CN104263851B - Hepatitis C virus genetic typing chip and typing method - Google Patents
Hepatitis C virus genetic typing chip and typing method Download PDFInfo
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Abstract
The invention discloses a hepatitis C virus genetic typing chip and typing method. The hepatitis C virus genetic typing chip comprises a chip carrier, an amplification primer and a probe nucleotide segment loaded on the chip carrier. The nucleotide sequences of the primer and the probe are shown as SEQ ID No:1 and SEQ ID No:8. According to epidemic conditions of the hepatitis C virus (HCV) and aiming at six subtypes of the HCV, including 1a, 1b, 2a, 3a and 3b, a nucleic acid liquid phase chip detection method capable of typing at the same time is established. A novel detection method is provided for clinical typing detection of hepatitis C and molecular epidemiological investigation; and references are provided for selection of anti-virus treatment plans.
Description
Technical field
The invention belongs to biology field, especially relate to a kind of HCV gene typing
Chip and classifying method.
Background technology
Hepatitis C virus (Hepatitis C Virus, HCV) is to cause non-a, non-b type liver after blood transfusion
Scorching main arch-criminal, whole world infection rate is up to 3%.After HCV infection, there are about 50%～80%
Transfer chronic infection to, and have up to 20%～30% can develop into after 20～30 years after infection
Liver cirrhosis and hepatocarcinoma.The health and lives of HCV infection serious threat people, causes huge
Society, economy and medical burden, be a serious social public health problem.
HCV genome is height heterogeneity, at least can be divided into 6 genotype (HCV1～6 types),
There is more than 100 gene hypotype.Accurately differentiate HCV hypotype, for being better understood by HCV
Virus evolution, inquires into the route of transmission of virus, selects clinical treatment, it is judged that change of illness state,
Evaluating antiviral therapy effect, vaccine is prepared etc. and to be had very important significance.
Summary of the invention
An object of the present invention be to design a kind of can be simultaneously to HCV gene typing
Nucleic acid liquid-phase chip, described chip includes chip carrier, amplimer and be carried on chip carrier
Probe nucleotide fragment；The nucleotide sequence of described primer and probe such as SEQ ID NO:1
To shown in SEQ ID NO:8.
Another object of the present invention is to provide a kind of nucleic acid liquid-phase chip pair using energy typing simultaneously
Hepatitis c virus gene carries out the method for typing, and the step that described method uses is:
A) magnetic carboxylic fluorescent encoding microsphere is coated nucleic probe；
B) set up with the gene standard substance plasmid of synthesis for template and optimize hepatitis C virus nucleic acid liquid
Phase chip genotyping detection method；
C) extracting sample rna, reverse transcription becomes cDNA, carries out hepatitis C virus nucleic acid liquid phase core
Sheet typing.
The present invention is according to the popularity of hepatitis C virus (HCV), for six kinds of Asias of HCV
Type, including 1a, 1b, 2a, 3a, 3b and 6a, sets up the nucleic acid liquid phase core of energy typing simultaneously
Chip detection method, can provide for the detection of hepatitis C Clinical typing, Molecule Epidemiology Investigation etc.
A kind of new detection method, the selection for antiviral therapy scheme provides reference frame.
Accompanying drawing explanation
Fig. 1 show hepatitis C virus (6a hypotype) nucleic acid liquid-phase chip genotyping detection method
Set up and optimization experiment result schematic diagram.
Fig. 2 show the spirit of hepatitis C virus (1b hypotype) nucleic acid liquid-phase chip genotyping detection method
Sensitivity experimental result schematic diagram.
Fig. 3 show the specificity experiments of hepatitis C virus nucleic acid liquid-phase chip genotyping detection method
Result schematic diagram.
Fig. 4 show the detection simulation mixing of hepatitis C virus nucleic acid liquid-phase chip genotyping detection method
Infect sample experiments result schematic diagram.
