CN101921871B - Hepatitis C sequencing and typing kit and detection method thereof - Google Patents
Hepatitis C sequencing and typing kit and detection method thereof Download PDFInfo
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Abstract
The invention discloses a hepatitis C sequencing and typing kit which comprises: (1) primers of specific amplified hepatitis C virus RNA reverse transcription product c DNA and (2) hepatitis C virus specific sequencing primers, wherein the nucleotide sequences of the primers of specific amplified hepatitis C virus RNA reverse transcription product c DNA are shown as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, the SEQ ID NO:1 and the SEQ ID NO:2 are a primer pair and the SEQ ID NO:3 and the SEQ ID NO:4 are another primer pair; and the nucleotide sequences of the hepatitis C virus specific sequencing primers are shown as SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7; the SEQ ID NO:4 is marked with a biotin at a 5' end; and all the primers are all used in a detection. The invention also discloses a detection method of the hepatitis C sequencing and typing kit. The hepatitis C sequencing and typing kit can be used for sequencing and typing hepatitis C, thereby providing more comprehensive reference information for clinical diagnosis and treatment.
Description
Technical field
The present invention relates to a kind of hepatitis C sequencing and typing kit and detection method thereof, belong to the detection technique field of virus subtype.
Background technology
The HCV genotype has areal variation, different genotype and disease progression, liver cancer take place and efficacy of interferon therapy relevant, all significant to the route of transmission of sporadic hepatitis C and vaccine development etc.
The HCV genotype distributes and has tangible areal variation.5 kinds of common genotype (1a, 1b, 2a, 2b, 3a) distribute extensively; Wherein, The 1a type is preponderated in the U.S., Brazil and the European northwestward, the 1b type in the Far East, European the western and eastern sees that 2a and 2b type are distributed in Asia and Europe more more; The 3a type in the west some countries and Thailand, Singapore see more, and in some crowds of East India and Bangladesh, become unique genotype that HCV infects.Other more rare genotypic distributions then are confined to certain areas, and 4 types are preponderated in the Middle East and North Africa, and the 5a type is confined to South Africa, and the 6a type is mainly seen in Hong Kong, Meccah and Vietnam.Many data show that the China's Mainland is main with 1b and 2a, and 1b is many.South 1b type accounts for more than 90%, and northern 2a type accounts for 46～70%.The genotype variety is not as China Hong Kong, Japan, but the polyinfection rate is apparently higher than other countries and area.
Different genotype is relevant with change of illness state.Numerous reports think that 1b type HCV and chronic hepatitis worsening, inductionization and hepatocellular carcinoma have substantial connection.Kobayashi etc. find 140 routine hepatitis C patients retrospective studies: the classification of 1b type liver histological, by stages deterioration advance rate and the average HCV2RNA of serum tire apparently higher than 2 types; Prompting 1b type can cause heavier hepatic necrosis property inflammation, hepatic fibrosis, and virus has stronger replication.Silini etc. discover that to 593 routine chronic hepatitis Cs, 219 routine liver cirrhosis and 166 routine hepatocellular carcinomas 1b type positive rate is significantly higher than chronic hepatitis C and liver cirrhosis among the hepatocellular carcinoma patient.But also there is report to think that 1b type and hepatic fibrosis and hepatocellular carcinoma do not have obvious relation.The relation of HCV genotype and hepatopathy severity does not obtain consensus as yet at present, and most scholars think that 1b type and hepatopathy progress and hepatocellular carcinoma have substantial connection.
Genotype is relevant with efficacy of interferon therapy.Interferon, rabbit is the anti-HCV active drug of generally acknowledging at present, is widely used in clinical.The data of home and abroad shows that the HCV genotype has a significant effect to IFN, and 1b type curative effect is starkly lower than other common genotype, and the long-term remission rate of IFN treatment 1b type and 2a type is respectively about 40% and 90%.Therefore have the people to advise, before interferon therapy, except considering patient age, stadium, degree of hepatic fibrosis and virus replication level, genotype should be as important prediction index.
