CN102676693A - RT-PCR detection kit and method for five subtypes of hepatitis c virus - Google Patents
RT-PCR detection kit and method for five subtypes of hepatitis c virus Download PDFInfo
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Abstract
The invention relates to a detection technology of disease pathogen genes, in particular to a method and a kit for detecting HCV (Hepatitis C Virus) 1b, 2a, 3a, 3b and 6a subtype virus nucleic acids. The technical scheme adopted by the invention is as follows: an RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection kit for five subtypes of hepatitis c virus. The kit comprises an RNA (Ribose Nucleic Acid) extraction solution, a PCR reaction solution, an RT-PCR enzyme system, a positive quality control material and a negative quality control material; the PCR reaction solution comprises a reverse transcription primer (SEQ ID NO.1),two hepatitis c virus specific amplification primers (SEQ ID NO.2 and SEQ ID NO.3), and five detection probes (SEQ ID NO.4 to SEQ ID NO.8). The invention has the benefits that the kit can be used for detecting separate infection and mixed infection of the HCV 1b, 2a, 3a, 3b and 6a subtypes and has the advantages of higher detection flux, high sensitivity and specificity, low cost, simpleness in operation and short detection time; general subtypes can be distinguished; and the detection result can be automatically judged by means of analytic software.
Description
Technical field
The present invention relates to a kind of disease pathogen technique of gene detection, relate in particular to a kind of real-time fluorescence RT-PCR that adopts and detect HCV 1b, 2a, 3a, 3b and 6a subtype virus nucleic acid exist in the sample method and test kit.
Background technology
(Hepatitis Cvirus HCV) is the main pathogens that causes NANB-PTH (NANBH) to hepatitis C virus, and global infection brings great harm to human health.The HCV route of transmission comprises: blood transfusion and organ transplantation propagation, the propagation of vein dopy, iatrogenic infection and transmission through sex etc.Estimate that according to The World Health Organization (WHO) HCV infection rate in whole world on average is about 3%, promptly about 1.7-2.0 hundred million HCV the infecteds, and still have 3,000,000～4,000,000 new cases every year.The HCV infection rate has very big-difference at country variant and area.Lasting existence in view of contagium and route of transmission also can not provide vaccine safely and effectively in addition at no distant date, estimates 2015, and the total number of persons of chronic HCV infection will increase by 4 times.China HCV infection rate is also quite high; 1992-1995 whole nation viral hepatitis seroepidemiological survey result shows the positive rate average out to 3.2% of China's anti-HCV; A little more than global M.L.; Because large population base, even calculate according to total population at that time, the absolute number that HCV infects also surpasses 3,000 ten thousand.
The development of hepatitis C natural history is a very complicated biological procedures, receives influence of various factors such as virus and host, like HCV virus load, genotype and host's age, sex, race and immunosuppression etc.Have that the infection of report high-level viremia and HCV gene 1b type maybe with PD or to develop into liver cancer relevant; In addition, older's HCV infection, the male sex, excessive drinking, smoking, obesity, fatty liver and merging HBV or HIV infection also are the correlative factors of quickening or promote disease progression.Factors such as HCV height variability, pantropic, immunological tolerance, immunologic injury are to cause HCV to infect the major reason of chronicity; Lasting chronic HCV infection can show as various dissimilar diseases clinically; Great disparity, advance rate speed differ from asymptomatic carrier to weight liver inflammation and fibrosis; Have also possibly develop into hepatocellular carcinoma (Hepatocellular carcinoma, HCC).In addition, HCV infects the outer disease of many livers of often occurring together, like cryoglobulinemia, glomerulonephritis, skin mucosal lesions and autoimmune illness etc.The time of developing into chronic hepatitis, liver cirrhosis and hepatocellular carcinoma after the HCV infection is roughly about 10 years, 20 years and 30 years.According to statistics, have 60% to transfer chronic hepatitis in the hepatitis C patients approximately, wherein have 20% patient to transfer liver cirrhosis or hepatocellular carcinoma (HCC) to again.Patient's number that the annual HCV in the whole world infects is in rising trend; And the chronicity incidence that HCV infects is apparently higher than hepatitis B; Be prone to liver cirrhosis, liver cancer; Be one of important cause of death, so the diagnosis of chronic hcv infection and treatment become the public health difficult problem that the mankind need to be resolved hurrily day by day.
