CN101377486A - HCV gene typing detecting reagent kit - Google Patents
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Abstract
The invention relates to a kit for detecting a hepatitis c virus genotype, particularly relates to that the nucleic acid reverse dot blot hybridization technology is used to prepare a kit for hepatitis c virus genotype detection. The invention is used for rapidly and accurately distinguishing the hepatitis c virus genotype in a clinical blood sample.
Description
Technical field
The present invention relates to detect the kit of hepatitis c virus genotype, particularly relate to the reverse nucleic acid dot hybridization technology of using, prepare the kit that a kind of hepatitis c virus genotype detects, be used for distinguishing rapidly and accurately clinical blood sample hepatitis c virus genotype.
Background technology
(hepatitis C virus is one of the hepatitis virus of serious harm human health HCV) to hepatitis C virus, is important liver diseases virulence factor.At present, there are 1.7 hundred million people's HCV infection of surpassing in the whole world, and wherein has every year the 100000 routine HCV patients of surpassing to develop into liver cancer, and then hemorrhage of digestive tract and ascites occur.According to WHO annual report in 2002, in calendar year 2001, chronic liver disease causes that 1,400 ten thousand example is dead, comprise 79.6 ten thousand examples by cirrhosis cause, 61.6 ten thousand examples cause by primary carcinoma of liver.And in calendar year 2001, because of chronic liver disease causes that it is to be infected by HCV to cause that 20% (surpassing 2.8 ten thousand) just arranged in the death cases.There have been at present a large amount of cases can prove that the increase of the dead quantity that is caused by liver cancer in most countries is because hepatitis C infection causes.
Hepatitis C virus is a kind of positivity single stranded RNA flavivirus, comprises about 9,400 nucleotide.The HCV genome has an independent open reading frame, and coding one has 3,010 amino acid whose polyprotein bodies, is divided into virus replication and virion after the translation and forms necessary structure and non-structural protein.HCV has variability highly, and (its aberration rate is up to 1.4-1.9 * 10
-3/ nucleotide/year) and itself have a feature of negative selection effect, high sudden change is because replicase does not have calibration function; Easy mistake property RDRP (error-prone RDRP, existence EP-RDRP) and reason such as selection effect just.Show as and itself have negative selection effect: range gene structure and function difference in the viral genome, some region height is conservative.The HCV that separates according to different regions, the whole world is all or part of genomic phylogenetic analysis of homophyletic not, and HCV can be divided into 6 types (HCV1-6 type) at least, the various many hypotypes (as 1a, 1b, 2a, 2b etc.) of dividing again.Differ 31-34% between the various nucleotide sequence, it is about 30% that amino acid sequence differs, and differ about 20-23% between the hypotype sequence.There are several HCV Strain mainly to be separated, are named as HCV7 from Southeast Asia, 8,9,10,11 types, except HCV10a type (being put into the HCV3 type at present), since the similarity on its phyletic evolution, all the other various HCV6 types that are put into.
At present the HCV methods of genotyping is mainly contained: restricted length polymorphism (RFLP), the type specificity primer PCR, type specificity probe nucleic acid hybridization analysis, direct method such as order-checking, but there is complex operation mostly in these methods, and detection time is long, cost an arm and a leg, sensitivity is low, and the not high shortcoming of specificity is not suitable for vast basic hospital and carries out.Therefore the inventor has prepared and has detected the genotypic kit of hepatitis C in a kind of short time in conjunction with known PCR-dot hybridization method.
Summary of the invention
The present invention relates to detect the kit of hepatitis c virus genotype in the clinical biological sample, a kind of kit that utilizes reverse nucleic acid dot hybridization technology to distinguish hepatitis c virus genotype in the clinical blood sample fast and accurately particularly is provided.