Through genotypic comparison and analysis, help to find the route of transmission of sporadic hepatitis C and control contagium.The frequent crowd of sexual behaviour such as prostitute, std patient and men homosexuality person investigated show that they all have higher H CV infection rate, be respectively 6.9%, 9% and 6.9～26%, general crowd or blood supply person's generally acknowledged level apparently higher than the locality.Chronic hepatitis C patient's spouse also has higher H CV infection rate, is 8.5～28%, and infection rate has the trend that increases along with the prolongation of property duration of contact.88 relatives of report such as Ideo 34 routine anti-2HCV positive cases have the anti-2HCV of 7 people positive 8%; All do not find anti-2HCV positive person among 26 relatives of the 8 negative patients of routine anti-2HCV in addition.And local general crowd HCV infection rate less than 1% in view of the above, thinks that hepatitis C exists family to a certain degree to propagate.307 relatives to the 104 routine third hepatopathy people investigate, and the anti-2HCV of 52 people is positive, and positive rate is 17%, surpasses local general crowd's infection rate.Infection rate is followed successively by parents' (54%), spouse's (28%), children's (6.9%) and other members (6.4%) from high to low in kinsfolk's distribution.And the age is big more, and is long more with patient's life-time, and the danger of infection is also big more.Thalter etc. carry out 2～19 months following up a case by regular visits to 8 babies that the positive mother of 8 routine HCV nucleic acid gives birth to, and wherein 7 babies confirm to have infected HCV, and prompting mother's Infection Status is influential to the hepatitis C vertical transmission.At present, above-mentioned propagation also lacks enough foundations, has only from the molecular biology angle, through the research of HCV genotype nucleotide sequence, further judges and confirms.
Owing to there is not the HCV vitro culture system, many traditional virusology sorting techniques can't be used, the classification of HCV will inevitably almost all depend between each strain whole genome sequence relatively or a certain fragments sequence relatively.
(1) the initial classification of nucleotide sequence analysis is based on the complete sequence basis relatively; Through analysis to total length 914kb nucleotide sequence; Understand its homology degree, developed into afterwards and utilize portion gene group sequence to compare analysis, this method is accurate and visual; But complicated operation is difficult to larger samples is observed.
(2) the type specificity primer extension method increases with every type specificity primer according to the characteristics of sequence variations in the nucleotide fragments zone, and different types can amplify fragment different in size.Classify in view of the above, this method is mainly used in C district and NC district.
(3) type specificity probe hybridization method is according to the HCV genome characteristics of range gene type, and the synthesis type specific probe carries out Southern hybridization with PCR product and type specificity probe, detects HCV genotype in the sample.Because various only has several Nucleotide to morph sometimes, the condition to hybridization has strict requirement like this, so this precision of method is not high, and less stable.
(4) restriction fragment length polymorphism (RFLP) carries out enzyme and cuts with the HCV gene product of multiple restriction enzyme after to amplification, because the different gene fragment has different restriction enzyme sites, so fragment that after enzyme is cut, can occur varying in size and polymorphum.This working method clinical value is bigger.
But no matter which kind of method of utilization, because genomic Nucleotide of HCV and amino acid variation are its bases, the result that various analytical procedure obtained has certain comparability.
HCV one has the heterogeneity virus strain of very high aberration rate, its RNA polymerase that in reproduction process, is relied on be one be easy to generate mispairing tendency (error-prone) the RNA polymerase that relies on of RNA, equally lack the mechanism of repairing with many other RNA viruses; Making out of true duplicate product can not obtain repairing; Thereby more mispairing occurs, show higher aberration rate, repeatedly duplicate and the result that makes a variation will cause the generation of multiple Different Variation strain; Show as unhomogeneity or otherness between the HCV strain; Therefore, the HCV strain isolated shows the different gene type, and ' terminal length of nucleotides differs its difference in each genotype 3.The method that adopts about the gene type of HCV, the gene fragment and the name of selection have nothing in common with each other; But in numerous methods of genotyping; Only credible and can be used for identifying new genotypic method; Select specific HCV constant gene segment C particularly to carry out sequencing and typing in the E1 district exactly, and the present invention carry out nucleotide sequence tetra-sodium sequencing analysis and identify each hypotype of HCV with shell type RT-PCR method specific amplification HCV gene fragment.
Summary of the invention
Technical problem to be solved by this invention is to overcome the deficiency that prior art exists, and a kind of hepatitis C sequencing and typing kit is provided.
The technical problem that the present invention also will solve provides the detection method of mentioned reagent box.
