CN108018376A - A kind of probe and method for hepatitis C virus parting and drug resistance site primer - Google Patents
A kind of probe and method for hepatitis C virus parting and drug resistance site primer Download PDFInfo
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Abstract
The invention discloses a kind of probe and method for hepatitis C virus parting and drug resistance site primer.The nucleotide sequence such as SEQ ID NO of the probe of the present invention：Shown in 1 17.Probe provided by the invention can be used in hepatitis C virus parting and drug resistance site primer, and can be used for a variety of (total quantitys>46 kinds) parting and drug resistance site primer of HCV virus hypotype, suitable medicine can be effectively screened, avoids, using immune medicine, accomplishing personalized treatment.
Description
Technical field
The present invention relates to hepatitis C virus detection technique field, more particularly to one kind to be used for hepatitis C virus parting and drug resistance site
The probe and method of detection.
Background technology
Hepatitis C virus (HCV) is the RNA virus of single-stranded positive, and full-length genome is about 9.6kb.HCV genomes are by both ends
Two noncoding regions (UTR) and an open reading frame (ORF) of centre composition.ORF areas are divided into C areas, E1 areas, E2 again
Area, NS1 areas, NS2 areas, NS areas, NS4A areas, NS4B areas, NS5A areas and NS5B areas.By the polymerase relied on when HCV is replicated
Lack proofreading function, easily undergone mutation plus itself to adapt to environment and escaping the immunosurveillance of host, therefore
Numerous mutant strains, i.e. genotype have been gradually formed in long-term evolutionary process.HCV genotype bases are divided into 6 kinds of genotype (1-6),
Each genotype is by being divided into the various hypotypes such as a/b/c, more than 80 kinds hypotypes at present.
Hepatitis C illness, severity extent caused by different genotype HCV judge and the use of antiviral treatment
Etc. there is significant difference.It is now recognized that HCV 1B types are carried out with heavy type hepatitis and the state of an illness, sexual development is related, accounts for the 80% of sum.In liver
Harden also common with 1b types in the patient of Decompensated stage, primary carcinoma of liver and after liver transplantation recurrence.Amoroso etc. (1998) is sent out
Existing 92% 1b type acute infection HCV patients can develop into chronic hepatitis C, and other genotype transition probabilities are then 35%-
50%.The research in (2015) such as Jiang Shupeng finds that different genotype HCV infection is related to the virus concentration of HCV in blood, gene 1
Type patient HCV concentration is higher than non-1 type patient；Different genotype HCV patients need to use different disease-resistant drugs, such as III type HCV
Complete response rate of the infected to IFN-α 2b is significantly higher than II type HCV infection person (P<0.005), III type HCV infection person couple
The unresponsive rate of IFN-α 2b is substantially less than II type HCV infection person (P<0.005).Telaprevir and Peg-IFN alpha-2b
(PEG-IFN)/ribavirin drug combinations are only limitted to the patient of 1 type of HCV infection gene；In April, 2014, European Hepatology Association
(EASL) delivered online newest《Treating hepatitis c guide》, various HCV genotypes are provided with corresponding therapeutic scheme, and
Point out before starting treatment, HCV genotype and 1 hypotype of gene (1a/1b) should be assessed, to determine the selection of therapeutic scheme.
The site mutation that the characteristics of natural mismatch rate of height and low RNA polymerase fidelity when HCV virus replicates produces,
Using medicine for a period of time after, formed virus drug resistance, cause virus quantity to rebound, limit DAA clinical practices.HIV researchs association
Can forum (Forum for Collaborative HIV Research) the HCV mutation position clinically relevant in 2012 annual reports
Point and drug resistance analysis.And in June, 2015 mechanism and center for Drug Evaluation and Research (Center for Drug
Evaluation and Research) etc. issued a drug resistance position related to more HCV DAA drug therapies in further detail
Point report, this report have been set out each relevant mutational site in HCV genotype NS3, NS5A, NS5B region, have used various DAA
The site mutation produced after medicine is corresponding with wild type replicon multiple analysis (fold-change), and various DAA medicines
Drug resistance site, HCV genotype and drug resistance analysis (fold-resistance) etc..