Detailed description of the invention
Below in conjunction with specific embodiments and the drawings, the present invention is described in further detail.
Material
1.1.1 Specimen origin in JIUYUE, 2012 in December, 2013 in the Chinese People's Liberation Army 100
Hospital and Suzhou City the 5th the People's Hospital's health check-up, outpatient service and healthy population 38 example being in hospital and anti-
-HCV positive patient 55 example (41 examples be chronic hepatitis, 14 examples be liver cirrhosis), anti-HCV
Basis for estimation " the third type diagnostic criteria in 2008 with HCV RNA positive patient lesion degree
WS2013 " diagnostic criteria.Wherein, male is 59 examples, and women is 34 examples, and the age is
34-57 year, average 48.3 years old.All specimen all adopt venous blood 2ml, separate serum ,-70 DEG C
Preserve.
1.1.2 main agents HCV-1a, 1b, 2a, 3a, 3b and 6a gene standard substance plasmid (on
March forward Bioisystech Co., Ltd in sea), magnetic carboxylic fluorescent encoding microsphere (Magnetic
COOH Beads, Luminex company of the U.S., 1.25 × 107Individual/mL), Streptavidin-
Phycoerythrin (treptavidin, R-phycoerythrin conjugate, SA-PE, 1mg/mL,
1mL, American I nvitrogen company), Shead Fluid (Luminex company of the U.S.), PCR
Master Mix (U.S. Fermentas),1st Strand cDNA Synthesis
Kit (Japan Takara).
1.1.3 (MAGPIX type, U.S. Luminex is public for instrument and equipment liquid-phase chip detection system
Department), PCR instrument (T100TM, Bio-Rad company of the U.S.).
1.1.4 the E1 gene order that primer and probe primer and probe are delivered all in accordance with Genbank,
Primer Primer5.0 software design is used to form.Primer, probe nucleotide sequence such as table 1
Shown in.In order to make pcr amplification product biotinylation, downstream primer 5 ' end has carried out biotin
Labelling.Carrying out coupling reaction for the ease of probe and carboxylated micro-spheres, all probes 5 ' are carried out
C6 amination is modified, simultaneously in order to ensure the hybridization efficiency of probe and PCR primer, at probe
5 ' end with the addition of 10 poly (T), poly (T) is effectively reduced sterically hindered as spacerarm,
Be conducive to the carrying out of probe hybridization.Primer and probe are by the Dalian limited public affairs of treasured biological engineering
Department's synthesis.
Table 1HCV liquid-phase chip typing detection primer and probe sequence
Note: 1, Y is degeneracy base code, represents C and T；2, N is degeneracy base code, represents A, T, C and G.
1.2 method
1.2.1 the activation of microsphere and nucleic probe coupling randomly select No. 26, No. 36, No. 43,
No. 46, No. 52 and No. 62 magnetic carboxylic fluorescent encoding microspheres are used for testing, and are coated respectively
6a, 1a, 1b, 2a, 3a and 3b hypotype probe, is labeled as 26-HCV-6a, 36-HCV-1a,
43-HCV-1b, 46-HCV-2a, 52-HCV-3a and 62-HCV-3b.With reference to U.S. Luminex
The operating instruction of company is carried out, and microsphere consumption is 50 μ L, and 100pmol/ μ L amination is modified
Nucleic probe consumption be 10 μ L.
1.2.2 hepatitis C virus nucleic acid liquid-phase chip genotyping detection method foundation and optimize with close
The gene standard substance plasmid become is that template is set up and optimizes hepatitis C virus nucleic acid liquid-phase chip
Genotyping detection method.