For solving the problems of the technologies described above, the technical scheme that the present invention adopts is following:
A kind of hepatitis C sequencing and typing kit, it comprises:
(1) primer of specific amplification hepatitis C virus RNA rt product cDNA; Its nucleotide sequence is shown in SEQ IDNO:1 to SEQ ID NO:4; Wherein SEQ ID NO:1 and SEQ ID NO:2 are that primer is right, and SEQ IDNO:3 and SEQ ID NO:4 are that primer is right;
(2) hepatitis C virus specificity sequencing primer, its nucleotide sequence is shown in SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7;
Wherein, SEQ ID NO:4 is at its 5 ' end mark vitamin H;
Above-mentioned primer uses in one-time detection jointly.
Concrete, above-mentioned hepatitis C sequencing and typing kit comprises following reagent:
(1) RNA extracts reagent: Trizol, chloroform, Virahol, absolute ethyl alcohol, DEPC treating water;
(2) rt reagent: 200U/ μ L M-MLV reversed transcriptive enzyme, 20U/ μ L RNase suppressor factor, 5 * RT-PCRBuffer, 10mM dNTP mix, 100 μ M random hexamer primer, DEPC treating water;
Wherein, 5 * RT-PCR Buffer is specially: 250mM pH 8.3Tris-HCl, 250mM KCl, 20mMMgCl
2, 50mM DTT;
(3) PCR reagent: 2 * Taq buffer, 10uM special primer SEQ ID NO:1～4,25mM MgCl
2, 10mM dNTPmix, 5U/ μ LTaq archaeal dna polymerase;
Wherein: 2 * Taq buffer is specially: 0.1% (v/v) NP-40,0.02% (v/v) gelatin, 0.06% (g/ml) BSA, 0.1% (v/v) Tween-20,0.06M pH8.9Tricine;
(3) strand purified reagent: streptavidin beads, 75% (v/v) ethanolic soln, 0.2M NaOH, 10mM pH7.6Tris-Acetate, binding buffer liquid, annealing buffer;
Wherein, binding buffer liquid is specially: 10mM Tris-HCl, 2M NaCl, 1mM EDTA, 0.1% (v/v) Tween20; Annealing buffer is specially: 20mM pH 7.6Tris-Acetate, 2mM magnesium acetate;
(4) sequencing reagent: archaeal dna polymerase, the ATP sulfurylase, luciferase, apyrase, substrate A PS, resorcinolphthalein and dNTP (dNTP is dATP S, dTTP, and dCTP, dGTP).
Utilize the detection method of above-mentioned hepatitis C sequencing and typing kit, comprise the steps:
The extracting of (1) third liver HCV RNA template;
(2) step (1) gained RNA template is become cDNA with the random primer rt, the primer by said specific amplification hepatitis C virus RNA rt product cDNA carries out pcr amplification again;
(3) pcr amplification product that step (2) is obtained carries out the strand purifying with the primer sequence of described mark vitamin H;
(4) step (3) gained strand purified product is checked order;
(5) interpretation of result.
In the step (1), the extracting of the described third liver HCV RNA template specifically comprises the steps:
(1a) with sample serum 200 μ L and 1mL Trizol, mixing adds 200 μ L chloroforms, mixing 15s, room temperature 5min, 4 ℃ of centrifugal 15min of following 12000g;
(1b) upper water moves into another centrifuge tube mutually, adds and upper water equal volume Virahol mixing 15s, room temperature 10min, 4 ℃ of centrifugal 10min of following 12000g;
(1c) abandon the supernatant that step (1b) obtains, 75% (v/v) ethanol (joining) 1mL of precooling on the rocks, 4 ℃ of centrifugal 5min of following 7500g with DEPC water;
(1d) abandon the supernatant that step (1c) obtains, dry air 5min adds 20uL DEPC treating water, and it is subsequent use to obtain RNA-70 ℃ of HCV.
Rt described in the step (2); Concrete grammar is: with 11 μ L RNA and 1 μ L 100uM random hexamerprimer, 4 μ L, 5 * RT-PCR Buffer, 1 μ L 20U/ μ L RNase suppressor factor, 2 μ L 10mM dNTP mix and 1 μ L200U/ μ L M-MLV reversed transcriptive enzyme; Mixing, 25 ℃ of 5min, 42 ℃ of 60min; 70 ℃ of 5min, it is subsequent use to obtain cDNA4 ℃ of preservation.