HCV methods of genotyping mainly include molecular method (including PCR sequencing PCR, specific primer PCR methods, specificity visit
Pin hybridization (LIPA), gene chips, restriction fragment length polymorphism analysis (RFLP) method etc.), serological method (including weight
Group western blot test (RIBA) and Dot Enzyme Immunoassay (EIA)).Wherein PCR sequencing PCR passes through the representational gene piece of PCR amplification
Section such as 5 ' NTR areas, C areas, NS5B areas, carry out nucleotide sequencing again, be " goldstandard " of HCV Genotypings.These methods
Main genotype can be effectively distinguished, but only the direct Sequencing of nucleotide is effective in hypotype is distinguished.
Existing HCV genotyping techniques are detected analysis, part mainly for some specific gene region in genome
Method there are it is unstable, accuracy is relatively low, and can only parting to genotype, and subtype typing ability is then very limited.Existing point
Type method basically can not also carry out drug resistance site primer.
The content of the invention
The present invention provides a kind of probe and method for hepatitis C virus parting and drug resistance site primer, available for a variety of
The parting and drug resistance site primer of HCV virus hypotype, can effectively screen suitable medicine, avoid using immune medicine
Thing, accomplishes personalized treatment.
According to the first aspect of the invention, the present invention provides a kind of spy for hepatitis C virus parting and drug resistance site primer
Pin, the nucleotide sequence such as SEQ ID NO of the probe：Shown in 1-17.
According to the second aspect of the invention, the present invention provides a kind of examination for hepatitis C virus parting and drug resistance site primer
Agent box, the kit include probe, the nucleotide sequence such as SEQ ID NO of above-mentioned probe：Shown in 1-17.
Further, mentioned reagent box further includes：For extracting the agent formulations of Hepatitis C virus RNA.
Further, mentioned reagent box further includes：Agent formulations for reverse transcription Hepatitis C virus RNA.
Further, mentioned reagent box further includes：For carrying out library to the cDNA obtained after Hepatitis C virus RNA reverse transcription
The component of structure.
Further, the above-mentioned component that library construction is carried out to cDNA includes：The component repaired for end, for 3 ' ends
End plus the component of A bases, for the component of connector connection, the component for PCR amplification.
Further, mentioned reagent box further includes：Component for library capture.
According to the third aspect of the invention we, the present invention provides a kind of base for hepatitis C virus parting and drug resistance site primer
Because of chip, which includes solid carrier and probe, the nucleotide sequence such as SEQ ID NO of above-mentioned probe：Shown in 1-17.
According to the fourth aspect of the invention, the present invention provides a kind of method for hepatitis C virus parting, and this method includes
Following steps：The extraction and processing of nucleic acid：To the plasma sample after whole blood or separation, disease is carried out using RNA virus extracts reagent
The extraction of malicious RNA；Then carry out reverse transcription and RNA is synthesized into double-strand cDNA；The structure in library：To the double-strand cDNA of above-mentioned acquisition
Carry out the structure in library；Library captures：Use nucleotide sequence such as SEQ ID NO：Capture probe shown in 1-17 is to building
Library carry out hybrid capture；Sequencing：To the library after capture, high-flux sequence is carried out；Information analysis：To acquisition it is original under
Then machine data are compared with existing HCV reference sequences into the removal of pedestrian's source sequence, obtain final sample HCV's
Complete genome sequence, and the parting of hepatitis C virus.
According to the fifth aspect of the invention, the present invention provides a kind of method for hepatitis C virus drug resistance site primer, should
Method includes the following steps：The extraction and processing of nucleic acid：To the plasma sample after whole blood or separation, extracted and tried using RNA virus
Agent carries out the extraction of viral RNA；Then carry out reverse transcription and RNA is synthesized into double-strand cDNA；The structure in library：To above-mentioned acquisition
Double-strand cDNA carries out the structure in library；Library captures：Use nucleotide sequence such as SEQ ID NO：Capture probe shown in 1-17
Hybrid capture is carried out to the library built；Sequencing：To the library after capture, high-flux sequence is carried out；Information analysis：To obtaining
Original lower machine data into the removal of pedestrian's source sequence, be then compared with existing HCV reference sequences, obtain final sample
The complete genome sequence of product HCV, and corresponding abrupt information and drug resistance site.
