CN102559930A

CN102559930A - Kit of detecting hepahtis C virus by fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction)

Info

Publication number: CN102559930A
Application number: CN2012100143966A
Authority: CN
Inventors: 李明; 丁渭; 陈华云
Original assignee: Daan Gene Co Ltd Zhongshan University
Current assignee: Daan Gene Co Ltd Zhongshan University
Priority date: 2012-01-16
Filing date: 2012-01-16
Publication date: 2012-07-11

Abstract

The invention relates to a kit of detecting a hepahtis C virus by fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction), in particular to a kit of quickly and qualitatively detecting hepahtis C virus infection by one-step real-time fluorescence quantitative RT-PCR technology. The kit has the characteristics of high sensitivity and specificity, can qualitatively detect hepahtis C virus nucleic acid RNA (ribonucleic acid) in a serum or blood plasma sample and is suitable for auxiliary diagnosis of the hepahtis C virus infection and monitoring of treatment effect of drug treatment of hepahtis C virus infectors.

Description

A kind of fluorescence quantitative RT-RCR detects the test kit of hepatitis C virus

Technical field

The present invention relates to the test kit that a kind of fluorescence quantitative RT-RCR detects hepatitis C virus, particularly relate to test kit quick with one-step method real-time fluorescent reverse-transcription polymerase chain reaction (RT-PCR) technology, the detection by quantitative infection with hepatitis C virus.This test kit has very high sensitivity and specificity; Realize hepatitis C virus nucleic acid RNA in serum or the plasma sample is carried out detection by quantitative through test kit of the present invention, be applicable to the auxiliary diagnosis of infection with hepatitis C virus and the curative effect monitoring of infection with hepatitis C virus person pharmacological agent.

Background technology

Hepatitis C (Hepatitis C virus; HCV) be a kind of invisible, infectivity, persistence, progressivity disease, the disease of mainly propagating that causes by infection with hepatitis C virus through blood, hepatitis C chronicity rate can be up to 50%～80%; Can cause necrosis of liver chronic inflammatory diseases and fibrosis; Part patient can develop into liver cirrhosis even hepatocellular carcinoma (HCC), and is very harmful to patient's health and lives, become serious society and public health problem.The infection of hepatitis C is worldwide distribution, is the main reason of America and Europe and Japan and other countries hepatopathy in whole latter stage.According to World Health Organization's statistics, the infection rate of global HCV is about 3%, about 3.5 ten thousand examples of annual New Development hepatitis C case.China CDC report hepatitis C case has been turned over 5 times in nearest 5 years, total number of the infected reaches 4,000 ten thousand, and can not get rid of the possibility that existence is failed to report.China's seroepidemiological survey data shows that China's general crowd's anti-HCV positive rate is 3.2%.Various places anti-HCV positive rate has certain difference, is the boundary with the Changjiang river, and the north (3.6%) is higher than south (2.9%), and southwest, East China, North China, northwest, Central-South and northeast are respectively 2.5%, 2.7%, 3.2%, 3.3%, 3.8% and 4.6%.The anti-HCV positive rate rises with age growth gradually, by 3.9% of 2.0% to 50～59 years old group of 1 years old group.No significant difference between the men and women.

HCV belongs to flaviviridae (flaviviridae); Its genome is the sub-thread positive chain RNA; Be prone to variation, can be divided into 6 genotype and different subtype at present, according to the method for the current international practice; Represent the HCV genotype with Arabic numeral, represent gene hypotype (like 1a, 2b, 3c etc.) with the English alphabet of small letter.It is popular that

HCV gene

1,2,3 types are the whole world, and be America, Europe and Japanese oligogene type, and wherein gene 1 type still is popular genotype the most widely, accounts for more than 70% of all HCV infection.The main popular type of China is gene 1b type and 2a type, and south all is the 1b type basically, and the 2a type increases gradually from south orientation north, and to northern some areas, 1b and 2a ratio are approaching basically.The 1a type distributes in the central plain area, the 2b type report that only sporadically appears.

The route of transmission of HCV comprises through blood propagates, spreads through sex intercourse and mother-to-baby transmission.(1) blood propagation: be main circulation way, mainly contain: propagate through blood transfusion and blood product.Because anti-HCV exists the quality instability of window phase, detection of anti-HCV reagent and minority the infected not to produce anti-HCV, therefore, can't sift out HCV positive person fully, a large amount of blood transfusions and hemodialysis PI HCV; Propagate through damaged skin and mucous membrane: this is present topmost circulation way, in certain areas, accounts for 60%～90% because of intravenous injection drug use causes HCV to propagate.Using non-once property syringe and syringe needle, the dental appliance without strict sterilization, scope, aggressive operation and acupuncture etc. also is the important channel of propagating through skin.Some possibly cause the traditional medical method of skin injury and blood also to propagate relevant with HCV; Shared shaver, toothbrush, the annular distance of tatooing and pierce one's ears etc. also are HCV potential menses circulation ways.(2) spread through sex intercourse: with HCV the infected's sexual intercourse and the dangerous higher of promiscuity person's HCV infection arranged.Simultaneously with other sexually transmitted disease (STD) persons, infected person immunodeficiency virus (HIV) person particularly, the danger of HCV infection is higher.(3) mother-to-baby transmission: in recent years, the HCV mother-to-baby transmission more and more received people's attention.It is 2% that the positive mother of anti-HCV propagates HCV to neonatal danger, if mother HCV RNA when childbirth is positive, then transmission danger property can be up to 4%～7%; When merging the HIV infection, transmission danger property increases to 20%.The high carrying capacity of HCV virus possibly increase transmission danger property.In addition, also have section H CV the infected's route of transmission not clear.