Pcr amplification method described in the step (2) is specially:
(2a) outside primer amplification:
50 μ L systems: 2 * Taq buffer, 25 μ L, 10mM dNTP mix 1 μ L, each 1 μ L of primer SEQ ID NO:1 and SEQID NO:2,25mM MgCl
24 μ L, 5U/ μ L Taq archaeal dna polymerase 0.3 μ L, cDNA 2.5 μ L, ddH
2O15.2 μ L.
Pcr amplification condition: 94 ℃ of preparatory sex change of 3min; 94 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 40s, 35 circulations; 72 ℃ of 5min;
(2b) inboard primer amplification:
50 μ L systems: 2 * Taq buffer, 25 μ L, 10mM dNTP mix 1 μ L, each 1 μ L of primer SEQ ID NO:3 and SEQ ID NO:4,25mM MgCl
25 μ L, 5U/ μ L Taq archaeal dna polymerase 0.3 μ L, outside P CR amplified production 2 μ L, ddH
2O 14.7 μ L;
Pcr amplification condition: 94 ℃ of preparatory sex change of 2min; 94 ℃ of 20s, 59 ℃ of 20s, 72 ℃ of 30s, 40 circulations; 72 ℃ of 5min.
The described pcr amplification product that step (2) is obtained of step (3) carries out the strand purifying with the primer sequence of described mark vitamin H, specifically comprises the steps:
The 50uL pcr amplification product that obtains in the step (2) shaken in a centrifuge tube with 3uL streptavidin beads and 47uL binding buffer liquid mix 10min; Grasp beads with the vaccuumprep tool in the Vaccuum prep workstation system then; With the vaccum prep tool that is adsorbed with beads successively at 75% (v/v) ethanolic soln; 0.2MNaOH solution cleans 10s in the 10mM pH 7.6Tris-Acetate solution, vacuum prep tool is put into the PSQ96 plate that contains 1uL sequencing primer and 49uL annealing buffer; Discharge beads; This PSQ96 plate is placed on is heated to 80 ℃ of 2min on the ThermoPlate, be cooled to room temperature again, promptly obtain to carry out the strand purified product of follow-up sequencing reaction.
The described interpretation of result of step (5) is specially: sequencing result is compared at HCV DB (http://hcv.lanl.gov/content/sequence/BASIC_BLAST/basic_blast.ht ml), obtains institute's this genotype of test sample.
Beneficial effect: the genotypic test kit of detection HCV of the present invention; This method can detect all hypotypes in the 1-6 type through rt, PCR and sequence measurement; The result of order-checking gained has high accuracy, and is quicker, easy than traditional Sanger PCR sequencing PCR, and all more special, sensitive, easy, quick, accurate, with low cost than other detection methods; Detect lower limit to 100copy/mL, and have very high flux.
Description of drawings
Fig. 1 utilizes test kit of the present invention and detection method (special sequencing primer SEQ ID NO:5) to detect the sequencing result figure of third liver.
Fig. 2 utilizes test kit of the present invention and detection method (special sequencing primer SEQ ID NO:6) to detect the sequencing result figure of third liver.
Fig. 3 utilizes test kit of the present invention and detection method (special sequencing primer SEQ ID NO:7) to detect the sequencing result figure of third liver.
Fig. 4 is the sequencing result figure that Sanger order-checking (sequencing primer SEQ ID NO:3) detects third liver, and the special sequencing primer SEQ of corresponding the present invention ID NO:5 detects the sequencing result of third liver.
Fig. 5 is the sequencing result figure that Sanger order-checking (sequencing primer SEQ ID NO:3) detects third liver, and the special sequencing primer SEQ of corresponding the present invention ID NO:6 detects the sequencing result of third liver.
Fig. 6 is the sequencing result figure that Sanger order-checking (sequencing primer SEQ ID NO:3) detects third liver, and the special sequencing primer SEQ of corresponding the present invention ID NO:7 detects the sequencing result of third liver.
Embodiment
According to following embodiment, can understand the present invention better.Yet, those skilled in the art will readily understand that embodiment only is used to explain the present invention, and the present invention that should also can not limit in claims to be described in detail.