1.2.2.1PCR expand with the gene standard substance plasmid synthesized as template, enter according to following system
Performing PCR reacts: PCR Master Mix (2 ×) 10 μ L, Primer HCV-E1-F0.8 μ L,
Primer HCV-E1-R0.8 μ L, template 1 μ L, use ddH2O supplies 20 μ L.Reaction condition
For: 95 DEG C of 10min；94℃30s,50℃30,72℃40sec,40cycles；72℃10min；
4 DEG C of preservations.Design primer ratio includes: F:R=1 μM: 10 μMs (1:10), 1.25 μMs: 10 μMs
(1:8),1.67μM:10μM(1:6),2.5μM:10μM(1:4),5μM:10μM(1:2),
10μM:10μM(1:1)。
1.2.2.2PCR product is abundant with the mixing microsphere hybridization of conjugated probes and liquid-phase chip detection
The mixing microsphere working solution of resuspended coupling nucleic acid probe；At each sample well, add 33 μ L cores
Acid hybridization buffer, is subsequently adding 10 μ L mixing microsphere working solutions, adds
The PCR primer of 1 μ L/3 μ L/5 μ L/7 μ L, each sample arranges 3 repetitions, adds TE and mends
Foot volume, to 50 μ L, fully mixes, reacts as follows: 1. 95 DEG C of degeneration 5min；②37℃
Hybridization 30min.With detection of nucleic acids buffer, 1mg/mL SA-PE is diluted to 4 μ g/mL,
Every hole adds 25 μ L, hatches 5min for 37 DEG C；Add 150 μ L and terminate reactant liquor (37 DEG C of preheatings),
Sample is placed on Luminex MAGPIX and tests, analyze the fluorescence signal value of sample
(MFI)。
1.2.3 the sensitivity experiment of hepatitis C virus nucleic acid liquid-phase chip genotyping detection method according to
Every kind of standard substance are diluted to 1 × 10 by the concentration of gene standard substance plasmid10Copies/ μ L, then
10 times of dilution methods are used to be diluted to 1 × 100Copies/ μ L, every PCR reaction adds template amount
1μL.The hepatitis C virus nucleic acid liquid-phase chip genotyping detection method using 1.2.2 to set up is carried out
Detection, the sensitivity of evaluation methodology.
1.2.4 the specificity experiments of hepatitis C virus nucleic acid liquid-phase chip genotyping detection method uses
1.2.2 the hepatitis C virus nucleic acid liquid-phase chip genotyping detection method set up is sick to hepatitis A
Poison, hepatitis B virus, treponema pallidum, chikungunya fever virus, dengue virus are carried out
Detection, it is 1 μ L that every PCR reaction adds template amount, the specificity of evaluation methodology.
1.2.5 hepatitis C virus nucleic acid liquid-phase chip genotyping detection method detection simulation mixed infection sample
Every kind of standard substance, according to the concentration of gene standard substance plasmid, are diluted to by product experiment
1×107Standard substance are combined by copies/ μ L, be simultaneously introduced in a reaction tube two kinds,
Three kinds, four kinds, five kinds, six kinds of plasmid standards.Use the hepatitis C virus that 1.2.2 sets up
Poison nucleic acid liquid-phase chip genotyping detection method detects, evaluation methodology detection mixed infection sample
Effect.
1.2.6 the clinical practice of hepatitis C virus nucleic acid liquid-phase chip genotyping detection method is just being collected
Ordinary person, hepatitis C patient blood sample, totally 93 parts, separate and obtain serum, extract sample
RNA, reverse transcription becomes cDNA.Use the hepatitis C virus nucleic acid liquid phase core that 1.2.2 sets up
Sheet genotyping detection method and real time fluorescent PCR method detect simultaneously.Tie with clinical diagnosis
Fruit is compared, and is analyzed laboratory test results, determines the hepatitis C virus that this institute is set up
The poison sensitivity of nucleic acid liquid-phase chip genotyping detection method, accuracy.Real-time fluorescence PCR side
Method is bought business-like test kit and is detected.Detect simultaneously for nucleic acid liquid-phase chip typing
The hepatitis C sample of method test positive, carries out PCR amplification, and PCR primer is cloned into
Carrier T, selects monoclonal, after PCR identifies the positive, delivers to Beijing promise match genome and grinds
Study carefully centered finite company to check order, sequencing result is analyzed, it is determined that HCV gene is sub-
Type, analysis result compares with liquid-phase chip detection method.