The laboratory commonly used that HCV infects is detected and is comprised serum biochemistry detection, detection of anti-HCV, HCV RNA detection and genotype chip detection etc.

1 serum biochemistry is learned and is detected

ALT, aspartic transaminase (AST) level change can reflect the hepatocellular damage degree, but the severity of the liver tissues inflammatory calibration that ALT, AST level and HCV infection cause and the state of an illness is not necessarily parallel; The ALT of acute hepatitis C patients and AST level are generally lower, but the higher person is also arranged.The serum albumin of acute hepatitis C patients, thrombogen mobility and cholinesterase activity reduce less, but when the long chronic hepatitis of the course of disease, liver cirrhosis or hepatitis gravis, can obviously reduce, and its reduction degree is directly proportional with the severity of disease.

Among the chronic hepatitis C patient, about 30%ALT level is normal, and about 40%ALT level is lower than 2 times of normal value upper limits.Though this type of patient of great majority has only slight liver injury, has the part patient can develop into liver cirrhosis.It is to occur one of important indicator of replying in the antiviral therapy that the ALT level descends.Prothrombin time can be used as the monitoring index of chronic hepatitis C conditions of patients progress, but still none or one group of serologic marker can carry out accurately by stages hepatic fibrosis so far.

2 detection of anti-HCV methods

Anti-HCV is exactly an antibody of HCV.EIA (enzyme immunoassay) is meant the antigen antibody reaction of carrying out with enzymic-labelled antibody or anti-antibody.Ultimate principle be earlier with known antigen or antibodies on certain solid phase carrier, and keep its immunocompetence.During mensuration; Sample to be checked and enzyme-labelled antigen or antibody antigen or the antibody by different step and surface of solid phase carriers absorption is reacted; Separate immune complex and free composition with washing methods, add the effect substrate catalysis colour developing of enzyme then, qualitatively or quantitatively determine.The domestic method of measuring anti-HCV is an indirect method: known antigens is adsorbed in solid phase carrier, adds sample to be checked (containing corresponding antibodies) and combine with it, after the washing, add enzyme mark anti-antibody and substrate and measure.

Anti-HCV enzyme immunoassay (EIA) is applicable to high risk population's examination, also can be used for HCV the infected's primary dcreening operation.Hepatitis C virus is infected in the positive prompting of detection of anti-HCV.But cloudy whether commentaries on classics of anti-HCV can not be as the index of antiviral curative effect.The method that the explanation of anti-HCV positive findings and assessment are adopted in the time of will combining to detect, necessary additional experiment, clinical characters etc. are comprehensively analyzed.

The detection method of 3HCV RNA

At the HCV acute infection period, the viral genome level in blood plasma or serum can reach 105～107 copy/ml.In HCV chronic infection person, there is very big-difference in the HCV rna level between Different Individual, and variation range is between 5 * 104～5 * 106 copy/ml, but the HCVRNA level is relatively stable in the same patient's the blood.

HCV RNA qualitative detection:, need through HCV RNA qualitative test conclusive evidence to anti-HCV male HCV persistent infection person.The specific degree of HCV RNA qualitative detection is more than 98%, as long as once viral qualitative detection is positive, can proves conclusively HCV and infect, but the one-time detection feminine gender can not be got rid of the HCV infection fully, answers rechecking.

HCV RNA detection by quantitative: quantitative polyase chain reaction (qPCR), branch DNA (bDNA), real-time fluorescence quantitative PCR method all can detect HCV RNA viruses carrying capacity.External HCV RNA detection by quantitative test kit has the Cobas V2.0, SuperQuant, LCx HCV RNA quantitative analysis method of pcr amplification etc., but the Versant HCVRNA 2.0 of bDNA is comparatively extensive with the application of 3.0 quantitative analysis methods.Domestic real-time fluorescence quantitative PCR method has obtained the official approval of State Food and Drug Administration (SFDA).Different HCV RNA detection by quantitative method usable copy/ml and two kinds of method for expressing of IU/ml; When converting between the two; Should adopt the reduction formula of different detection methods, like copy number/ml reduction formula of the SuperQuant of the IU/ml of the Cobas V2.0 of Roche Holding Ag and U.S.'s National Institute of Genetics be: IU/ml=0.854 * copy number/ml+0.538.