Therefore an object of the present invention is to provide a kind of kit that is used for detecting clinical biological sample hepatitis c virus genotype, this kit comprises extraction reagent (2) the reverse transcription reagent of (1) blood serum sample RNA to be checked; (3) PCR reagent; (4) hybond membrane and reverse dot blot hybridization reaction reagent.It is characterized in that:
Hepatitis C (HCV) viral nucleic acid (RNA) reverse transcriptase primer:
Reverse transcription (RT) primer: 5 '-GCTCATGGTGCACGGTCTACGAGACCT-3 ' (SEQ ID NO:1)
The upstream and downstream primer that uses in the PCR amplification system is respectively:
The PCR upstream primer: 5 '-TCTAGCCATGGCGTTAGTATGAGTGT-3 ' (SEQ ID NO:2)
The PCR downstream primer: 5 '-CACTCGCAAGCACCCTATCAGGCAGT-3 ' (SEQ ID NO:3)
The oligonucleotide probe that uses in the hybridization reaction is respectively:
Probe I C:5 '-GCCGTAACCGTCACAATCCGT-3 ' (SEQ ID NO:4)
Probe PC1:5 '-ATTTGGGCGTGCCCCCGC-3 ' (SEQ ID NO:5)
Probe PC2:5 '-CGGAACCGGTGAGTACACC-3 ' (SEQ ID NO:6)
Probe 1:5 '-CAATGCCTGGAGATTTGGGCG-3 ' (SEQ ID NO:7)
Probe 1b-1:5 '-CCGCGAGACTGCTAGCCG-3 ' (SEQ ID NO:8)
Probe 1b-2:5 '-GCGAGACCGCTAGCCGAGT-3 ' (SEQ ID NO:9)
Probe 2-1:5 '-AGTAGCGTTGGGTTGCGAAAG-3 ' (SEQ ID NO:10)
Probe 2-2:5 '-GTCCTTTCTTGGATAAACCCAC-3 ' (SEQ ID NO:11)
Probe 2a-1:5 '-GCCGGGAAGACTGGGTCCT-3 ' (SEQ ID NO:12)
Probe 2a-2:5 '-CCCACTCTATGCCCGGCCA-3 ' (SEQ ID NO:13)
Probe 3-1:5 '-CCCGCGAGATCACTAGCCG-3 ' (SEQ ID NO:14)
Probe 3-2:5 '-AATCGCTGGGGTGACCGGG-3 ' (SEQ ID NO:15)
Probe 3b:5 '-CCCGCTCAATGCCCGGAAAT-3 ' (SEQ ID NO:16)
Probe 6:5 '-GACCGGGTCCTTTCCATTGG-3 ' (SEQ ID NO:17)
According to an enforcement preferred version of the present invention, designed primer and probe are at the HCV (1-6) that has delivered among the international gene pool Genebank, choose 5 ' the UTR district design primer and the probe of high conservative.
According to an enforcement preferred version of the present invention, be no less than two bases at the probe of 1 type and known hepatitis C virus 2,3,4,5,6 type corresponding gene group sequence differences.
According to a preferred embodiment of the invention, the extraction reagent of wherein said blood serum sample RNA to be checked comprises RNA extract A, RNA extract B, DEPC water.
According to a preferred embodiment of the invention, wherein said reverse transcription (RT) reagent comprises reverse transcriptase, reverse transcription reaction system, positive criteria product, negative quality-control product.
According to a preferred embodiment of the invention, the PCR reaction mixture formed by dNTPs, heat-resisting Taq enzyme etc. of wherein said pcr reagent.
According to a preferred embodiment of the invention, wherein said hybridization reaction reagent comprise hybridization solution I (1.8 * SSC-0.1%SDS) and II (0.5 * SSC-0.1%SDS), solution I (the streptavidin solution of 250U/ml coupling horseradish peroxidase), II (sodium citrate solution of 0.1mol/L), III (the tetramethyl biphenyl amine aqueous solution of 2mg/ml) and IV (3% superoxol), hybridization is with film bar and standard reference material.
According to a preferred embodiment of the invention, said detection is to use the reversal point blot hybridization device, water bath with thermostatic control or the airshower that have porous filter plate and decompression pumping part to finish.
According to a preferred embodiment of the invention, wherein the micromolecule of said labeled primer can be a biotin, digoxin etc.
According to a preferred embodiment of the invention, wherein said matrix can be nylon membrane, nitrocellulose filter cellulose acetate membrane.
According to a preferred embodiment of the invention, wherein said suitably is the method for position with probe stationary at the film bar, comprise probe is passed through ultraviolet-crosslinkable, and the solid phase support material of handling with the crosslinked EDC of the probe of amino labeled (N-ethyl-N '-[dimethyl aminopropyl]-carbodiimide, Sigma company) such as cellulose nitrate, nylon or cellulose acetate.
Traditional blot hybridization method is that target DNA is fixed on nylon membrane or the nitrocellulose filter, is in contact with one another and hybridizes with the probe of mark and target DNA to be checked, develops or colour developing back result of determination.Each in this way hybridization reaction can only detect the complementary series that whether contains a certain probe among the DNA to be measured, promptly once can only judge a kind of genotype.(as HLA-A, HLA-B, HLA-DR site) maybe will detect a plurality of target sequences (as thalassemia mutator, abo blood group etc.) if certain gene locus has multiple allele, and be just very numerous and diverse with the disposable detection of this dot blot method, even be difficult to finish.The reverse dot blot hybridization method then is that design earlier is at each allelic specific probe, and with probe points be added to hybridization matrix on, and then with DNA sample to be measured (generally be to use 5 '-end be marked with the product behind biotin or the equimolecular primer PCR amplified target of the digoxin dna fragmentation) hybridization with it, sample to be tested will combine with the probe with complementary series like this, after unconjugated DNA is removed in washing, can detect the target sequence of specificity combination.Therefore, use this method can once detect the normal and relevant various mutations form in a certain site simultaneously, or the normal or sudden change of different loci.