1.2.7 hepatitis C virus nucleic acid liquid-phase chip genotyping detection method experimental result criterion
Establish according to Luminex business recommendations criterion and documents and materials, in conjunction with this research experiment
Situation, the criterion of hepatitis C virus nucleic acid liquid-phase chip typing test experience result sets
As follows: (1) data validity analysis: the fluorescence signal value of blank group is not higher than 100；
(2) sample to be tested analysis judges: testing result Cutoff value is set as PCR Blank fluorescence
4 times of signal value, when the fluorescence signal value of 1. sample to be tested is not higher than PCR Blank fluorescence letter
Number value 2 times time, it is determined that for negative sample；2. the fluorescence signal value >=Cutoff of sample to be tested
During value, it is determined that for positive sample；3. when 2 times≤fluorescence of PCR Blank fluorescence signal value
During signal value ＜ Cutoff value, be set to gray scale interval, it is determined that for suspicious specimen, need into
Row repeats experiment or takes other detection methods to verify further.
2 results
The foundation of 2.1 hepatitis C virus nucleic acid liquid-phase chip genotyping detection methods and optimization experiment knot
Fruit is analyzed by the gene standard substance plasmid of six hypotypes of hepatitis C virus carries out detection,
The optimum reaction condition determining hepatitis C virus nucleic acid liquid-phase chip genotyping detection method is: F:
R primer ratio is 1:10 and 1:8；PCR primer sample-adding amount is 3 μ L, 5 μ L or 7 μ L.Root
According to experimental conditions and the convenience of experimental implementation, selecting F:R primer ratio is 1:10, PCR
Product sample-adding amount is 5 μ L, 95 DEG C of degeneration 5min, and 37 DEG C of hybridization 30min, SA-PE concentration are
4 μ g/mL are optimum reaction condition.With 6a hypotype experimental result to the foundation of detection method and excellent
Change illustrates, and Fig. 1 is it can be seen that along with the rising of primer ratio F:R, fluorescence signal
It is worth on a declining curve.When F:R primer ratio is 1:10 and 1:8, fluorescence signal value is higher,
And background signal value is relatively low, it is thus achieved that preferably experimental result.When primer ratio is 1:2 and 1:1
Time, while fluorescence signal value reduces, sample segment microsphere background signal value but raises.When drawing
When thing ratio is 1:6 and 1:4, fluorescence signal value reduces less obvious, but sample segment microsphere
Background signal value is also increased significantly.Along with the increase of PCR primer sample-adding amount, fluorescence signal
Value is in the trend raised and tend to be steady.PCR primer sample-adding amount when 1 μ L increases to 3 μ L,
The amplitude that fluorescence signal value increases is maximum, when PCR primer sample-adding amount increases to 5 μ L from 3 μ L
During with 7 μ L, fluorescence signal value has increase, but the amplitude being to increase is the most inconspicuous.To sum up
Described, hepatitis C virus (6a hypotype) nucleic acid liquid-phase chip genotyping detection method optimal
Reaction condition is: F:R primer ratio is 1:10 and 1:8；PCR primer sample-adding amount be 3 μ L,
5 μ L or 7 μ L.
The sensitivity experiment experiment knot of 2.2 hepatitis C virus nucleic acid liquid-phase chip genotyping detection methods
Fruit shows, for HCV1a, 3a and 6a hypotype nucleic acid liquid-phase chip genotyping detection method
Sensitivity is 1 × 105copies/PCR；For HCV1b, 2a and 3b hypotype nucleic acid liquid phase core
The sensitivity of sheet genotyping detection method is 1 × 104copies/PCR.Wherein, for HCV1a
Hypotype and the 1 × 10 of 6a hypotype4The testing result of copies plasmid standard sample is positioned at gray area
In；For HCV1a hypotype 1 × 104Copies and in 1 × 103Copies plasmid standard
The testing result of sample is positioned at gray scale interval, can be further confirmed that by other detection method.
