' ends mark CY5 of HBV-H-P, 3' ends mark MGB.
4. the fluorescent PCR kit of eight kinds of Genotypings of hepatitis type B virus according to claim 1, which is characterized in that
The standard sequence of hepatitis type B virus A-H type genotype is further included in the kit.
5. the nondiagnostic detection method of eight kinds of Genotypings of hepatitis type B virus, which is characterized in that the described method includes following
Step：
（1）Synthesize primer and probe described in claim 1；
（2）According to instrument each probe both ends are marked with fluorescent reporter group and the corresponding fluorescence described in claim 2 respectively
Quenching group；
（3）Prepare positive criteria product；
（4）Extract sample to be tested DNA；
（5）Prepare PCR reaction systems, PCR reaction systems are divided into two, for the primer of hepatitis type B virus ABCD type genotype
It adds in the first reaction system with probe, is added in for the primer and probe of hepatitis B virus E FGH type genotype to second instead
Answer system；
（6）Run PCR；
（7）Data analysis.
6. the nondiagnostic detection method of eight kinds of Genotypings of hepatitis type B virus according to claim 5, feature exist
In the step（3）The preparation method of middle positive criteria product includes eight kinds of gene hypotypes of hepatitis type B virus A-H respectively for structure
Detect the plasmid of target gene standard sequence：It is respectively synthesized eight kinds of gene hypotype detection target gene marks of hepatitis type B virus A-H
Quasi- sequence is simultaneously cloned into P18-T carriers, is named as PMT-HBV-A, PMT-HBV-B, PMT-HBV-C, PMT-HBV-D, PMT-
HBV-E, PMT-HBV-F, PMT-HBV-G, PMT-HBV-H.
7. the nondiagnostic detection method of eight kinds of Genotypings of hepatitis type B virus according to claim 5, feature exist
In the step（2）The mark of middle probe is respectively：The 5' ends flag F AM of HBV-A-P, 3' ends mark MGB, the 5' of HBV-B-P
The 5' ends mark ROX of end mark HEX, 3' ends mark MGB, HBV-C-P, the 5' ends mark CY5 of 3' ends mark MGB, HBV-D-P,
3' ends mark MGB；The 5' ends mark HEX of the 5' ends flag F AM of HBV-E-P, 3' ends mark MGB, HBV-F-P, 3' ends mark
The 5' ends mark ROX of MGB, HBV-G-P, the 5' ends mark CY5 of 3' ends mark MGB, HBV-H-P, 3' ends mark MGB.
8. the nondiagnostic detection method of eight kinds of Genotypings of hepatitis type B virus according to claim 7, feature exist
In the step（6）Fluorescence channel detects and selects when running PCR：FAM, HEX, ROX and CY5 passage are selected in detection.
9. the nondiagnostic detection method of eight kinds of Genotypings of hepatitis type B virus according to claim 5, feature exist
In the step（6）Response procedures when running PCR are 95 DEG C of 2min；Again by 95 DEG C of 15s, 60 DEG C of 1min, cycle 40 times.
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| CN107022543A (en) * | 2017-05-25 | 2017-08-08 | 郑州市第六人民医院 | One kind extracts hbv nucleic acid with magnetic bead, extracts reagent, extracting method, quantitative detection kit |
| CN113549707A (en) * | 2020-04-24 | 2021-10-26 | 利多(香港)有限公司 | Method, oligonucleotide and kit for HBV genotype detection |
| CN112813204A (en) * | 2021-03-10 | 2021-05-18 | 北京康美天鸿生物科技有限公司 | Fluorescent PCR kit for genotyping hepatitis B virus |
| CN114250325A (en) * | 2021-12-31 | 2022-03-29 | 广西壮族自治区疾病预防控制中心 | Primer, probe, kit and method for rapidly detecting 9 genotypes of hepatitis B virus |
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| CN1584053A (en) * | 2004-06-03 | 2005-02-23 | 佛山市第一人民医院 | Hepatitis B virus gene parting method |
| CN101402993A (en) * | 2007-11-05 | 2009-04-08 | 中国科学院武汉病毒研究所 | Parting method for hepatitis B virus gene |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1584053A (en) * | 2004-06-03 | 2005-02-23 | 佛山市第一人民医院 | Hepatitis B virus gene parting method |
| CN101402993A (en) * | 2007-11-05 | 2009-04-08 | 中国科学院武汉病毒研究所 | Parting method for hepatitis B virus gene |
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| Novel Method for Genotyping Hepatitis B Virus on the Basis of TaqMan Real-Time PCR;Sebastian Malmstrom等;《JOURNAL OF CLINICAL MICROBIOLOGY》;20100430;第48卷(第4期);第1105-1111页 * |
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