Related kit is to the somatotype detection of hepatitis C virus among the present invention in order to finish, and at first routine prepares the HCV RNA in person's blood to be checked.Use RNA to extract the kit extraction and obtain HCV RNA.Extract the RNA that obtains as template with this, and carry out reverse transcription reaction with synthetic oligonucleotides reverse transcriptase primer and obtain cDNA; Use the cDNA that obtains by said method as template then, and carry out pcr amplification with synthetic PCR upstream and downstream oligonucleotides PCR primer.Behind the resulting DNA cloning product of degenerative treatments, make it respectively and the oligonucleotide probe hybridization that is enough to disclose different hepatitis C virus (HCV) genotype and hypotype.At last, judge different genotype and the gene hypotypes of hepatitis C virus (HCV) according to the change color after the dyeing of hybridization product.Now, be described in detail as follows respectively at several key steps of the inventive method.
1, the design of primer probe
Pertinent literature confirms, 5 ' UTR district gene order is carried out somatotype and can be embodied the full genetic component type situation of HCV in the HCV genome, and this research is according to the sequence of known hepatitis C virus (HCV), and 5 ' the UTR district that chooses high conservative after the comparison is as the target area, design 2 HCV general probes, article 1,1 type general probe, 2 1b hypotype probes, 22 type general probes, article 2,2a hypotype, article 2,3 type general probes, 1 3b type probe, 16 type probe; Can detect by general probe whether the HCV infection is arranged, other 11 probes then can detect concrete type and hypotype and infect; Synthetic probe separates and purifying through 20% polyacrylamide gel electrophoresis.
The type of all designs or subtype sepcific probe, all the hepatitis C virus genom sequence than its alloytype or hypotype has two differences more than the base at least.This design makes that the purpose amplified fragments hybridization of corresponding type or hypotype probe and its alloytype or hypotype hepatitis C virus is more difficult, thereby has farthest avoided non-specific generation.
2, the preparation of hybrid vector
Film as hybrid vector can be made by solid phase support material such as cellulose nitrate, nylon or cellulose acetates.New synthetic probe is fixed on the appropriate location of film bar in a suitable manner.Wherein two general probes are diluted to a pipe, wherein PC1 concentration is 10pmol/ μ l, PC2 concentration is 20pmol/ μ l, to synthesize liquid dot to a position, a pipe back, form general probe point (PC point), fully wash with NaOH solution-treated film bar and with distilled water after will having diluted probe point sample and airing, be stored in then 4 ℃ standby.
According to a preferred embodiment of the invention, wherein said suitable mode, comprise probe is passed through ultraviolet-crosslinkable, and the solid phase support material of handling with the crosslinked EDC of the probe of amino labeled (N-ethyl-N '-[dimethyl aminopropyl]-carbodiimide, Sigma company) such as cellulose nitrate, nylon or cellulose acetate.
3, the extraction of nucleic acid
Adopt the HCV RNA in the conventional RNA extract extraction individual human blood sample.For example, extract 2 milliliters in person under inspection's venous blood, inject aseptic dry glass tube with disposable sterilized injector, room temperature (22～25 ℃) placement 30～60 minutes or centrifugal after separate out serum.Draw upper serum 100 μ l, be transferred to 1.5ml sterilization centrifuge tube, and add the abundant mixing of 20 μ l RNA extract A, add the abundant mixing of RNA extract B of 400 μ l again.Room temperature was placed 5 minutes, 6000rpm, and centrifugal 1 minute, the careful suction went supernatant, the pre-cooled ethanol with 75% (preparation of DEPC water) to wash precipitation twice.65 degrees centigrade drying precipitated 10 minutes.The hepatitis C reverse transcription system that adds 18 μ l in sediment tube just can be carried out next step reverse transcription reaction.
4, reverse transcription system and the optimization of experimental conditions of RNA
In reverse transcription system and optimization of experimental conditions process, by the efficient of reverse transcription under the condition of more a plurality of temperature, optimized choice goes out best reverse transcription temperature conditions, in addition, and our primer and concentration thereof, and Mg to using in the reverse transcription process
2+, other concentration of reactants such as template have all made suitable adjustment and optimization.
4, the optimization of nucleic acid amplification system
In pcr amplification, at first utilize touchdown PCR to grope annealing temperature, and grope reaction conditions so as to improving the specificity and the high efficiency of nucleic acid amplification.Particularly, we are to the primer that uses in the pcr amplification and its concentration and Mg
2+, other concentration of reactants such as template have all made suitable adjustment and optimization.
5, the optimization of reverse dot blot hybridization system
Reverse dot blot hybridization is in conjunction with PCR-Dot blot hybridization technique and filter hybridization, its operating process comprises: for example, it is centrifugal at first will to put into the 2ml that preheating hybridization solution I is housed as the HCV Genotyping film bar of preceding preparation, during hybridization, earlier 95 ℃ of sex change pcr amplification products are 5 minutes, put in the ice cooling then more than 2 minutes, again in the presence of the hybridization solution I of about 56 ℃ of preheatings, in 56 ℃ shaking bath, make about 60 minutes of the probe hybridization of pcr amplification product and 5 ' end amino labeled.After hybridization reaction is finished, through washing film and development step showing existing of hybridization spot, and judge the genotype that each is individual according to the probe type that is developed the color.