The height of HCV virus load does not have absolute relevance with the progress of the severity of disease and disease, but can be used as the observation index of antiviral curative effect assessment.In HCV RNA detects, should note to have false positive and false negative result.

4 genotype chip detection technology

Gene chip is that the detection cake core of microtronics and molecular biology bonded new and high technology HCV gene type progressively is applied to clinical.Its ultimate principle is: with bioinformatics method design HCV 5 ' UTR type specificity probe.Press Design Mode, with the glass surface of robotization microarray point sample in the APTES-PDC modification.Mix fluorescence molecule with commodity PCR test kit patient's blood sample is originally increased, detect and analytical results with laser co-focusing fluorescent scanning appearance.

The strain isolated of in the worldwide HCV being cloned at present can be divided into 6 genotype (genotype), about 50 hypotypes (subtype).The genotypic detection of HCV especially judges that to hepatitis C patients the state of an illness, guiding treatment, prediction curative effect and prognosis are significant for epidemiology, variation trend, the route of transmission of research HCV.

In sum, along with immunology and development of molecular biology, HCV diagnostic method not only susceptibility, specificity, repeatability improves constantly, and the operation easy day by day, just develop towards robotization, standardized direction.Several different methods interpenetrates, brings out one's strengths to make up for one's weaknesses, combines to use, and is another trend of development.

The real-time fluorescence PCR technology is a kind of rapidly nucleic acid detection technique of development in recent years, uses a kind of pcr amplification appearance that has nuclear power coupling devices (CCD), reflects each round-robin level of amplification of PCR in real time through the dynamic change that detects fluorescent signal.CCD can periodically send the exciting light of specific wavelength according to certain procedure, collect to detect fluorescent signal, and is aggregated into workstation through software analysis and obtains amplification curve.One-step method real-time fluorescent RT-PCR technology is a kind of (Yuqi Z of real-time fluorescence PCR; Min Y; Johann WM, et al.2002.Quantification of Human Immunodeficiency Virus Type 1Proviral DNA by Using TaqMan Technology.J Cli Microbiol.40 (2): 675-678; Drosten C; Seifried E; Roth WK; Et al.2001, TaqMan 5_-nuclease human immunodeficiency virus type 1 PCR assay with phage-packaged competitive internal control for high-throughput blood donor screening.J Clin Microbiol, 39:4302-4308; Schuurman R; Descamps D; Weverling GJ; Et al.1996, Multicenter comparison of three commercial methods for quantification of human immunodeficiency virus type 1 RNA in plasma.J Clin Microbiol, 34:3016-3022; Christopherson C; Kidane Y; Conway B et al.2000.PCR-based assay to quantify human immunodeficiency virus type 1 DNA in peripheral blood mononuclear cells.J Clin Microbiol; 38:630-634.), it is the method for a kind of direct rapid detection RNA, compares with the real-time fluorescence PCR that detects DNA; Difference is that the former has increased reversed transcriptive enzyme in reaction system, simultaneously many reverse transcription reaction steps; Something in common is that both all have the probe of a two ends difference mark fluorescent reporter group and quenching group in reaction system, and when probe structure was complete, the energy that the fluorescence report group sends fluorescence shifted to quenching group, presents quenching effect.If the existence of target sequence is arranged in the amplification procedure, the process middle probe molecule of amplification is hydrolyzed cut-out gradually, and fluorescence report group and quenching group dissociate each other, have blocked the two FRET effect, and the fluorescence report group sends fluorescent signal.Along with the carrying out of amplification, fluorescent signal presents linear the enhancing along with the segmental amplification of purpose.

The real time fluorescent quantitative method is carried out pcr amplification to its product cDNA then at first to HCV target nucleic acid RNA rt (RT).In the PCR reaction system, add fluorophor, utilize the fluorescent signal whole PCR process of monitoring in real time, the method for through typical curve unknown template being carried out quantitative analysis at last.This technology not only realized PCR from sxemiquantitative to quantitative leap, and compare with conventional PCR, it have high specificity, level of automation high, efficiently solve characteristics such as PCR pollution problem.

This test kit adopts the TaqMan fluorescent probe technique; The relative conserved regions of choosing HCV genome encoding district is a target region; Design specific primers and fluorescent probe; Wherein purpose fluorescent probe 5 ' end mark fluorescent is launched group FAM, and the non-fluorescent quenching group B of 3 ' end mark HQ1 utilizes the fluorescent PCR detector to detect fluorescent signal at the FAM passage.The mark quality control system is used for the quality control of whole nucleic acid extraction and reaction system in being provided with simultaneously.Interior target area is chosen consistent with the HCV target area; And at the probe location synthetic one section oligonucleotide; The designs specificity fluorescent probe, with the equal no cross reaction of any species, wherein fluorescent probe 5 ' end mark fluorescent is launched group HEX; And mark system of quality control in forming with interior mark plasmid is common, can detect fluorescence at the VIC passage.For thing to be checked, if detected result shows typical S type fluorescence growth curve, then positive sample, on the contrary then negative.Through nucleic acid extraction system simple to operation, in conjunction with real-time fluorescence PCR detection system operation single stage method RT-PCR amplification program, this test kit has been realized the high-level efficiency that detects, highly sensitive and high special.