Probe provided by the invention can be used in hepatitis C virus parting and drug resistance site primer, and available for a variety of (total
Quantity>46 kinds) parting and drug resistance site primer of HCV virus hypotype, suitable medicine can be effectively screened, is avoided using
Immune medicine, accomplishes personalized treatment.
Embodiment
The present invention is described in further detail below by embodiment.
Genome area of the present invention based on 46 kinds of HCV gene hypotypes carries out probe design, and skill is sequenced by capture, two generations
Testing result that is more accurate, stablizing that art can provide, accurately parting can go out most HCV hypotypes；The present invention can be done resistance at the same time
Medicine site primer, more accurately guidance is provided for medication.
First, probe design and synthesis：
From NCBI (http://www.ncbi.nlm.nih.gov/) download what 46 kinds of HCV gene hypotypes were designed as probe
Reference sequences；Repetitive sequence between each genome is removed, only retains a copy；Consider probe length, Tm values, repeat sequence
The information such as row ratio, G/C content, specificity, target area overburden depth, it is 90bp to design every probe length, it is allowed to most
Big G/C content is 60%.Synthesized after probe design using oligonucleotides (oligo) synthesizer, and configure supporting use examination
Agent.
Based on designed by common two kinds of HCV hypotype 2a and the 1b genomes of China, and with the relevant probe sequence in drug resistance site
Core sequence is classified as, it is specific as shown in table 1.
Table 1
2nd, probe uses experimental technique：
1. the extraction and processing of nucleic acid
Sample is the blood plasma after whole blood or separation, the extraction of plasma viral is carried out using RNA virus extracts kit, then
Reverse transcription is carried out, synthesizes double-stranded DNA, and is RT-PCR (reverse transcription PCR amplification), for subsequent experimental.
2. the structure in library
The structure in Hiseq libraries is carried out to the cDNA of above-mentioned acquisition using trace dna Library development flow.
3. chip captures
Hybrid capture is carried out to the library built using HCV virus capture probe (table 1).
4. sequencing
To the library after capture, using Hiseq microarray datasets, high-throughout sequencing is completed using the sequencing length of PE 90.
5. information analysis strategy
To the original lower machine data of acquisition first into the removal of pedestrian's source sequence, then to existing HCV reference sequences into
Row compares, and obtains the complete genome sequence of final sample HCV and corresponding abrupt information.
Below by way of the specific embodiment technical solution that the present invention will be described in detail and technique effect, it should be appreciated that real
Apply example to be only exemplary, do not form limiting the scope of the invention.
Embodiment
The present embodiment is detected using 40 HCV patient's blood samples, and overall flow includes RNA extractions, RNA reversions
Record as cDNA, two generation library constructions, the sequencing of upper machine and information analysis.
First, RNA extractions (being extracted using QIAampultraSens Virus Kit)：
1. each sample takes 1mL blood plasma to be placed in 2mL centrifuge tubes, 0.8mL Buffer (buffer solution) AC, and 5.6 are added
μ g vector rnas solution (carrier RNA solution), overturns and mixes, and is incubated 10 minutes；
2. completing to centrifuge after being incubated, 1200 × g of rotating speed 3 minutes, removes supernatant；
3. add 300 μ L buffer (buffer solution) AR (ahead of time 60 DEG C incubation) and 20 μ L Proteinase Ks, concussion mixing until
Magnetic bead is resuspended completely, is placed on 40 DEG C of couveuses and is incubated 10 minutes, gentle centrifugation；
4. adding 300 μ L buffer (buffer solution) AB, centrifugation is mixed；
5. taking 700 μ L of mixed liquor to go to purification column (QIAamp spin column), 4000 × g is centrifuged 1 minute, removed
Liquid after filter；
6. adding 500 μ L buffer (buffer solution) AW1,6000 × g is centrifuged 1 minute, removes filtered fluid, and by purification column
It is transferred on a new 2mL collecting pipe, adds 500 μ L buffer (buffer solution) AW2,20000 × g is centrifuged 3 minutes；
7. purification column is transferred in new 1.5mL collecting pipes, 30 μ L buffer (buffer solution) AVE dissolving RNA are added,
6000 × g centrifuges 1min；Add 30 μ L buffer (buffer solution) AVE, 6000 × g centrifugations 1min；
8. remove purification column, the purified product after being filtered.