By detection example shown in Figure 3 as can be seen, when if chromogenic reaction control probe 1 (SEQ ID NO:4) and probe 2 (SEQID NO:5-6) all develop the color, a point develops the color or colour developing simultaneously in probe 4 (SEQ ID NO:8) and the probe 5 (SEQ ID NO:9), and decidable HCV genotype is the 1b type; When chromogenic reaction control probe 1 (SEQ ID NO:4) and probe 2 (SEQID NO:5-6) all develop the color, probe 3 (SEQ ID NO:10) colour developing, decidable HCV genotype is 1 type; When if chromogenic reaction control probe 1 (SEQ ID NO:4) and probe 2 (SEQ ID NO:5-6) all develop the color, a point develops the color or colour developing simultaneously in probe 6 (SEQID NO:10) and the probe 7 (SEQ ID NO:11), and decidable HCV genotype is 2 types; When if chromogenic reaction control probe 1 (SEQ ID NO:4) and probe 2 (SEQ ID NO:5-6) all develop the color, a point in probe 8 (SEQ ID NO:12) and the probe 9 (SEQ ID NO:13) develops the color or colour developing simultaneously, and decidable HCV genotype is the 2a type; When if chromogenic reaction control probe 1 (SEQ ID NO:4) and probe 2 (SEQ ID NO:5-6) all develop the color, probe 3 (SEQ ID NO:7), probe 6 (SEQ ID NO:10) and probe 10 (SEQ ID NO:17) also all develop the color, and decidable HCV genotype is 6 types; When if chromogenic reaction control probe 1 (SEQ ID NO:4) and probe 2 (SEQ IDNO:5-6) all develop the color, a point develops the color or colour developing simultaneously in probe 11 (SEQ ID NO:14) and the probe 12 (SEQ ID NO:15), and decidable HCV genotype is 3 types; If when chromogenic reaction control probe 1 (SEQ ID NO:4) and probe 2 (SEQ ID NO:5-6) all developed the color, probe 13 (SEQ ID NO:16) also developed the color, decidable HCV genotype is the 3b type; If chromogenic reaction control probe 1 (SEQ ID NO:7) and probe 2 (SEQ ID NO:4-6) colour developing are arranged, illustrate that the pathogen that detects is HCV; If have only chromogenic reaction control probe 1 (SEQ ID NO:4) colour developing, other probe does not develop the color the negative or pcr amplification failure of HCV then is described; If each type probe is arranged: the type in 1 type (3,4,5), 2 types (6,7,8,9), 3 types (11,12,13), 6 types (10) is put one or more type points and is developed the color simultaneously, then be judged as mixed infection (during 10 colour developings of because HCV6 type---probe,---probe 3 and HCV2 type---colour developing of probe 6 all with the HCV1 type, this kind situation can only be judged to be the HCV6 type, but not mixed infection).(referring to Fig. 3).In chromogenic reaction, use Color Appearance System to develop the color, for example, the label biotin is combined with streptomysin avidin-horseradish peroxidase bond (POD) specificity, make substrate tetramethyl chain aniline (TMB) generate blue precipitation.Then, by computer image analysis or naked eyes sentence read result.In the hybridization process color, if use streptavidin coupling alkaline phosphatase equally can with the biotin combination, and make substrate 5-bromo-4-chloro-3-indolylphosphate para-totuidine salt (BCIP) generate dark blue precipitate and finish process color among the present invention.
In addition, (for example, hybridization temperature is set in 56 ℃, concentration and probe concentration is between 5pmol/L～20pmol/L) by groping different hybridization temperatures and concentration and probe concentration repeatedly in the present invention, the result that can make detection and has better detection stability (repeatability) more accurately and reliably.
Among the present invention, because the inventor optimizes the amplification system and the hybridization system of target nucleic acid fragment, the probe and the primer of high specificity have been designed and synthesized, use shaking bath as reversal point blot hybridization device in detecting simultaneously, thereby guaranteed the reliability of the quick and testing result of method.
As previously mentioned, because reverse dot blot hybridization the method simultaneously sudden change or the polymorphism of a plurality of bases in the testing goal gene, so in the detection of pathogen and complex inheritance disease, more easy superiority is arranged.In the diagnosis and treatment of third liver, the generation of hepatitis c virus genotype chronic hepatitis and some severe hepatopathy (cirrhosis, liver cancer etc.) and develop closely related, and different genotype also has certain relation to medicine (as interferon-alpha), so the HCV genotype detection becomes the focus of clinical medical personnel's research.HCV genotype detection method of the present invention obviously can provide a kind of quick, easy, specificity and all higher laboratory inspection means of accuracy for the diagnosis of hepatitis C.