1b hypotype sensitivity experiment result is as in figure 2 it is shown, remaining 5 hypotype experimental result is omited.
The specificity experiments knot of 2.3 hepatitis C virus nucleic acid liquid-phase chip genotyping detection methods
Really use the hepatitis C virus nucleic acid liquid-phase chip genotyping detection method set up to A type
Hepatitis B virus hepatitis virus, hepatitis C virus, treponema pallidum, Chikungunya fever
Virus, dengue virus and hepatitis c virus gene standard substance plasmid detect, Fig. 3
Test result indicate that, for hepatitis c virus gene standard substance plasmid and hepatitis c virus-like
Product, testing result is positive.Wherein hepatitis c virus-like product, 3 is HCV1b hypotype,
1 is HCV1a hypotype, and 1 is HCV2a hypotype, also 1 be HCV1b and
2a hypotype mixed infection.HCV3a, 3b and 6a hypotype does not detects.Sick for hepatitis A
Poison, hepatitis B virus, treponema pallidum, chikungunya fever virus and dengue virus sample,
Testing result is negative.Experimental result illustrates, the hepatitis C virus nucleic acid that this institute is set up
The specificity of liquid-phase chip genotyping detection method is preferable.Meanwhile, the detection to HCV-002 is tied
Fruit is it is also shown that mixed infection sample can accurately be detected by this method.
2.4 hepatitis C virus nucleic acid liquid-phase chip genotyping detection method detection simulation mixed infection samples
Every kind of standard substance, according to the concentration of gene standard substance plasmid, are diluted to by product experimental result
1×107Standard substance are combined by copies/ μ L, be simultaneously introduced in a reaction tube two kinds,
Three kinds, four kinds, five kinds, six kinds of plasmid standards.Use hepatitis C virus nucleic acid liquid phase core
Sheet genotyping detection method detects, and Fig. 4 test result indicate that, the method can be to arbitrarily
Two kinds of hypotypes, any three kinds of hypotypes, any four hypotype, any five kinds of hypotypes and six kinds of hypotypes
Nucleic acid accurately detects.This experiment uses standard substance plasmid simulation mixed infection sample, it is thus achieved that
Preferably testing result, illustrates that mixed infection sample can accurately be detected by this method.
The clinical practice experimental result of 2.5 hepatitis C virus nucleic acid liquid-phase chip genotyping detection methods
Collect normal person, hepatitis C patient blood sample, totally 93 parts, separate and obtain serum, carry
Taking sample rna, reverse transcription becomes cDNA.Use the hepatitis C virus nucleic acid that 3.2.9 sets up
Liquid-phase chip genotyping detection method and real time fluorescent PCR method detect simultaneously.Experiment inspection
Survey result is as shown in table 2, and the hepatitis C virus nucleic acid liquid-phase chip that this institute is set up divides
The testing result of type detection method is compared with clinical diagnosis result, and sensitivity is 74.55%,
Specificity is 86.84%, and diagnosis efficiency is 79.57%；Detect with real-time fluorescence RT-PCR method
Result compares, and sensitivity is 76.47%, and specificity is 83.33%, and diagnosis efficiency is 79.57%.