The HCV molecular biological characteristics:
Hepatitis C virus (HCV) is a kind of sub-thread positive chain RNA virus, because genotype is different, isolating HCV genome length is also inconsistent from various places, is generally about 9400 Nucleotide.Genome 5 ' with 3 ' end be respectively non-coding region (Untranslated Regeion, UTR), middle body be a big open reading sign indicating number framework (Open Reading Frame, ORF).Structural area is divided into core protein gene district (C district) and capsid protein gene district (E1 and E2) again; Non-structural area is divided into NS1, NS2, NS3, NS4, NS5 etc. again.The NS5 district is divided into two subprovinces again: NS5a district and NS5b district.The conservative degree of each region sequence is followed successively by from high to low: 5 ' UTR district＞C district＞NS3 district＞NS4 district, NS5 district＞NS2 district＞NS1/E2, E1＞3 ' UTR district.
HCV RNA has the variability of height, and its aberration rate is up to 1.4-1.9 * 10
-3/ Nucleotide/year.Its reason mainly contains: 1. replicative enzyme does not have correct functioning: HCV duplicates the participation that does not need reversed transcriptive enzyme; Directly at RNA RNA-dependent polymerase (RNA-dependent RNA polymerae; RDRP) duplicate under the guidance, and repair ability behind RDRP shortage 3 ' → 5 ' exonuclease correct functioning and other nucleic acid replications, random mutation can take place in virus replication; Mistake in duplicating is mixed and can't be proofreaied and correct, and finally causes the height variation of HCV; 2. easy mistake property RDRP (error-prone RDRP, existence EP-RDRP): the EP-RDRP that in the HCV reproduction process, exists also is to cause the highly major reason of variation; 3. positive selective action: because the effect of host immune selective pressure, the variation that enables effectively to activate the gene of host immune response is higher than other gene.
The HCV gene type:
HCV RNA is made up of a plurality of zones, and the sequence in 5 ' UTR district is the most conservative, and kind is that intensity of variation and evolution rate are all very low, can be used for distinguishing the oligogene type.At present according to the isolating HCV in different areas, the whole world all or part of genomic phylogenetic analysis of homophyletic not, HCV presents the hereditary variability of three levels: type (types), hypotype (subtypes) and accurate virus strain (quasispecies).HCV can be divided into 6 genotype (HCV1-6 type) at least, comprises to surpass 100 gene hypotypes (like 1a, 1b, 1c, 2a, 2b etc.).Differ 31-34% between the various nucleotide sequence, and differ about 20-23% between the hypotype sequence, differ 1-10% between the accurate virus strain sequence.Maximum difference sequences concentrates on E1 and E2 district, and for example NS3 is then much conservative relatively with some nonstructural protein genes for the C gene.The most conservative zone of sequence is 5 ' UTR district between various.There are several kinds of HCV virus strain mainly to be separated, are named as HCV7 from South East Asia, 8,9,10,11 types, except HCV10a type (being put into the HCV3 type at present), all the other are various because the similarity on its phyletic evolution is put into the HCV6 type.
The clinical meaning of HCV gene type: help to understand the HCV type and evolve, inquire into HCV route of transmission and distribution characteristics; The genotypic research of HCV provides clue for understanding various appearance and variation in evolution, also helps to understand the epidemiology situation of global hepatitis C.
HCV1b and 2a are comparatively common all over China, are positioned at the most of areas in northwest, northeast, the southeast and Central Plains, and main HCV type is genotype 1b, is genotype 2a secondly; And in Delta of the Pearl River area, 6a replaces 2a gradually becomes the second common hypotype; It is main then appearing with 1b in the southwest, and the characteristics that several genes types such as 2a, 3a, 3b, 6a distribute have significantly different with other areas, interior ground; Infect in rare HCV 4 types of China, 5 types, 1a, the 2b report that also only sporadically appears, more common polyinfection is 1b/2a.Therefore the main HCV gene of present domestic popular type is 1b, 2a, 3a, 3b and 6a.
About the meaning of HCV gene type in the hepatic diseases prediction is the topic of quite being disputed in the HCV research.But in the whole process that HCV infects, the performance of the disease of HCV has tangible biology difference in the host.Behind some population infection HCV severe complications can appear at short notice, like hepatic fibrosis and liver cancer; Even and some crowd's infection time does not for a long time have any complication.Therefore, probably there is the factor of virus or host aspect, comprises the HCV genotype, can cause the process of this disease infection.Present most researchers supports that extremely the HCV genotype is the principal element that influences the state of an illness.