Embodiment 1: the preparation of test kit.
(1) RNA extracts reagent:
Trizol: purchase company in invitrogen,
Chloroform: purchase high brilliant Fine Chemical Co., Ltd in Hangzhou,
Virahol: purchase two woods chemical reagent factories in Hangzhou,
Absolute ethyl alcohol: purchase Long March chemical reagent ltd in Hangzhou,
DEPC treating water: purchase company in fermentas.
(2) rt reagent:
200U/ μ L M-MLV reversed transcriptive enzyme: purchase company in fermentas,
20U/ μ L RNase suppressor factor: purchase company in fermentas,
5 * RT-PCR Buffer:250mM Tris-HCl (pH 8.3), 250mM KCl, 20mM MgCl
2, 50mMDTT (purchasing company) in fermentas,
10mM dNTP mix: purchase ancient cooking vessel state Bioisystech Co., Ltd in Shanghai,
100 μ M random hexamer primer: purchase company in invitrogen,
DEPC treating water: purchase company in fermentas.
(3) PCR reagent:
2 * Taq buffer:0.1% (v/v) NP-40; 0.02% (v/v) gelatin (purchasing Sigma company) in the U.S., 0.06% (g/mL) BSA (purchasing Sigma company), 0.1% (v/v) Tween-20 (purchasing Sigma company) in the U.S. in the U.S.; 0.06M pH8.9Tricine (purchasing company) in Merck
Special primer: SEQ ID NO:1～4,10uM,
25mM MgCl
2: purchase Sigma company in the U.S.,
10mM dNTP mix: purchase ancient cooking vessel state Bioisystech Co., Ltd in Shanghai,
5U/ μ LTaq archaeal dna polymerase: available from U.S. Fermentas company.
(3) strand purified reagent:
Streptavidin beads (being dissolved in 20% ethanol): purchase company in GE,
75% (v/v) ethanolic soln: purchase Long March chemical reagent ltd in Hangzhou,
0.2M NaOH: purchase in Shishewei Chemical Co., Ltd., Shanghai,
10mM Tris-Acetate (pH 7.6): Tris-base purchases the Sigma company in the U.S., and anhydrous acetic acid is purchased the chemical reagent ltd in Hangzhou,
Binding buffer liquid: (Tris-base purchases the Sigma company in the U.S. by 10mM Tris-HCl; Hydrochloric acid is purchased the chemical reagent ltd in Hangzhou); 2M NaCl (purchasing) in Shishewei Chemical Co., Ltd., Shanghai; 1mM EDTA (purchasing the chemical reagent ltd in Hangzhou), 0.1% (v/v) Tween 20 (purchasing the Sigma company in the U.S.) forms
Annealing buffer: by 20mM Tris-Acetate (pH 7.6) (Tris-base purchases the Sigma company in the U.S., and anhydrous acetic acid is purchased the chemical reagent ltd in Hangzhou), 2mM magnesium acetate (purchasing in Shishewei Chemical Co., Ltd., Shanghai) is formed.
(4) sequencing reagent:
Archaeal dna polymerase, ATP sulfurylase, luciferase and apyrase: purchase company in QIAGEN,
Substrate A PS and resorcinolphthalein: purchase company in QIAGEN,
Four kinds of dNTP (dATP S, dTTP, dCTP, dGTP): purchase company in QIAGEN.
Embodiment 2: detection method.
The extracting of (1) third liver HCV RNA template specifically comprises the steps:
(1a) with sample serum 200 μ L and 1mLTrizol, mixing adds 200 μ L chloroforms, mixing 15s, room temperature 5min, 4 ℃ of centrifugal 15min of following 12000g (whizzer is Microfuge 22R Centrinofuge, and BECKMAN company is as follows);
(1b) upper water moves into another centrifuge tube mutually, adds and upper water equal volume Virahol mixing 15s, room temperature 10min, 4 ℃ of centrifugal 10min of following 12000g;
(1c) abandon the supernatant that step (1b) obtains, 75% (v/v) ethanol (joining) 1mL of precooling on the rocks, 4 ℃ of centrifugal 5min of following 7500g with DEPC water;
(1d) abandon the supernatant that step (1c) obtains, dry air 5min adds the 20uLDEPC treating water, and it is subsequent use to obtain RNA-70 ℃ of HCV.