Summary of the invention

The present invention relates to the test kit that a kind of fluorescence quantitative RT-RCR detects hepatitis C virus, particularly relate to test kit quick with one-step method real-time fluorescent reverse-transcription polymerase chain reaction (RT-PCR) technology, the detection by quantitative infection with hepatitis C virus.This test kit has very high sensitivity and specificity; Realize hepatitis C virus nucleic acid RNA in serum or the plasma sample is carried out detection by quantitative through test kit of the present invention, be applicable to the auxiliary diagnosis of infection with hepatitis C virus and the curative effect monitoring of infection with hepatitis C virus person pharmacological agent.Its ultimate principle is to utilize the fluorescent PCR technology; With relative conserved regions in the HCV gene is target region; Design specific primers and fluorescent probe; After the sample nucleic acid purification, through the single stage method RT-PCR that contains reversed transcriptive enzyme (c-MMLV enzyme), warm start Taq enzyme, RNA enzyme inhibitors (RNasin) HCV RNA is carried out fast quantification and detect.Simultaneously, test kit also has internal standard substance matter, is used for the whole process of nucleic acid extraction is monitored, and reduces the appearance of false negative result.And the detection fluorescent signal, the instrument software system draws out real-time amplification curve automatically, realizes the detection by quantitative to unknown sample according to threshold cycle values (CT value).

In order to realize the present invention, we have adopted following technical scheme:

1) uses suitable foranalysis of nucleic acids software respectively the nucleotide sequence of known HCV gene to be carried out homology relatively, finding out on the basis of homology segment, further use suitable primer-design software to select and design oligonucleotides primer and probe.Because institute's designed primer and probe all have the sequence that is complementary to the HCV gene; And there is not homology with the nucleotide sequence of other pathogenic agent; The restriction enzyme site that does not also comprise any common endonuclease; So false negative and the false positive of having avoided hepatitis C virus to detect have improved the safety and the accuracy that detect.The mark quality control system is used for the quality control of whole reaction system in being provided with simultaneously.Interior target area is chosen consistent with the HCV target area, and at the probe location synthetic one section oligonucleotide, the designs specificity fluorescent probe is with the equal no cross reaction of any species.

2) use synthetic required Oligonucleolide primers and the probe of DNA synthesizer, separate processing with carrying out ammonia behind molecular sieve and fast protein liquid chromatography method (FPLC) purifying.Same synthetic required probe sequence; Ammonia is separated after the processing respectively at 6-FAM amidite and the HEX of its 5 ' end mark as fluorescence generation group (reporter group), and its 3 ' end mark by active connecting arm coupling on as fluorescent quenching or suppress the BHQ1 of group.With the fluorescently-labeled probe of FPLC purification by chromatography.Then, polyacrylamide gel (20%) electrophoretic method and spectrophotometry physical characterization institute synthetic primer and the probe under the use sex change condition.

3) be suitable for the reaction system that single stage method RT-PCR increases.Reaction system comprises reversed transcriptive enzyme; Warm start Taq enzyme; 2 '-deoxynucleoside triphosphate; RNA enzyme inhibitors (RNasin); Can with the forward primer of article one chain combination of double-stranded target polynucleotide; Can with the reverse primer of the second chain combination of double-stranded target polynucleotide; Can combine with the target polynucleotide and two ends are combined with the oligonucleotide probe of fluorescence generation group and fluorescent quenching group respectively; Can combine with interior mark Nucleotide and two ends are combined with target oligonucleotide probe in the detection of fluorescence generation group and fluorescent quenching group respectively; The damping fluid that contains mg ion.The mark quality control system is used for the quality control of whole nucleic acid extraction and reaction system in being provided with simultaneously.

4) from sample to be tested, extract nucleic acid RNA, add respectively in the aforesaid reaction system,, the nucleic acid RNA of unknown sample is carried out detection by quantitative from the amplified fluorescence curve directly through single stage method RT-PCR amplification.

Test kit based on above technical scheme invention comprises: (1) nucleic acid extracting reagent, HCV reaction solution A, HCV reaction solution B, HCV reaction liquid C, HCV inner mark solution, negative quality control product, HCV strong positive quality control product, the critical positive quality control product of HCV, the HCV positive are quantitatively with reference to article 1-4 and (2) separation and concentrate the packing box of packing these reagent bottles or pipe.