Have research to be reported among the chronic HCV infection patient, compare with other genotype, HCV 1b type infects with more serious hepatic diseases, disease progression is relevant more rapidly.Discovery HCV 1b genotype such as Zein are higher than other genotype far away at the infection rate that liver cirrhosis and liver lose in the compensatory chronic active hepatitis C patients that requires liver transplantation.Evidence suggests that in the higher Japan of HCV 1b infection rate, the incidence of hepatocellular carcinoma is higher than HCV 1b the infection rate low Europe or the U.S. far away, and time of origin also more early.Though the genotype that some research has been mentioned more than having denied and the dependency of the state of an illness; But discoveries such as Ze in; In the U.S., the genotypic patient age of HCV infection 1b generally is higher than the patient who infects other types, genotypic patient infection's time of HCV 1b more early and the course of disease longer; Identical result of study also has report in France and Spain.Therefore, HCV 1b genotype is the mark of more serious HCV dependency hepatic diseases.
Since HCV comes to light; People reply (the sustained viral response to influencing the lasting virusology of Interferon, rabbit; SVR) factor has been done big quantity research; Like HCV genotype, virus load, patient's type, age and degree of hepatic fibrosis etc., wherein the HCV genotype is considered to result of treatment is had the greatest impact.A large amount of clinical trials show, it is poorer that HCV 1b and 1a genotype are replied (SVR) than HCV 2 types or 3 types to the lasting virusology of interferon therapy.Report HCV such as Zein 2 type infected patients have 60-70% to reply after 6 months at interferon therapy, and HCV 1 type the infected has only 10-15% that responsing reaction is arranged.This difference often occurs aspect replying in the persistence of interferon therapy, and does not receive the influence of HCV rna level before liver histological characteristics or the treatment.Some add discovering of ribavirin combination therapy chronic hcv patient about Interferon, rabbit: non-1 type patient HCV has than higher SVR, and 48 weeks of 1 type HCV patient just reach the effect in non-24 weeks of 1 type HCV patient; 2, the SRV of Interferon, rabbit is led is 2～3 times of 1 type HCV infected patient to 3 type HCV infected patients; Interferon, rabbit adds ribavirin, and to treat SVR that 4 type HCV infect similar or be lower than 1 type, and the SRV that treats 5 types approaches 2 and 3 types; The SVR that 6 types of treating infect acquisition is between 1 type and 2,3 infects.
In a word; Identical treat-ment there are differences the result of treatment that different genotype HCV infects really; Although not clear and definite yet at present its theoretical foundation certainly, detects the HCV genotype and will help HCV to infect clinical diagnosis and predicted treatment effect; Be adjustment dosage and treatment time, formulate individuation antiviral therapy scheme guidance is provided.
In recent years, along with the develop rapidly of molecular diagnostic techniques, RT-polymerase chain reaction (RT-PCR) technology has been widely used in the research of rna virus cdna level, possibility is provided also for the Rapid identification of mikrobe.The RT-PCR technology overcomes that traditional method is time-consuming, effort and can't satisfy the shortcoming of handling the needs of great amount of samples during the viral prevalence simultaneously, has become the important means of RNA viruses quick diagnosis.Single stage method RT-PCR carries out the RNA-cDNA-PCR reaction continuously as long as adding RNA and Auele Specific Primer can be implemented in the same reaction tubes; Need not add any reagent midway; Easy handling when handling a large amount of sample; Help to reduce residual contamination, because between cDNA synthesizes and increases, need not open the pipe lid.Single stage method can obtain higher sensitivity, minimumly can reach the total RNA of 0.1pg.
Type specificity nucleic probe real-time fluorescence PCR (Real time PCR): fluorescent PCR is on the basis of primer specific, to adopt the sequence-specific fluorescent probe of masterplate further to improve the specificity that PCR detects; Special product of every amplification only discharges the optical dye of a molecule; Instrument detecting be the result of specific amplified; Non-special product effectively improves the specificity that detects to not influence of detection signal.Utilize the principle of FRET simultaneously, make the detection sensitivity of PCR get raising greatly.Detection probes generally is Taqman or MGB probe, and amplification region adopts 5 ' conservative UTR of HCV, and detected result is consistent with the LiPA test kit of Innogenetics company.This method can be distinguished the oligogene type preferably.And the quantitative product of HCV all adopts the fluorescent PCR method basically on the home market at present, can realize seamless connection with the quantitative product of HCV so adopt the fluorescent PCR method to carry out the HCV gene type.