2nd, RNA reverse transcriptions (reagent source is in INVITROGEN companies)
1. added in each 10 μ L RNA of sample 3 μ L 5X First strand buffer (chain synthesize buffer solution) into
Break Row, 94 DEG C in PCR instrument, 10min, is immediately placed on ice；
2. 0.5 μ L N6 primers (0.1 μ g/ μ L) are added in RNA one step up.65 DEG C of 5min, are immediately placed on ice in PCR instrument
On.
3. prepare reaction mixture according to following proportioning：Multiple samples are prepared according to about 10% surplus capacity.
4. adding thing mixed above (Mix) into RNA, reacted after mixing in PCR instrument according to following procedure：
5. a chain cDNA (totally 16 μ L) for synthesis is placed on ice, following reagent Mix is prepared：Multiple samples are according to about 10%
Surplus capacity is prepared.
6. adding thing mixed above (Mix) into a chain cDNA, after mixing, 16 DEG C of constant temperature blending instruments are placed in
(Thermomixer) 2h (300rpm intermittent control shakings 15s, static 2min) on.After the completion of using Ampure XP Beads purify
CDNA products, are dissolved in 43 μ L EB solution.
3rd, library construction
1. (reagent source is in ENZYMATICS companies) is repaired in end
Prepare reaction mixture according to following proportioning：
20 DEG C in constant temperature blending instrument (Thermomixer), 30min is reacted.After the completion of reaction, with 90 μ L Ampure XP
Beads (1.8x) repairs product to end and purifies, and is dissolved in 21 μ L EB solution.
The ends of 2.cDNA 3 ' add ' A ' base (reagent source is in ENZYMATICS companies)
Prepare reaction mixture according to following proportioning：
In constant temperature blending instrument (Thermomixer), 37 DEG C of reaction 30min.This step is without purifying.
3.DNA Adaptor (connector) connections and connection product purifying (reagent source is in Huada gene company)
Prepare reaction mixture according to following proportioning：
In constant temperature blending instrument (Thermomixer), 20 DEG C of reaction 20min.Then with 47.7 μ L Ampure XP
Beads (1.8x) purifies connection product, and sample finally is dissolved in 25 μ L EB solution.
Pay attention to：This step volume of dissolution is adjusted according to sample state.As sample initial amount is low or second-rate, this step is dissolved in
15.5 μ L EB solution simultaneously all carry out PCR reactions.
4.PCR (reagent source is in INVITROGEN companies)
Primer (primer) 1.0 and sequence label primer (Index primer) are dissolved in pure water and its concentration is reached 100 μ
Mol/L, is put in -20 DEG C, is preserved as storage liquid；Appropriate primer1.0 and Index primer are taken to be diluted to 10 μ according to demand
Mol/L working solutions, freeze in -20 DEG C.If upper step DNA is dissolved in 25 μ L EB solution (solution), 15.1 μ L are taken to carry out PCR anti-
Should, remaining is positioned over -20 DEG C of refrigerators and preserves.
PCR system and reaction condition：
Amplification system is as follows：
Pay attention to：In addition to DNA and sequence label primer (10 μM), remaining reagent can be configured to reaction mixture (Mix), multiple
Sample is prepared by about 10% surplus capacity.DNA and sequence label primer (10 μM) need to be corresponded to and be individually added into.
Response procedures：94℃2min；94 DEG C of 15s, 62 DEG C of 30s, 72 DEG C of 30s, 15 circulations；72℃10min；16℃∞.
Purified after PCR reactions using magnetic bead, be dissolved in 15 μ L EB solution.
4th, library capture (reagent source is in Hua Da gene)
1. synthesize the probe (table 1) of HCV hypotype reference sequences；
2. taking 500ng to be placed in new 1.5mL centrifuge tubes respectively in all libraries, it is evaporated, is supplemented using traditional vacuum
3.4 μ L water dissolve again.
3. add 2.5 μ L of block#1, block#2 2.5 μ L, 1.5 μ L and Index Block N of public P1Block
(200 μM) 1.5 μ L, are transferred in PCR pipe, and 95 DEG C are denatured 5 minutes, and 65 DEG C are incubated 5 minutes.
4. in addition one Hybridization Buffer liquid mixture of configuration, system are as shown in table 2：
Table 2
5. Hybridization Buffer liquid mixture is taken 13 μ L respectively into step 3 system, HCV probes 5 μ L, RNase are added
1.5 μ L of 0.5 μ L of Block and water, mix.Hybridize 24 it is small when.