As previously mentioned, for detecting concrete genotype of hepatitis C virus in the individual blood sample (HCV) and gene hypotype quickly and easily, the invention provides corresponding hepatitis C virus (HCV) Genotyping reverse dot blot hybridization detection kit.Specifically, kit of the present invention provides:
(1) RNA that is made up of RNA extract A, RNA extract B, DEPC water extracts reagent;
(1) reverse transcription (RT) reagent of forming by reverse transcriptase, reverse transcription reflection system, positive criteria product, negative quality-control product.
(2) comprise that (totally 10 manage, every pipe contains PCR reaction buffer, Mgcl to the PCR reaction mixture
2(1.6mmol/L), dNTPs (0.2mmol/L), forward and reverse primer (0.1umol/L), and the pcr reagent of Taq enzyme (3u/ μ l, 1 pipe);
(3), also comprise in addition as 10 of the film bars of hybridization reaction carrier by 20 * SSC of routine, sodium dodecylsulphonate (SDS), hybridization detectable that the streptomysin avidin-horseradish peroxidase bond (POD) is formed.
Kit of the present invention also is furnished with chromogenic reaction control probe and standard reference material except that the pairing specific probe in each site is provided.
In the kit that the present invention relates to, owing to used at each type or the designed primer amplification target nucleic acid sequence of hypotype HCV nucleotide sequence, and designed colour developing contrast probe and detection HCV genotype or gene hypotype oligonucleotide probe and carried out nucleic acid hybridization analysis, so HCV genotype and hypotype can accurately and effectively be differentiated and distinguish to this kit.The kit that the present invention relates to can not only be used to detect single HCV to be infected, and can detect the mixed infection of HCV qualitatively, thereby for the clinical individualized treatment of hepatitis C provides the Molecular Virology evidence, for clinical application provides significant reference.In general, use the kit that relates among the present invention, only need about 10 hours time from the processing of sample to interpretation genotype detection result, compare with traditional hybridizing method, that the invention of this method obviously also has is simple to operate, advantage quickly and easily.Therefore, method of the present invention and kit are specially adapted to the generaI investigation of the fast detecting and the great amount of samples of clinical samples.
Table 1 HCV Genotyping type decision table
| Sample number | Probe colour developing | Genotype | |
| 1 | 1, No. 2 probes all develop the color | HCV infects | |
| 2 | 1, No. 2 all colour developings, and No. 3 probe colour developings, all the other do not have colour developing | 1 type infects | |
| 3 | 1, No. 2 all colour developings, and 4, No. 5 probes simultaneously or one of them colour developing | The 1b type infects | |
| 4 | 1, No. 2 colour developings and 6, No. 7 probes simultaneously or one of them colour developing, all the other do not have colour developing | 2 types infect | |
| 5 | 1, No. 2 colour developings and 8, No. 9 probes are simultaneously or one of them colour developing | The 2a type infects | |
| 6 | 1, No. 2 colour developings and 3,6, No. 10 probes develop the color simultaneously | 6 types infect | |
| 7 | 1, No. 2 colour developings and 11, No. 12 probes are simultaneously or one of them colour developing | 3 types infect | |
| 8 | 1, No. 2 colour developings and No. 13 probes also develop the color | The 3b type infects |
Description of drawings
Fig. 1 shows the Pareto diagram of employed probe on the film bar.No. 1 probe has the sequence shown in the SEQ ID NO:4, is the colour developing probe, is used to control the whole process of chromogenic reaction; No. 2 positive control probe has the sequence shown in the SEQ ID NO:5-6, positive contrast probe, if colour developing, then representing the biological specimen that is detected is HCV; No. 3 probe has the sequence shown in the SEQ ID NO:7, is 1 type specific probe, is judged to be 1 type during colour developing; No. 4 probe has the sequence shown in the SEQ ID NO:8, and No. 5 probe has the sequence shown in the SEQ ID NO:9, is 1b type specific probe, and single or demonstration simultaneously all is judged to be the 1b type; No. 6 probe has the sequence shown in the SEQ ID NO:10, and No. 7 probe has the sequence shown in the SEQ ID NO:11, is 2 type specific probes, and single or demonstration simultaneously all is judged to be 2 types; No. 8 probe has the sequence shown in the SEQ ID NO:12, and No. 9 probe has the sequence shown in the SEQ ID NO:13, is 2a type specific probe, and single or demonstration simultaneously all is judged to be the 2a type; No. 10 the control probe has the sequence shown in the SEQ ID NO:17, is 6 type Idiotype probes, and it and No. 3 and No. 6 probes are judged to be 6 types when developing the color simultaneously; No. 11 the control probe has the sequence shown in the SEQ ID NO:14, and No. 12 the control probe has the sequence shown in the SEQ ID NO:15, is 3 type specific probes, and single or demonstration simultaneously all is judged to be 3 types; No. 13 the control probe has the sequence shown in the SEQ ID NO:16, is 3b type specific probe, and colour developing is to be judged to be the 3b type.