According to a preferred embodiment of the invention, wherein the PCR reaction system comprises HCV reaction solution A, HCV reaction solution B and HCV reaction liquid C.

According to another preferred embodiment of the present invention; Wherein HCV reaction solution A (primer probe mixed solution) is made up of forward primer, reverse primer, purpose oligonucleotide probe, interior mark oligonucleotide probe, and the forward and the reverse primer that it is characterized in that being used for the target polynucleotide amplification are respectively 5 '-CATGGCGTTAGTAYGAGTGTCG-3 ' (SEQ ID NO:1) and 5 '-TCCYGGCAATTCCGGTGTAC-3 ' (SEQ ID NO:2).

According to another preferred embodiment of the present invention; The oligonucleotide probe that its characteristic also is to be used for the target polynucleotide amplification is 5 '-X-TCCGCAGACCACTATGGCTCTCCCG-Y-3 ' (SEQ ID NO:3); Wherein X/Y representes the fluorescent mark group, comprise can combine with target polynucleotide and two ends respectively bonded the oligonucleotide probe of fluorescence generation group and fluorescent quenching group is arranged.

According to another preferred embodiment of the present invention, its characteristic is that also the primer concentration and probe concentration that is used for the target polynucleotide amplification is 5～15pmol/ person-portion, and preferred primer concentration is that 10pmol/ person-portion, concentration and probe concentration are the 5pmol/ person-portion.

According to another preferred embodiment of the present invention; The oligonucleotide probe of mark polynucleotide amplification is 5 '-X-TCCACGGCAACTCTAGTCGCCCTGC-Y-3 ' (SEQ ID NO:4) in being used in the primer probe mixed solution; Wherein X/Y representes the fluorescent mark group, comprise can combine with interior mark polynucleotide and two ends respectively bonded the oligonucleotide probe of fluorescence generation group and fluorescent quenching group is arranged.

According to another preferred embodiment of the present invention, the concentration and probe concentration of mark polynucleotide amplification was 3～10pmol/ person-portion in its characteristic also was to be used in the primer probe mixed solution, and preferred concentration and probe concentration is the 5pmol/ person-portion.

According to another preferred embodiment of the present invention, HCV reaction solution B is made up of reversed transcriptive enzyme (c-MMLV enzyme), warm start Taq enzyme, RNasin, dNTPs, enzyme diluent.

According to another preferred embodiment of the present invention; Its characteristic is that also the c-MMLV enzyme dosage is that 1.5～3.5U/ person-portion, warm start Taq enzyme dosage are that 3～7U/ person-portion, RNasin consumption are that 15～25U/ person-portion, dNTPs concentration are 0.20mmol/L, and the enzyme diluent is general commercially available enzyme diluent.

According to another preferred embodiment of the present invention, the HCV reaction liquid C comprises single stage method RT-PCR reaction buffer and DEPC water, and wherein single stage method RT-PCR reaction buffer comprises 10mmol/L Tris-HCl (pH8.0), 50mmol/L KCl, 3.0mmol/LMgCl ₂

According to a preferred embodiment of the invention, the negative blood plasma of negative quality control product, the critical positive quality control product of HCV strong positive quality control product and HCV is and contains the segmental virus-like particle of HCV purpose, and concentration is respectively 5.0 * 10 ⁶With 5.0 * 10 ³IU/ml; Positive quantitatively being with reference to article 1-4 of HCV contained the segmental recombinant plasmid of HCV purpose, and concentration is 1.0 * 10 ⁴-1.0 * 10 ⁷IU/ml; Plasmid and virus-like particle form employed upstream and downstream primer sequence SEQ ID NO:1 and SEQ ID NO:2 by the PCR product of upstream and downstream primer amplification through commercially available carrier cloning structure.

According to another preferred embodiment of the present invention, the optimal reaction temperature and the time that wherein are used for pcr amplification are: 50 ℃ of rt 15min, 1 circulation; 95 ℃ of 15min, 1 circulation; Last 94 ℃ of 15s, 55 ℃ of 45s, 45 circulating collection fluorescent signals.

According to a preferred embodiment of the invention, wherein the sample to be checked of test kit is samples such as serum or blood plasma.

The minimum concentration that one-step method real-time fluorescent RT-PCR test kit provided by the invention can detect hepatitis C virus is 250IU/ml, explains that this test kit has extraordinary sensitivity.

The present invention is directed to hepatitis c virus gene design specific primers and probe, the linearity range of detection is 1.0 * 10 ³IU/ml～1.0 * 10 ⁷IU/ml, or other viruses (hepatitis B virus, human immunodeficiency virus, Epstein-Barr virus, nerpes vinrus hominis etc.) no cross reaction that infection symptoms similar identical with infection site.The sensitivity of test kit is 99.47%, and specificity is 98.89%.