6. prepare Dynabeads magnetic beads：
A) Dynabeads M-280Streptavidin magnetic beads are acutely vibrated to mixing with whirlpool mixed instrument.
B) each hybridization reaction takes what 50 μ L Dynabeads M-280Streptavidin magnetic beads had been marked in 1.5mL
In centrifuge tube.
C) 200 μ L Binding Buffer (combination buffer) are added.
D) mixing is acutely vibrated with whirlpool mixed instrument.
E) centrifuge tube is put on magnetic frame, supernatant is removed after liquid becomes clarification.
F) it is repeated 2 times " step c) arrives step e) ".
G) 200 μ L Binding Buffer (combination buffer) are added and magnetic bead is resuspended.
7. by hybridize 24 it is small when after mixed system be transferred in Dynabeads, room temperature be incubated 30 minutes.
8. wash magnetic bead：
A) sample is removed from evenly mixing device, brief centrifugation is to ensure not having liquid residue in tube cover；
B) centrifuge tube transfer is placed on Dynal magnetic frames, stands 1-2min and clarified to liquid in pipe, remove supernatant
Liquid；
C) centrifuge tube is kept on magnetic frame, adds 500 μ L SureSelect Wash Buffer#1 and magnetic bead is resuspended, and
Sample is mixed in vibrating 10s on whirlpool mixed instrument；
D) samples of incubation 15min at room temperature；
E) sample cell is placed on Dynal magnetic frames after centrifuging, 1-2min is stood to clarifying, removes supernatant；
F) keeping centrifuge tube, magnetic is resuspended in the SureSelect Wash Buffer#2 for adding 65 DEG C of 500 μ L on magnetic frame
Pearl, and mix sample in vibrating 10s on whirlpool mixed instrument；
G) sample is placed on 65 DEG C of incubation 10min in dry bath；
H) centrifuge tube is overturned to mix sample, brief centrifugation；
I) centrifuge tube is placed on Dynal magnetic frames, stands 1-2min to clarifying, remove supernatant；
J) it is repeated 2 times " step f) arrives step i) "；Wash Buffer#2 are removed as far as possible clean；
K) 33.5 μ L Nuclease-Free Water (water of nuclease free) are added.
9.Post-PCR (rear-PCR) reaction system (reagent source is in Agilent companies)：
Post PCR Mix are prepared according to table 3 below, 16.5 μ L Mix are often added in pipe sample, fully mix of short duration centrifugation
Afterwards, it is put into PCR instrument.
Table 3
| Reagent | Volume (μ L) |
| DNA with magnetic bead | 33.5 |
| 5 × Herculase II Reaction Buffer (reaction buffer) | 10 |
| dNTP(100mM) | 0.5 |
| HS-EXON-FC-1.1(10μM) | 2.5 |
| HS-EXON-FC-1.2(10μM) | 2.5 |
| Herculase II Fusion DNA Polymerase (fusion dna polymerase) | 1 |
| Cumulative volume | 50 |
Post-PCR reaction conditions：98℃2min；98 DEG C of 20s, 60 DEG C of 30s, 72 DEG C of 30s, 16 circulations；72℃5min；4℃
Insulation.
10. using magnetic beads for purifying after completing PCR, finally it is dissolved in 30 μ L Elution Buffer (elution buffer), it is complete
Into capture.
5th, upper machine sequencing
Library uses on the machine Hiseq 4000 and is sequenced, and using PE91 sequencing strategies, final obtain is more than each samples of 1G
This data volume.
6th, data analysis
1) two generation sequencing datas are handled, and remove the influence because error tape is sequenced.Including：Remove the sequence for including sequence measuring joints
Row；Remove low-quality sequence；Include the sequence of more than 5 percent uncertain bases；Remove the repetition introduced during PCR
Sequence.The result form of output is FASTQ, quality system Phred+64.
2) host sequences are removed.Since the host of HCV virus is the mankind, the part mankind can be included in the result of sequencing
DNA, so demand removes the sequence from the mankind by way of comparison.Use comparison software SOAP2 (http://
Soap.genomics.org.cn/#down2), using reference gene group hg19 (http://
Hgdownload.cse.ucsc.edu/goldenPath/hg19/bigZips/), due to HCV virus genome and human gene
Group differs greatly, it is allowed to 13 mispairing；Analogue data assessment result shows the genome of HCV different genotypes in same parameter
The lower ratio compared to human genome is less than 0.1%.