Real-time fluorescence RT-PCR testing result is compared with clinical diagnosis result, and sensitivity is
76.36%, specificity is 76.32%, and diagnosis efficiency is 76.34%.Use nucleic acid liquid-phase chip
The testing result of method has higher with clinical diagnosis result and real-time fluorescence RT-PCR testing result
Concordance.Hepatitis C sample for nucleic acid liquid-phase chip genotyping detection method test positive
Totally 41 parts, wherein, 1a hypotype is 9 examples, and 1b hypotype is 20 examples, and 2a hypotype is 2 examples,
3b hypotype is 2 examples, and 6a hypotype is 5 examples, 1b and 2a mixed infection hypotype is 3 examples.To sample
After product carry out sequencing analysis, wherein, 1a hypotype is 7 examples, and 1b hypotype is 22 examples, 2a hypotype
Being 4 examples, 3b hypotype is 2 examples, and 6a hypotype is 6 examples.Nucleic acid liquid-phase chip typing detection side
Method result and sequencing testing result have higher concordance, but sequencing can only be to single hypotype
It is analyzed, not accurate enough to mixed infection sample detection result.Hepatitis C virus nucleic acid liquid
Phase chip genotyping detection method is high-flux detection method, can be by one-time detection simultaneously to six kinds
Hypotype carries out examination detection, relative to the real-time fluorescence that can only detect an index every time
RT-PCR method, can be greatly shortened operating time and detection workload, hepatitis C can be conducive to sick
The typing detection of poison.
Sequence table (SEQUENCE LISTING)
<110>Zhou Youliang
Hu Chunling
Wang Haitao
Chen Feng
Wang Chaohui
<120>HCV gene typing chip and classifying method
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GGTTGCTCYTTYTCTATCTT
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<400> 2
TCATCATATCCCANGCCAT
<210> 3
<211> 26
<212> DNA
<213>artificial sequence
<400> 3
TTTTTTTTTT-ACTAGGGACGGCAAAC
<210> 4
<211> 25
<212> DNA
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<400> 4
TTTTTTTTTT-AGAACAACGCCTCCC
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TTTTTTTTTT-AGAACACCAGTAAGAG
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TTTTTTTTTT-AGCTAGTCTAGGGTG
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<211> 27
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<213>artificial sequence
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TTTTTTTTTT-GTCTGGTCTAGAGCACA
<210> 8
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<213>artificial sequence
<400> 8
TTTTTTTTTT-GGCTCTTACCTACG
Claims (1)
1. a HCV gene typing chip, including chip carrier, amplimer and the probe nucleotide fragment being carried on chip carrier；The nucleotide sequence of described primer and probe is as shown in SEQ ID NO:1 to SEQ ID NO:8；F:R primer ratio is 1:10 or 1:8；Described F primer is forward primer, and R primer is downstream primer.
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| CN201410336476.2A CN104263851B (en) | 2014-07-15 | 2014-07-15 | Hepatitis C virus genetic typing chip and typing method |
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| CN201410336476.2A CN104263851B (en) | 2014-07-15 | 2014-07-15 | Hepatitis C virus genetic typing chip and typing method |
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| CN104263851A CN104263851A (en) | 2015-01-07 |
| CN104263851B true CN104263851B (en) | 2017-01-11 |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101377486A (en) * | 2007-08-29 | 2009-03-04 | 中山大学达安基因股份有限公司 | HCV gene typing detecting reagent kit |
| CN101660002A (en) * | 2008-08-27 | 2010-03-03 | 中山大学达安基因股份有限公司 | HCV genotyping DNA microarray chip |
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2014
- 2014-07-15 CN CN201410336476.2A patent/CN104263851B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101377486A (en) * | 2007-08-29 | 2009-03-04 | 中山大学达安基因股份有限公司 | HCV gene typing detecting reagent kit |
| CN101660002A (en) * | 2008-08-27 | 2010-03-03 | 中山大学达安基因股份有限公司 | HCV genotyping DNA microarray chip |
Non-Patent Citations (2)
| Title |
|---|
| 丙型肝炎病毒基因分型与临床分子诊断;张太松;《分子诊断与治疗杂志》;20100205;第2卷(第5期);289-293 * |
| 丙型肝炎病毒核酸液相芯片分型检测方法的建立及应用;周有良;《国际检验医学杂志》;20140715;第35卷(第13期);1710-1715 * |
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| CN104263851A (en) | 2015-01-07 |
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