The present invention compared with prior art has the following advantages:

(1) use single stage method RT-PCR that HCV RNA is carried out fast quantification and detect, sensitivity, specificity improves greatly.

(2) totally-enclosed reaction is extracted after the viral nucleic acid RNA, directly is used for RT-PCR and detects, and has avoided polluting and has taken place.

(3) test kit has internal standard substance matter, can be used for the whole process of nucleic acid extraction and pcr amplification is monitored, thus the appearance of minimizing false negative result.

Description of drawings

Fig. 1 shows the detected result of the positive quantitatively reference of HCV strong positive quality control product, the critical positive quality control product of HCV, negative quality control product and HCV article 1-4.HCV strong positive quality control product, the critical positive quality control product amplification curve of HCV have FAM detection path amplification curve that obvious increased logarithmic phase is arranged, and be typical S type amplification curve, and the definite value of strong positive and critical positive quality control product are respectively 1.0 * 10 ⁶-2.0 * 10 ⁷IU/ml and 1.0 * 10 ³-5.0 * 10 ⁴In the IU/ml scope. Can clearly be judged to be the positive.Negative quality control product FAM detection path amplification curve does not have the logarithm rise period, and VIC detection path amplification curve is an increased logarithmic phase; Can clearly be judged to be feminine gender.HCV is positive quantitatively to have obvious increased logarithmic phase with reference to article 1-4FAM detection path amplification curve, is typical S type curve, CT value＜40, and R ²>=0.97, all meet the quality control judgement criteria.

Fig. 2 shows the detected result of HCV RNA specificity with reference to article.10 parts of specificitys with reference to the amplification curve of article straight or oblique do not have down and with baseline intersect, do not have the Ct value, can clearly be judged to be feminine gender.10 choose normal human serum (N) and other haematogenous pathogenic micro-organisms, comprise hepatitis B virus (HBV), human immunodeficiency virus-1 (HIV-1); Hepatitis D virus (HDV), hepatitis E virus (HEV), Epstein-Barr virus (EBV); Human cytomegalic inclusion disease virus (HCMV); Treponema pallidum (TP), herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2).

The detected result of Fig. 3 display sensitivity.With enterprise quantitative with reference in the article with reference to article L4 (1 * 10 ³IU/ml) be benchmark, with the dilution of successively decreasing of normal human serum or blood plasma, the final concentration of dilution is respectively 1000IU/ml, 500IU/ml, 250IU/ml, 100IU/ml, 50IU/ml.Detected result shows that the minimum concentration of the HCV sample that detects is 250IU/ml.

Embodiment

The following example is intended to illustrate rather than limit the present invention.

Embodiment 1: the detection method of hepatitis C virus test kit

(1) collection of specimens, transports and preserve

Collection of specimens:

Serum-extract 2 milliliters in person under inspection's venous blood with disposable sterilized injector; Inject aseptic dry glass tube, room temperature (22～25 ℃) was placed 30～60 minutes, and blood specimen can spontaneous complete aggegation be separated out serum; Or direct usage level whizzer, centrifugal 5 minutes of 1500rpm; Draw upper serum, be transferred to 1.5ml sterilization centrifuge tube.

Blood plasma-extract 2 milliliters in person under inspection's venous blood with disposable sterilized injector; Injection contains the Glass tubing of EDTA-2Na (EDTA Disodium) or sodium citrate anticoagulant; Putting upside down Glass tubing immediately gently mixes 5～10 times; Make the abundant mixing of antithrombotics and venous blood, can isolate blood plasma after 5～10 minutes, be transferred to 1.5ml sterilization centrifuge tube.

Sample is preserved and transported: the sample of being gathered can be used for test immediately, and-20 ℃ of preservation perives are 3 months;-70 ℃ of following prolonged preservation should be avoided multigelation.Sample transports and adopts 0 ℃ of curling stone.

(2) nucleic acid extraction

Sample to be tested and negative quality control product, HCV strong positive quality control product, the critical positive quality control product of HCV are carried out synchronous processing.

A). in the aseptic centrifuge tube of 1.5ml, add the Proteinase K of 50 μ l;

B). these 200 μ l of sampling add in the centrifuge tube;

C). add the cracking working fluid (employing virus cracking liquid that has promptly contained Carrier RNA) of 200 μ l again, the tight pipe lid of lid, vortex oscillation 15 seconds is with abundant mixing, high speed centrifugation 10 seconds (producing bubble when preventing incubation), 72 ℃ 10 minutes.Simultaneously can elutriant be placed 72 ℃ of preheatings;

D). add 250 μ l absolute ethyl alcohols again, the tight pipe lid of lid vibrated 15 seconds;

E). mixed solution all is drawn to centrifugal post, and under the room temperature 12, centrifugal 1 minute of 000rpm is filled to new collection tube with centrifugal post;

F). the inhibition of 500 μ l is removed liquid add centrifugal post, under the room temperature 12, centrifugal 1 minute of 000rpm is filled to new collection tube with centrifugal post;