(2) step (1) gained RNA template is become cDNA with the random primer rt, the primer by said specific amplification hepatitis C virus RNA rt product cDNA carries out pcr amplification (S1000Thermal Cycler, BIO-RAD company) again;
Wherein, The rt concrete grammar is: with 11 μ L RNA and 1 μ L 100uM random hexamer primer, 4 μ L5 * RT-PCR Buffer, 1 μ L 20U/ μ L RNase suppressor factor, 2 μ L 10mM dNTP mix and 1 μ L 200U/ μ LM-MLV reversed transcriptive enzyme; Mixing, 25 ℃ of 5min, 42 ℃ of 60min; 70 ℃ of 5min, it is subsequent use to obtain cDNA4 ℃ of preservation.
Wherein, the pcr amplification method is specially:
(2a) outside primer amplification:
50 μ L systems: 2 * Taq buffer, 25 μ L, 10mM dNTP mix 1 μ L, each 1 μ L of primer SEQ ID NO:1 and SEQID NO:2,25mM MgCl
24 μ L, 5U/ μ L Taq archaeal dna polymerase 0.3 μ L, cDNA 2.5 μ L, ddH
2O15.2 μ L.
Pcr amplification condition: 94 ℃ of preparatory sex change of 3min; 94 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 40s, 35 circulations; 72 ℃ of 5min;
(2b) inboard primer amplification:
50 μ L systems: 2 * Taq buffer, 25 μ L, 10mM dNTP mix 1 μ L, each 1 μ L of primer SEQ ID NO:3 and SEQ ID NO:4,25mM MgCl
25 μ L, 5U/ μ L Taq archaeal dna polymerase 0.3 μ L, outside P CR amplified production 2 μ L, ddH
2O 14.7 μ L;
Pcr amplification condition: 94 ℃ of preparatory sex change of 2min; 94 ℃ of 20s, 59 ℃ of 20s, 72 ℃ of 30s, 40 circulations; 72 ℃ of 5min.
(3) pcr amplification product that step (2) is obtained carries out the strand purifying with the primer sequence of described mark vitamin H, specifically comprises the steps:
The 50uL pcr amplification product that obtains in the step (2) shaken in a centrifuge tube with 3uL streptavidin beads and 47uL binding buffer liquid mix 10min; Use Vaccuum prep workstation (the PyroMark ID of system then; QIAGEN company) vaccuum prep tool in grasps beads, with the vaccum prep tool that is adsorbed with beads successively at 75% (v/v) ethanolic soln, 0.2M NaOH solution; Clean 10s in the 10mM pH 7.6Tris-Acetate solution; Vacuum prep tool is put into the PSQ96 plate (purchasing the company in QIAGEN) that contains 1uL sequencing primer and 49uL annealing buffer, discharge beads, this PSQ96 plate is placed on the ThermoPlate (purchasing the company in QIAGEN) is heated to 80 ℃ of 2min; Be cooled to room temperature again, promptly obtain to carry out the strand purified product of follow-up sequencing reaction.
(4) step (3) gained strand purified product is checked order, operate according to instrument (PyroMark ID, QIAGEN company) specification sheets.
(5) interpretation of result is specially: sequencing result is compared at HCV DB (http://hcv.lanl.gov/content/sequence/BASIC_BLAST/basic_blast.ht ml), obtains institute's this genotype of test sample.
Fig. 1 is the special sequencing primer SEQ of the 1st routine third liver specimen ID NO:5 sequencing result figure, and sequencing result is: TGCCAGGACGACCGGGTCCTTTCTTGGATCAACCCG,
Fig. 2 is the special sequencing primer SEQ of the 1st routine third liver specimen ID NO:6 sequencing result figure, and sequencing result is: TCAACCCGCTCAATGCCTGGAGATTTGGGCG
Fig. 3 is the special sequencing primer SEQ of the 1st routine third liver specimen ID NO:7 sequencing result figure, and sequencing result is: GAGACTGCTAGCCGAGT
Sequencing result is compared at HCV DB (http://hcv.lanl.gov/content/sequence/BASIC_BLAST/basic_blast.ht ml) respectively, in conjunction with 3 parts order-checking comparison result, the highest this genotype of the test sample 1b of the institute type that is of score.