Fig. 2 display target DNA cloning fragment is through 2% agarose gel electrophoresis figure, and wherein the 1-6 swimming lane is specific target dna fragmentation (220bp), and the M swimming lane is the standard molecular weight sign.
Fig. 3 shows the reverse dot blot hybridization testing result of five kinds of different genotype samples.Have the positive test symbol of 9 routine samples, wherein be 1 type 1,2, No. 3, No. 1 is 1 type, and No. 2 and No. 3 is the 1b type; 4, be 2 types 5, No. 6, No. 4 is 2 types, and No. 5 and No. 6 is the 2a type; No. 7 is 3 types, and No. 8 is the 3b type, and No. 9 is 6 types; (referring to table 1 and Fig. 3).
Embodiment
The following example is intended to illustrate rather than limit the present invention.
Embodiment 1: the preparation of sample target nucleic acid
The reverse transcription of embodiment 2:RNA becomes cDNA
Get the 1.5mL centrifuge tube that does not contain RNase, get reverse transcription reaction liquid by 19 μ L * (N+1), add the reverse transcriptase of 1 μ L * (N+1) again, mixing is mixed with the reverse transcription system, divide and to install to that N (N=detected sample number+2) is individual not to be contained in the blank 0.2mL centrifuge tube of RNase, install to each minute then and add the RNA template that 5 μ L prepare in the centrifuge tube of reverse transcription system, after instantaneous (3 seconds) are centrifugal, each reaction tube is put into the PCR instrument.Carry out reverse transcription (RT) reaction by following condition: 40 ℃ of constant temperature 60 minutes, 95 ℃ of deactivations then 3 minutes, 4 ℃ of constant temperature then.
Embodiment 3: the pcr amplification of target nucleic acid
Get single part of some pipes of PCR reactant liquor of single tube, directly add 3 μ l and reverse the cNDA template that records, fully mixing after instantaneous (3 seconds) are centrifugal, is put into the PCR instrument with each reaction tube.By the amplification of following condition: 93 ℃ of pre-sex change 6 minutes, then by 93 ℃ 45 seconds, 57 ℃ 45 seconds, 10 circulations of 72 ℃ of amplifications in 45 seconds, 93 ℃ 30 seconds, 55 ℃ 40 seconds, 72 ℃ of amplification 30 circulations, totally 40 circulations in 45 seconds; Last 72 ℃ of insulations 7 minutes.Amplified production detects (seeing accompanying drawing 2) through 2% agarose gel electrophoresis.
4: seven kinds of genotypic sample reverse dot blot hybridizations of embodiment detect
Before the hybridization, get hybridization solution I (1.8 * SSC-0.1%SDS) mix with hybridization solution II and be preheated to 56 ℃ standby.After 8 minutes, place mixture of ice and water to place 8-10 minute 98 ℃ of degenerative treatments of pcr amplification product.Get 1000 μ l hybridization solution I+2 μ l solution I (1000:2) mixed solutions then as standby in conjunction with 4 ℃ of preservations of liquid, and get solution II: solution III: the potpourri of solution IV (1900:200:1) (19ml solution II+2ml solution+10 μ l solution IV) is standby as colour developing liquid lucifuge.
Earlier the shaking bath temperature is transferred to 56 ℃ before the hybridization, treat that water temperature just can carry out hybrid experiment after constant.At first get the 2.0mL centrifuge tube of corresponding number according to the number (comprising the contrast of detected sample harmonizing yinyang) of detected sample, in each centrifuge tube, add mark good single part of film bar, the hybridization solution I that adds the 1.5mL preheating then to each pipe respectively, again the pcr amplification product after the sex change is added in the centrifuge tube, will add with it product one to one according to film bar mark during application of sample.After closing lid centrifuge tube is put into 56 ℃ of shaking baths, incubation 60 minutes, and with slowly shaking.Abandon hybridization solution I, the film bar is put into the 50mL centrifuge tube, clean film bar (add 15mL hybridization solution II in each 50mL centrifuge tube approximately, clean each 3 minutes 3 times) with the hybridization solution II that preheating is good.At room temperature add in conjunction with liquid (adding 15mL approximately in conjunction with liquid in each 50mL centrifuge tube), place biochemistry to wave on the instrument, abandon in conjunction with also, add room temperature hybridization solution I and clean the film bar and (add 15mL hybridization solution I in each 50mL centrifuge tube approximately in conjunction with 10 minutes, clean each 3 minutes 3 times).Add solution II (adding 15mL approximately in each 50mL centrifuge tube) subsequently, place biochemistry to wave on the instrument, abandon solution II, add freshly prepared colour developing liquid (adding 15mL approximately in each 50mL centrifuge tube), developed the color about 10 minutes in conjunction with 5 minutes.Abandon colour developing liquid,, take out the film bar, blot, just can scan or directly read the result with paper handkerchief with pure water washing 2 times.