G). the de-ionized liquid of 500 μ l is added centrifugal post, and under the room temperature 12, centrifugal 1 minute of 000rpm is filled to new collection tube with centrifugal post;

H). the de-ionized liquid with 500 μ l adds centrifugal post once more, and under the room temperature 12, centrifugal 1 minute of 000rpm is filled to new collection tube with centrifugal post;

I). under room temperature 14, centrifugal 3 minutes of 000rpm is to remove remaining ethanol with centrifugal post-collection tube;

J). centrifugal post is taken out, be positioned over new 1.5ml centrifuge tube.Open centrifugal post lid, place 2 minutes (use the dry type thermostatted, can not use water-bath) for 72 ℃;

K). the careful elutriant 50 μ l that add 72 ℃ of preheatings directly over the film of centrifugal post, cover tight centrifugal column jecket lid, after room temperature leaves standstill 1 minute, 14, centrifugal 1 minute of 000rpm.Be viral nucleic acid solution in the centrifuge tube, suggestion is used immediately, preserves like need, places-20 ℃.

(3) PCR detects

A) preparation system

With 2 μ l HCV reaction solution A, 3 μ l HCV reaction solution B, 25 μ l HCV reaction liquid C thorough mixing, packing 30 μ l are to each PCR reaction tubes then.

B) application of sample

In above-mentioned HCV reaction tubes, add sample to be tested nucleic acid, negative quality control product, HCV strong positive quality control product, the critical positive quality control product of HCV after extracting respectively and directly add the positive quantitatively reference of HCV article 20 μ l.The tight pipe lid of lid, 8,000rpm is transferred to the pcr amplification appearance after the centrifugal several seconds.

Amplification program is provided with

The parameter of ABI7300/7500 fluorescent PCR amplification appearance is provided with as follows:

Reporter?Dye1：FAM

Quencher?Dye：none

Reporter?Dye2：VIC

Quencher?Dye：none

Passive?Reference：none

The parameter of LightCycler480 fluorescent PCR amplification appearance is provided with as follows:

Select the combination of FAM (483-533) and VIC (523-568) filter disc in " Customizes " module,

The unification of amplification temperature parameter is provided with as follows:

50 ℃ 15 minutes;

95 ℃ 15 minutes;

45 circulations (94 ℃ 15 seconds → 55 ℃ 45 seconds), fluoroscopic examination is chosen in 55 ℃ of 45 seconds these links;

Preserve file, operation.

(4) interpretation of result

Reaction finishes back saving result automatically; (user can adjust according to practical situation voluntarily according to Start value, End value and the Threshold value of analyzing back image adjustment Baseline; The Start value can be 3～15, the End value can be located at 5～20, at Log collection of illustrative plates window the Value value of Threshold are set, and make baseline be positioned at the amplification curve exponential phase; It is straight or be lower than threshold line to adjust the amplification curve of negative quality control product); Click Analysis and obtain analytical results automatically, watch the result, record unknown sample numerical value (C) at the Report interface.

(5) result judges

If the FAM sense channel does not have the logarithm rise period, and at the VIC sense channel increased logarithmic phase is arranged, the HCV RNA concentration of then declaring sample is less than detection sensitivity.

If FAM fluorescent signal amplification is obvious, amplification curve has obvious increased logarithmic phase, is typical S type curve, and Ct value＜45, then judges by following method:

If the C＜1.00E+003 of sample, then HCV RNA concentration＜1 * 10 of this sample ³IU/ml;

If the 1.00E+003≤C≤1.00E+007 of sample, then the HCV RNA concentration=CIU/ml of this sample;

If the C＞1.00E+007 of sample, then HCV RNA concentration＞1 * 10 of this sample ⁷IU/m1.Accurate quantification result if desired detects after can sample being diluted to linearity range with negative quality control product again.The HCV RNA concentration of this sample=(C * extension rate) IU/ml then.

Embodiment 2: the use of quality control product and positive quantitatively reference article in the hepatitis C virus test kit

Quality control product and positive quantitatively reference article comprise HCV strong positive quality control product in the hepatitis C virus test kit, the critical positive quality control product of HCV, and negative quality control product and HCV are positive to be used for the clinical trial quality control quantitatively with reference to article 1-4, and working method is with sample to be checked.

Quality control:

Negative quality control product: FAM detection path amplification curve does not have the logarithm rise period, and VIC detection path amplification curve is an increased logarithmic phase;

The HCV positive quality control product: FAM detection path amplification curve has obvious increased logarithmic phase, be typical S type amplification curve, and the definite value of strong positive and critical positive quality control product is respectively 1.0 * 10 ⁶-2.0 * 10 ⁷IU/ml and 1.0 * 10 ³-5.0 * 10 ⁴In the IU/ml scope.