Be the checking result reliability, same sample carried out classical Sanger with the PCR product of step (2) gained with common sequenator (ABI3100, American I nvitrogen company) check order that sequencing primer uses inboard PCR primer SEQ IDNO:3.
Fig. 4～6 are the Sanger sequencing result of the 3 parts order-checking in corresponding this test kit, and Fig. 4 sequencing result is: TGCCAGGACGACCGGGTCCTTTCTTGGATCAACCCG; Fig. 5 sequencing result is: TCAACCCGCTCAATGCCTGGAGATTTGGGCG; Fig. 6 sequencing result is: GAGACTGCTAGCCGAGT.Fit like a glove with this test kit 3 special sequencing primers (SEQ ID NO:5～SEQ ID NO:7) sequencing result.
Claims (2)
1. hepatitis C sequencing and typing kit is characterized in that it comprises:
(1) primer of specific amplification hepatitis C virus RNA rt product cDNA; Its nucleotide sequence is shown in SEQ IDNO:1 to SEQ ID NO:4; Wherein SEQ ID NO:1 and SEQ ID NO:2 are that primer is right, and SEQ IDNO:3 and SEQ ID NO:4 are that primer is right;
(2) hepatitis C virus specificity sequencing primer, its nucleotide sequence is shown in SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7;
Wherein, SEQ ID NO:4 is at its 5 ' end mark vitamin H;
Above-mentioned primer uses in one-time detection jointly.
2. hepatitis C sequencing and typing kit according to claim 1 is characterized in that it comprises following reagent:
(1) RNA extracts reagent: Trizol, chloroform, Virahol, absolute ethyl alcohol, DEPC treating water;
(2) rt reagent: 200U/ μ L M-MLV reversed transcriptive enzyme, 20U/ μ L RNase suppressor factor, 5 * RT-PCRBuffer, 10mM dNTP mix, 100 μ M random hexamer primer, DEPC treating water;
(3) PCR reagent: 2 * Taq buffer, 10 μ M special primer SEQ ID NO:1～4,25mM MgCl
2, 10mM dNTP mix, 5U/ μ L Taq archaeal dna polymerase;
(3) strand purified reagent: streptavidin beads, volume percent is 75% ethanolic soln, 0.2M NaOH, 10mM pH 7.6 Tris-Acetate, binding buffer liquid, annealing buffer;
(4) sequencing reagent: archaeal dna polymerase, ATP sulfurylase, luciferase, apyrase, substrate A PS, resorcinolphthalein and dNTP.
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| CN102719558A (en) * | 2012-05-30 | 2012-10-10 | 杭州艾迪康医学检验中心有限公司 | Kit for detecting hepatitis C virus (HCV) genotype |
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| CN102154510B (en) * | 2011-02-25 | 2013-01-16 | 湖南圣湘生物科技有限公司 | Nucleic acid quantitative detection kit for hepatitis C virus (HCV) |
| CN102286644B (en) * | 2011-08-26 | 2013-04-10 | 李艳 | Kit for genotyping hepatitis C virus (HCV) |
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| CN103710464B (en) * | 2013-12-30 | 2015-03-11 | 湖南圣维尔医学检验所有限公司 | HCV (Hepatitis c virus) genotype detection kit |
| CN104263850B (en) * | 2014-06-19 | 2017-06-06 | 重庆医科大学 | MHV genotyping detection method and kit based on SNaPshot technologies |
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| CN1651579A (en) * | 2004-12-03 | 2005-08-10 | 中国科学院上海微系统与信息技术研究所 | Gene chip internal reference, preparation method and application thereof |
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| CN1477209A (en) * | 2003-07-16 | 2004-02-25 | 中国科学院上海微系统与信息技术研究 | Method for quickly-detecting hepatilis C virus and its gene type |
| CN1651579A (en) * | 2004-12-03 | 2005-08-10 | 中国科学院上海微系统与信息技术研究所 | Gene chip internal reference, preparation method and application thereof |
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| CN102719558A (en) * | 2012-05-30 | 2012-10-10 | 杭州艾迪康医学检验中心有限公司 | Kit for detecting hepatitis C virus (HCV) genotype |
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