3) HCV genotype identifications.By the genome for comparing HCV different genotypes/hypotype (genotype/subtype)
Sequence, the mode for calculating genome coverage are identified；It is general to require more than 90% coverage, more than 100 layers of sequencing depth,
The genome type of optimal overburden depth is selected as the genome type of the sample；The type included at present in database includes
1a、1b、2a、2b、3a、4a、5a、6a.Under same alignment parameters, assessed using analogue data, the correct gene of the results show
Set type is all 99+% coverages, and other types ratio is below 30%, and the different assembling result coverage rates of same type exist
More than 90%.The comparison software used is BWA (http://bio-bwa.sourceforge.net/)；Calculate overburden depth
Software is soap.coverage (http://soap.genomics.org.cn/#down2).
4) genome assembles.Detected for follow-up variant sites, can be used for the sample genotype mirror of mixed type
It is fixed.The assembling of the parameter progress genome of qualification is automatically selected by analysis, normal assembling result Genome Size should be in 8k
~10k, the HCV virus for needing to determine whether different genotype for sample of the assembling result more than 10k mix.Genome group
Dress uses software SOAPdenovo2 (https://github.com/aquaskyline/SOAPdenovo2).
5) variant sites (SNP) detect.At present mainly for common HCV infection genotype 1a, 1b, select respectively wild
Type genome H77 and Con1 are as reference.In order to ensure the variant sites accuracy and recall rate found out, software mummer is selected
Two groups of softwares of+SOAPpileup and BWA+GATK are analyzed, and are merged for result and are given confidence level label.With reference to base
It is as follows with the source of related software because organizing：
H77(http://www.ncbi.nlm.nih.gov/nuccore/22129792/)；
Con1(http://www.ncbi.nlm.nih.gov/nuccore/AJ238799)；
mummer(http://mummer.sourceforge.net/)；
GATK(https://software.broadinstitute.org/gatk/)。
6) gene annotation.HCV virus inhibitor is mainly for these three genes of NS3/NS5A/NS5B at present.According to variation
The result of detection is annotated to corresponding gene, marks corresponding amino acid variation information on gene.It is primarily upon and HIV suppression
Nonsynonymous mutation on relevant three genes of agent.
7) drug resistance site primer.Drug resistance site information derives from the documents and materials delivered, and mainly includes HCV 1a, 1b
Two types.Variation testing result after annotation and drug resistance site database are subjected to analysis annotation, obtain each sample
Drug resistance site primer as a result, the genotype including each sample, whether have drug resistance site, drug resistance site specifically make a variation situation,
Drug information of tolerance etc..
7th, information analysis result
40 sample genotyping results are shown in Table 4, and analysing content includes Rawdata (initial data), cleandata (close by quality
Lattice data), Effectivedata (valid data for matching HCV sequences), HCVgenotype (HCV partings) and
Coverage (reference sequences are by the ratio of data cover).
Table 4
After drug resistance site primer, find there was only 2 samples there are drug resistance site, the results are shown in Table 5.
Table 5
Above content is to combine specific embodiment further description made for the present invention, it is impossible to assert this hair
Bright specific implementation is confined to these explanations.For general technical staff of the technical field of the invention, do not taking off
On the premise of from present inventive concept, some simple deduction or replace can also be made, should all be considered as belonging to the protection of the present invention
Scope.