By result shown in Figure 3 as can be seen, the colour developing situation of each probe of generation specific hybrid is all high-visible.Array format interpretation shown in one goes out single and mixed infection with reference to the accompanying drawings.
Embodiment 4: different thermostats carries out reverse dot blot hybridization and detects.
Employing is equipped with porous filter plate and the decompression reversal point blot hybridization device of pumping part and air bath hybrid heater that the sample of above-mentioned seven kinds of different genotype and gene hypotype is detected, testing result illustrates that with the testing result unanimity of water conservancy diversion hybridization instrument water bath with thermostatic control and airshower can be used for sample is detected equally.
Embodiment 5: the reverse dot blot hybridization Genotyping testing result of different genotype sample is judged
Use method of the present invention and kit, 400 parts of DNA samples that pick up from hepatitis C patients are carried out the detection of HCV Genotyping site, wherein 380 routine samples are able to correct somatotype.In order further to confirm the accuracy of the inventive method, all above-mentioned 400 routine samples have been carried out sequencing.The result shows that the result and the sequencing result that use kit of the present invention to detect are identical substantially, and specificity reaches 95.3%, simultaneously sample is carried out quantitative copy number analysis, and it is 10 that 45 examples are wherein arranged
4Copies/ml, 4 examples are 10
3Copies/ml, equal accurate somatotypes after height copied serum and carry out gradient dilution, finds that various sample limit of identification all can reach 10
4Copy/ml.
Sequence table
＜110〉Da
＜120〉HCV gene typing detecting reagent kit
<140>
<141>
<160>17
<210>1
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification.
<400>1
<210>2
<211>22
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification.
<400>2
<210>3
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification.
<400>3
<210>4
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, to be used as hybridization probe.
<400>4
<210>5
<211>18
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, to be used as hybridization probe.
<400>5
<210>6
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, to be used as hybridization probe.
<400>6
<210>7
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, to be used as hybridization probe.
<400>7
<210>8
<211>18
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, to be used as hybridization probe.
<400>8
<210>9
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, to be used as hybridization probe.
<400>9
<210>10
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, to be used as hybridization probe.
<400>10
<210>11
<211>22
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, to be used as hybridization probe.
<400>11
<210>12
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, to be used as hybridization probe.
<400>12
<210>13
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, to be used as hybridization probe.
<400>13
<210>14
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, to be used as hybridization probe.
<400>14
<210>15
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, to be used as hybridization probe.
<400>15
<210>16
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, to be used as hybridization probe.
<400>16
<210>17
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, to be used as hybridization probe.
<400>17
Claims (3)
1, a kind of kit that is used to detect hepatitis c virus genotype, this kit comprises: (1) RNA extracts reagent; (2) reverse transcription reagent; (3) PCR reagent; (4) hybond membrane and reverse dot blot hybridization reaction reagent is characterized in that the applied primer of kit is respectively:
Reverse transcription (RT) primer: 5 '-GCTCATGGTGCACGGTCTACGAGACCT-3 ' (SEQ ID NO:1)
The PCR upstream primer: 5 '-TCTAGCCATGGCGTTAGTATGAGTGT-3 ' (SEQ ID NO:2)
The PCR downstream primer: 5 '-CACTCGCAAGCACCCTATCAGGCAGT-3 ' (SEQ ID NO:3).
2, according to the kit of claim 1, its feature is that also employed oligonucleotide probe is:
Probe I C:5 '-GCCGTAACCGTCACAATCCGT-3 ' (SEQ ID NO:4)
Probe PC1:5 '-ATTTGGGCGTGCCCCCGC-3 ' (SEQ ID NO:5)
Probe PC2:5 '-CGGAACCGGTGAGTACACC-3 ' (SEQ ID NO:6)
Probe 1:5 '-CAATGCCTGGAGATTTGGGCG-3 ' (SEQ ID NO:7)
Probe 1b-1:5 '-CCGCGAGACTGCTAGCCG-3 ' (SEQ ID NO:8)
Probe 1b-2:5 '-GCGAGACCGCTAGCCGAGT-3 ' (SEQ ID NO:9)
Probe 2-1:5 '-AGTAGCGTTGGGTTGCGAAAG-3 ' (SEQ ID NO:10)
Probe 2-2:5 '-GTCCTTTCTTGGATAAACCCAC-3 ' (SEQ ID NO:11)
Probe 2a-1:5 '-GCCGGGAAGACTGGGTCCT-3 ' (SEQ ID NO:12)
Probe 2a-2:5 '-CCCACTCTATGCCCGGCCA-3 ' (SEQ ID NO:13)
Probe 3-1:5 '-CCCGCGAGATCACTAGCCG-3 ' (SEQ ID NO:14)
Probe 3-2:5 '-AATCGCTGGGGTGACCGGG-3 ' (SEQ ID NO:15)
Probe 3b:5 '-CCCGCTCAATGCCCGGAAAT-3 ' (SEQ ID NO:16)
Probe 6:5 '-GACCGGGTCCTTTCCATTGG-3 ' (SEQ ID NO:17).