HCV is positive quantitatively with reference to article: FAM detection path amplification curve has obvious increased logarithmic phase, is typical S type curve, CT value＜40, and R ²>=0.97;

Need more than to require in once testing, satisfying simultaneously, otherwise this experiment is invalid, need carry out again.

Detected result (seeing accompanying drawing 1): the amplification curve of negative quality control product is not S-type; The amplification curve of positive quality control product is obvious S type curve; HCV is positive quantitatively to have obvious increased logarithmic phase with reference to article 1-4FAM detection path amplification curve; Be typical S type curve, the positive Quality Control requirement that quantitatively all meets test kit of positive and negative quality control product and HCV with reference to article 1-4, therefore the detected result of sample to be checked is effective.

Above content is to combine concrete preferred implementation to the further explain that the present invention did, and can not assert that practical implementation of the present invention is confined to these explanations.For the those of ordinary skill of technical field under the present invention, under the prerequisite that does not break away from the present invention's design, can also make some simple deduction or replace, all should be regarded as belonging to protection scope of the present invention.

Claims

1. a fluorescence quantitative RT-RCR detects the test kit of hepatitis C virus; Test kit comprises: (1) nucleic acid extracting reagent, HCV reaction solution A, HCV reaction solution B, HCV reaction liquid C, HCV inner mark solution, negative quality control product, HCV strong positive quality control product, the critical positive quality control product of HCV, the positive quantitatively reference of HCV article 1-4; (2) packing box of separation and concentrated these reagent bottles of packing or pipe; Wherein the PCR reaction system comprises HCV reaction solution A, HCV reaction solution B and HCV reaction liquid C, it is characterized in that middle forward of HCV reaction solution A (primer probe mixed solution) and reverse primer are respectively:

5’-CATGGCGTTAGTAYGAGTGTCG-3’(SEQ?ID?NO：1)

5’-TCCYGGCAATTCCGGTGTAC-3’(SEQ?ID?NO：2)；

HCV reaction solution A oligonucleotide probe is 5 '-X-TCCGCAGACCACTATGGCTCTCCCG-Y-3 ' (SEQ ID NO:3); Wherein X/Y representes the fluorescent mark group, comprise can combine with target polynucleotide and two ends respectively bonded the oligonucleotide probe of fluorescence generation group and fluorescent quenching group is arranged;

Oligonucleotide probe is 5 '-X-TCCACGGCAACTCTAGTCGCCCTGC-Y-3 ' (SEQ ID NO:4) among the HCV reaction solution A; Wherein X/Y representes the fluorescent mark group, comprise can combine with interior mark polynucleotide and two ends respectively bonded the oligonucleotide probe of fluorescence generation group and fluorescent quenching group is arranged.

2. according to the test kit of claim 1, its characteristic is that also the concentration of forward and reverse primer is the 10pmol/ person-portion, and the concentration of purpose oligonucleotide probe, interior mark oligonucleotide probe is the 5pmol/ person-portion.

3. according to the test kit of claim 1; HCV reaction solution B is made up of reversed transcriptive enzyme (c-MMLV enzyme), warm start Taq enzyme, RNasin, dNTPs, enzyme diluent; Wherein the c-MMLV enzyme dosage is that 1.5～3.5U/ person-portion, warm start Taq enzyme dosage are that 3～7U/ person-portion, RNasin consumption are that 15～25U/ person-portion, dNTPs concentration are 0.20mmol/L, and the enzyme diluent is a diluent for general commercially available enzyme.

4. according to the test kit of claim 1, the HCV reaction liquid C comprises single stage method RT-PCR reaction buffer and DEPC water, and wherein single stage method RT-PCR reaction buffer comprises 10mmol/L Tris-HCl (pH8.0), 50mmol/L KCl, 3.0mmol/LMgCl ₂

5. according to the test kit of claim 1, its characteristic is that also the optimal reaction temperature of pcr amplification and time is: 50 ℃ of rt 15min, 1 circulation; 95 ℃ of 15min, 1 circulation; Last 94 ℃ of 15s, 55 ℃ of 45s, 45 circulating collection fluorescent signals.

6. root is according to the test kit of claim 1, and its characteristic also is the negative blood plasma of negative quality control product, and the critical positive quality control product of HCV strong positive quality control product and HCV is and contains the segmental virus-like particle of HCV purpose, and concentration is respectively 5.0 * 10 ⁶With 5.0 * 10 ³IU/ml; Positive quantitatively being with reference to article 1-4 of HCV contained the segmental recombinant plasmid of HCV purpose, and concentration is 1.0 * 10 ⁴-1.0 * 10 ⁷IU/ml; Plasmid and virus-like particle form employed upstream and downstream primer sequence SEQ ID NO:1 and SEQ ID NO:2 by the PCR product of upstream and downstream primer amplification through commercially available carrier cloning structure.

7. root is according to the test kit of claim 1, and its characteristic is that also the sample to be checked of test kit is samples such as serum or blood plasma.