SEQUENCE LISTING
<110>Shenzhen Hua Da gene limited company
<120>A kind of probe and method for hepatitis C virus parting and drug resistance site primer
<130> P2016-1-0111.CN
<160> 17
<170> PatentIn version 3.3
<210> 1
<211> 90
<212> DNA
<213>Probe sequence
<400> 1
catataccag gcctgctccc tgcccgagga ggcccgcact gccatacact cgctgactga 60
gagactttac gtaggagggc ccatgttcaa 90
<210> 2
<211> 90
<212> DNA
<213>Probe sequence
<400> 2
catataccag gcctgctccc tgcccgagga ggcccgcact gccatacact cgctgactga 60
gagactttac gtaggagggc ccatgttcaa 90
<210> 3
<211> 90
<212> DNA
<213>Probe sequence
<400> 3
cctgcgtcaa tggcgtgtgt tggactgtct atcatggtgc cggctcaaag acccttgccg 60
gcccaaaggg cccaatcacc caaatgtaca 90
<210> 4
<211> 90
<212> DNA
<213>Probe sequence
<400> 4
caaagggccc aatcacccaa atgtacacca atgtggacca ggacctcgtc ggctggcaag 60
cgccccccgg ggcgcgttcc ttgacaccat 90
<210> 5
<211> 90
<212> DNA
<213>Probe sequence
<400> 5
tggaccagga cctcgtcggc tggcaagcgc cccccggggc gcgttccttg acaccatgca 60
cctgcggcag ctcggacctt tacttggtca 90
<210> 6
<211> 90
<212> DNA
<213>Probe sequence
<400> 6
tgcacctgcg gcagctcgga cctttacttg gtcacgaggc atgccgatgt cattccggtg 60
cgccggcggg gcgacagcag ggggagccta 90
<210> 7
<211> 90
<212> DNA
<213>Probe sequence
<400> 7
ttggtcacga ggcatgccga tgtcattccg gtgcgccggc ggggcgacag cagggggagc 60
ctactctccc ccaggcccgt ctcctacttg 90
<210> 8
<211> 90
<212> DNA
<213>Probe sequence
<400> 8
gacagcaggg ggagcctact ctcccccagg cccgtctcct acttgaaggg ctcttcgggc 60
ggtccactgc tctgcccctc ggggcacgct 90
<210> 9
<211> 90
<212> DNA
<213>Probe sequence
<400> 9
tccactgctc tgcccctcgg ggcacgctgt gggcatcttt cgggctgccg tgtgcacccg 60
aggggttgcg aaggcggtgg actttgtacc 90
<210> 10
<211> 90
<212> DNA
<213>Probe sequence
<400> 10
tgggcatctt tcgggctgcc gtgtgcaccc gaggggttgc gaaggcggtg gactttgtac 60
ccgtcgagtc tatggaaacc actatgcggt 90
<210> 11
<211> 90
<212> DNA
<213>Probe sequence
<400> 11
taagagatgt ttgggattgg atatgcacgg tgttgactga tttcaagacc tggctccagt 60
ccaagctcct gccgcgattg ccgggagtcc 90
<210> 12
<211> 90
<212> DNA
<213>Probe sequence
<400> 12
ctaggacctg tagtaacacg tggcatggaa cattccccat taacgcgtac accacgggcc 60
cctgcacgcc ctccccggcg ccaaattatt 90
<210> 13
<211> 90
<212> DNA
<213>Probe sequence
<400> 13
gccccctgac taattctaaa gggcagaact gcggctatcg ccggtgccgc gcgagcggtg 60
tactgacgac cagctgcggt aataccctca 90
<210> 14
<211> 90
<212> DNA
<213>Probe sequence
<400> 14
ttttctgcgt ccaaccagag aaggggggcc gcaagccagc tcgccttatc gtattcccag 60
atttgggggt tcgtgtgtgc gagaaaatgg 90
<210> 15
<211> 90
<212> DNA
<213>Probe sequence
<400> 15
aggactgcac gatgctcgta tgcggagacg accttgtcgt tatctgtgaa agcgcgggga 60
cccaagagga cgaggcgagc ctacgggcct 90
<210> 16
<211> 90
<212> DNA
<213>Probe sequence
<400> 16
tcaggaaact tggggtaccg cccttgcgag tctggagaca tcgggccaga agtgtccgcg 60
ctaggctact gtcccagggg gggagggctg 90
<210> 17
<211> 90
<212> DNA
<213>Probe sequence
<400> 17
atccagctgg ttcgttgctg gttacagcgg gggagacata tatcacagcc tgtctcgtgc 60
ccgaccccgc tggttcatgt ggtgcctact 90
Claims (10)
- A kind of 1. probe for hepatitis C virus parting and drug resistance site primer, it is characterised in that the nucleotides sequence of the probe Row such as SEQ ID NO：Shown in 1-17.
- 2. a kind of kit for hepatitis C virus parting and drug resistance site primer, it is characterised in that the kit includes visiting Pin, the nucleotide sequence such as SEQ ID NO of the probe：Shown in 1-17.