3, according to the kit of claim 1, its feature is that also the micromolecule of labeled primer is selected from biotin, digoxin etc.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100299460A CN101377486A (en) | 2007-08-29 | 2007-08-29 | HCV gene typing detecting reagent kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100299460A CN101377486A (en) | 2007-08-29 | 2007-08-29 | HCV gene typing detecting reagent kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101377486A true CN101377486A (en) | 2009-03-04 |
Family
ID=40421131
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2007100299460A Pending CN101377486A (en) | 2007-08-29 | 2007-08-29 | HCV gene typing detecting reagent kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101377486A (en) |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102286644A (en) * | 2011-08-26 | 2011-12-21 | 李艳 | Kit for genotyping hepatitis C virus (HCV) |
| CN102127603B (en) * | 2010-01-19 | 2013-01-09 | 中山大学达安基因股份有限公司 | Kit for detecting genotyping and IL28-site polymorphism of hepatitis C virus (HCV) |
| CN102899422A (en) * | 2011-07-25 | 2013-01-30 | 深圳国际旅行卫生保健中心 | HCV core protein gene RT-PCR typing and detection method |
| CN103710464A (en) * | 2013-12-30 | 2014-04-09 | 湖南圣湘生物科技有限公司 | HCV (Hepatitis c virus) genotype detection kit |
| CN104263851A (en) * | 2014-07-15 | 2015-01-07 | 周有良 | Hepatitis C virus genetic typing chip and typing method |
| CN104593509A (en) * | 2015-01-29 | 2015-05-06 | 中国水产科学研究院珠江水产研究所 | Specific-nucleic-acid-probe dot-blot-hybridization detection kit for tilapia streptococcus agalactiae |
| CN105986040A (en) * | 2015-01-28 | 2016-10-05 | 苏州新波生物技术有限公司 | Kit for HCV virus genotyping detection and SNP locus detection of IL28B, use method and application thereof |
| CN105986038A (en) * | 2015-01-28 | 2016-10-05 | 苏州新波生物技术有限公司 | Kit for HCV virus genotyping detection, use method and application thereof |
| CN106951731A (en) * | 2017-03-28 | 2017-07-14 | 上海至本生物科技有限公司 | A kind of large fragment insertion or the Forecasting Methodology and system of missing |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102127603B (en) * | 2010-01-19 | 2013-01-09 | 中山大学达安基因股份有限公司 | Kit for detecting genotyping and IL28-site polymorphism of hepatitis C virus (HCV) |
| CN102899422A (en) * | 2011-07-25 | 2013-01-30 | 深圳国际旅行卫生保健中心 | HCV core protein gene RT-PCR typing and detection method |
| CN102899422B (en) * | 2011-07-25 | 2014-07-02 | 深圳国际旅行卫生保健中心 | HCV core protein gene RT-PCR typing and detection method |
| CN102286644A (en) * | 2011-08-26 | 2011-12-21 | 李艳 | Kit for genotyping hepatitis C virus (HCV) |
| CN102286644B (en) * | 2011-08-26 | 2013-04-10 | 李艳 | Kit for genotyping hepatitis C virus (HCV) |
| CN103710464A (en) * | 2013-12-30 | 2014-04-09 | 湖南圣湘生物科技有限公司 | HCV (Hepatitis c virus) genotype detection kit |
| CN104263851A (en) * | 2014-07-15 | 2015-01-07 | 周有良 | Hepatitis C virus genetic typing chip and typing method |
| CN104263851B (en) * | 2014-07-15 | 2017-01-11 | 周有良 | Hepatitis C virus genetic typing chip and typing method |
| CN105986040A (en) * | 2015-01-28 | 2016-10-05 | 苏州新波生物技术有限公司 | Kit for HCV virus genotyping detection and SNP locus detection of IL28B, use method and application thereof |
| CN105986038A (en) * | 2015-01-28 | 2016-10-05 | 苏州新波生物技术有限公司 | Kit for HCV virus genotyping detection, use method and application thereof |
| CN104593509A (en) * | 2015-01-29 | 2015-05-06 | 中国水产科学研究院珠江水产研究所 | Specific-nucleic-acid-probe dot-blot-hybridization detection kit for tilapia streptococcus agalactiae |
| CN106951731A (en) * | 2017-03-28 | 2017-07-14 | 上海至本生物科技有限公司 | A kind of large fragment insertion or the Forecasting Methodology and system of missing |
| CN106951731B (en) * | 2017-03-28 | 2019-04-12 | 至本医疗科技（上海）有限公司 | A kind of prediction technique and system large fragment insertion or lacked |
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