Summary of the invention
The purpose of this invention is to provide a kind of technique of gene detection that the highly sensitive and the technological rapid and convenient of single stage method RT-PCR of fluorescent PCR technology are combined, single stage method RT-PCR is that rt and PCR carry out in a common system.Single stage method RT-PCR is easy handling when handling a large amount of sample, because between cDNA synthesizes and increases, need not open the pipe lid, helps to reduce residual contamination, has simplified the experimental implementation step.And the cDNA of single stage method RT-PCR rt gained all is used for pcr amplification, so can obtain higher sensitivity.Based on above characteristics, single stage method RT-PCR will have significant advantage in clinical detection is used.
The present invention adopts a HCV Auele Specific Primer rt to get HCV genome part fragment, and this fragment has covered the detection site of all hypotypes to be measured, is that template is carried out pcr amplification and fluoroscopic examination with this fragment again.
Technical scheme of the present invention is the RT-PCR detection kit that five kinds of hypotypes of a kind of hepatitis C virus are provided, and this test kit comprises: the RNA extracting solution, and the PCR reaction solution, RT-PCR enzyme system, positive quality control product, negative quality control product, said PCR reaction solution comprises:
Primer L-pri-C1:5 '-ACTGCCTGATAGGGTGCTTGCGAG-3 ';
Primer L-pri-C2:5 '-AATTGCCAGGACGACCGGGTCCTT-3 ';
Primer L-pri-C3:5 '-GAAAGCGTCTAGCCATGGCGTTAGT-3 ';
Probe L-pro-C1:5 '-CCCGCTCAATGCCTGGAGATTTGGGC-3 ';
Probe L-pro-C2:5 '-GGAATTGCCGGGAAGACTGGGTCCTTTC-3 ';
Probe L-pro-C3:5 '-ACCGGAATCGCTGGGGTGACCGGGTCC-3 ';
Probe L-pro-C4:5 '-AACCCGCTCAATGCCCGGAAATTTGG-3 ';
Probe L-pro-C5:5 '-CCCGCTCAATGCCTGGAGATTTGGGCGT-3 ';
Said probe 5 ' end is used fluorochrome label, and 3 ' end is used the cancellation fluorochrome label.
Preferably, said fluorescent probe 5 ' end flag F AM or HEX, 3 ' end mark TAMARA or BHQ1.
Preferably, said RT-PCR enzyme is to comprise M-MLV reversed transcriptive enzyme, RNase suppressor factor, Taq archaeal dna polymerase.
Preferably, said PCR reaction solution also comprises pure water, RT-PCR buffer, MgCl
2, dATP, dTTP, dGTP, dCTP.
Another technical scheme of the present invention is the RT-PCR detection method of five kinds of hypotypes of a kind of hepatitis C virus, and this method comprises following concrete steps:
The RNA of a, extraction testing sample;
B, the RNA that extracts with step a are template, adopt single stage method RT-PCR method to carry out the PCR fluoroscopic examination with said primer of claim 1 and probe, judge the sample detection result according to amplification curve.
Preferably, said step a is specially
1) draw 600 μ l RNA and extract the 1.5ml centrifuge tube that 1 liquid changes cleaning over to, add 100 μ l testing samples again, the mixing that promptly vibrates left standstill 5 minutes;
2) add 500 μ l RNA and extract 2 liquid, mixing promptly vibrates;
3) the cracking mixed solution that absorption 600 μ l steps 2) obtains is transferred in the adsorption column of RNA purification column centrifugal 1 minute of 8000rpm;
4) liquid in the collection tube of RNA purification column is outwelled repeating step 3);
5) liquid in the collection tube of RNA purification column is outwelled, added 500 μ lRNA in the adsorption tube and extract 3 liquid, centrifugal 1 minute of 8000rpm;
6) liquid in the collection tube of RNA purification column is outwelled, added 500 μ lRNA in the adsorption tube and extract 4 liquid, centrifugal 1 minute of 8000rpm;
7) liquid in the collection tube of RNA purification column is outwelled, added 500 μ lRNA in the adsorption tube and extract 5 liquid, centrifugal 1 minute of 8000rpm;
8) liquid in the collection tube of RNA purification column is outwelled, adsorption tube is reinstalled in the collection tube, centrifugal 1 minute of 12000rpm abandons the collection tube of RNA purification column, and the adsorption tube of RNA purification column is put in the clean RNA collection tube;
9) extract 6 liquid toward the Dropwise 35 μ l RNA of adsorption tube central authorities, leave standstill 2 minutes after, centrifugal 2 minutes of 12000rpm collects stream and wears liquid ,-20 ℃ of preservations are subsequent use.