- 3. kit according to claim 2, it is characterised in that the kit further includes：For extracting hepatitis C virus The agent formulations of RNA.
- 4. kit according to claim 2, it is characterised in that the kit further includes：For reverse transcription hepatitis disease The agent formulations of malicious RNA.
- 5. kit according to claim 2, it is characterised in that the kit further includes：For to Hepatitis C virus RNA The cDNA obtained after reverse transcription carries out the component of library construction.
- 6. kit according to claim 5, it is characterised in that the component that library construction is carried out to cDNA includes： The component repaired for end, the components of A bases is added for 3 ' ends, for the component of connector connection, for PCR amplification into Point.
- 7. the kit according to claim 5 or 6, it is characterised in that the kit further includes：For library capture Component.
- A kind of 8. genetic chip for hepatitis C virus parting and drug resistance site primer, it is characterised in that the genetic chip bag Include solid carrier and probe, the nucleotide sequence such as SEQ ID NO of the probe：Shown in 1-17.
- A kind of 9. method for hepatitis C virus parting, it is characterised in that described method includes following steps：The extraction and processing of nucleic acid：To the plasma sample after whole blood or separation, viral RNA is carried out using RNA virus extracts reagent Extraction；Then carry out reverse transcription and RNA is synthesized into double-strand cDNA；The structure in library：The structure in library is carried out to the double-strand cDNA of above-mentioned acquisition；Library captures：Use nucleotide sequence such as SEQ ID NO：Capture probe shown in 1-17 carries out the library built miscellaneous Hand over capture；Sequencing：To the library after capture, high-flux sequence is carried out；Information analysis：Removal to the original lower machine data of acquisition into pedestrian's source sequence, then with existing HCV reference sequences into Row compares, and obtains the complete genome sequence of final sample HCV, and the parting of hepatitis C virus.
- A kind of 10. method for hepatitis C virus drug resistance site primer, it is characterised in that described method includes following steps：The extraction and processing of nucleic acid：To the plasma sample after whole blood or separation, viral RNA is carried out using RNA virus extracts reagent Extraction；Then carry out reverse transcription and RNA is synthesized into double-strand cDNA；The structure in library：The structure in library is carried out to the double-strand cDNA of above-mentioned acquisition；Library captures：Use nucleotide sequence such as SEQ ID NO：Capture probe shown in 1-17 carries out the library built miscellaneous Hand over capture；Sequencing：To the library after capture, high-flux sequence is carried out；Information analysis：Removal to the original lower machine data of acquisition into pedestrian's source sequence, then with existing HCV reference sequences into Row compares, and obtains the complete genome sequence of final sample HCV, and corresponding abrupt information and drug resistance site.
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| CN201610929662.6A CN108018376B (en) | 2016-10-31 | 2016-10-31 | Probe and method for hepatitis C virus typing and drug-resistant site detection |
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| CN108018376A true CN108018376A (en) | 2018-05-11 |
| CN108018376B CN108018376B (en) | 2020-11-03 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113025756A (en) * | 2021-03-31 | 2021-06-25 | 广州金域医学检验中心有限公司 | Detection method for detecting HCV1 type drug-resistant mutant gene and application thereof |
| CN113151595A (en) * | 2021-03-31 | 2021-07-23 | 广州金域医学检验中心有限公司 | Amplification primer for detecting HCV6 type drug-resistant mutant gene, detection method and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113025756A (en) * | 2021-03-31 | 2021-06-25 | 广州金域医学检验中心有限公司 | Detection method for detecting HCV1 type drug-resistant mutant gene and application thereof |
| CN113151595A (en) * | 2021-03-31 | 2021-07-23 | 广州金域医学检验中心有限公司 | Amplification primer for detecting HCV6 type drug-resistant mutant gene, detection method and application |
| CN113025756B (en) * | 2021-03-31 | 2024-02-06 | 广州金域医学检验中心有限公司 | Detection method for detecting HCV type 1 drug-resistant mutant gene and application |
| CN113151595B (en) * | 2021-03-31 | 2024-02-06 | 广州金域医学检验中心有限公司 | Amplification primer for detecting HCV6 type drug-resistant mutant gene, detection method and application |
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| CN108018376B (en) | 2020-11-03 |
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