Beneficial effect of the present invention can detect the several genes sequence simultaneously for test kit of the present invention, and it is higher to detect flux, sensitivity, specificity height; Can distinguish common hypotype, detected result can judge that cost is low automatically by analysis software; Simple to operate; Detection time is short, is suitable for promoting the use of in common Molecular Biology Lab and clinical labororatory, has the good clinical application prospect.Once experiment can detect domestic common 5 the oligogene hypotypes (1b, 2a, 3a, 3b, 6a) of HCV simultaneously, test set is required simple.
Description of drawings
Fig. 1: negative with reference to the pcr amplification curve of article CN1 at three reaction solutions;
Fig. 2: negative with reference to the pcr amplification curve of article CN2 at three reaction solutions;
Fig. 3: negative with reference to the pcr amplification curve of article CN3 at three reaction solutions;
Fig. 4: positive with reference to the pcr amplification curve of article CP1 at three reaction solutions;
Fig. 5: positive with reference to the pcr amplification curve of article CP2 at three reaction solutions;
Fig. 6: positive with reference to the pcr amplification curve of article CP3 at three reaction solutions;
Fig. 7: positive with reference to the pcr amplification curve of article CP4 at three reaction solutions;
Fig. 8: positive with reference to the pcr amplification curve of article CP5 at three reaction solutions;
Fig. 9: positive with reference to the pcr amplification curve of article CP6 at three reaction solutions;
Figure 10: positive with reference to the pcr amplification curve of article CP7 at three reaction solutions;
Figure 11: the lowest detection amount is with reference to the pcr amplification curve of article CS1 at three reaction solutions.
Embodiment
By specifying technology contents of the present invention, structural attitude, realized purpose and effect, give explanation below in conjunction with embodiment and conjunction with figs. are detailed.
1 test kit of embodiment is formed
The present invention adopts a HCV Auele Specific Primer rt to get HCV genome part fragment, and this fragment has covered the detection site of all hypotypes to be measured, is that template is carried out pcr amplification and fluoroscopic examination with this fragment again.
Detection kit is used 1 reverse transcriptase primer: SEQ ID NO.1; Article 2, hepatitis C virus specific amplimer: SEQ ID NO.2 and SEQ ID NO.3; 5 of detection probes: SEQ IDNO.4 is to SEQ ID NO.8; Be equipped with RT-PCR Buffer, archaeal dna polymerase (Taq), reversed transcriptive enzyme, RNa se suppressor factor, dATP, dTTP, dCTP, four kinds of compositions such as mononucleotide monomer of dGTP, detect through reverse transcription reaction, PCR amplification in vitro and fluorescent signal and carry out the hepatitis C virus detection.
One, raw material sources preparation
1, primer and probe
Primer and probe are synthetic by Shanghai Ying Jun Bioisystech Co., Ltd.The synthetic back that finishes of sequence is checked by the associate, is diluted to desired concn then as required.Wherein different probe 5 ' ends is flag F AM or HEX fluorescence report group respectively, the equal mark TAMRA fluorescent quenching group of 3 ' end.Primer and probe sequence are seen table 1.
Table 1
Product functional quality control criterion article
Positive quality control product when using positive reference article CP1 to use as product, the negative quality control product when using feminine gender to use as product with reference to article CN1.
Form by three parts through determining each hypotype RT-PCR reaction solution production technique of this test kit, i.e. preparation before the preparation, preparation and packing.
Confirm that according to production technique and reaction system result of study each the hypotype RT-PCR reaction solution preparation of this test kit is single as shown in table 2.
Table 2
Symbol in the table 2 represent need not add this reagent
Get three liquid mixing bottles, multiply by preparation people umber, mix by the amount of preparing single every kind of reagent.Use the single passage pipettor that above-mentioned each hypotype RT-PCR reaction solution is managed packing by 380 μ L/.Each hypotype RT-PCR reaction solution of packing 1000 person-portions needs 3 hours approximately.
2, the RT-PCR enzyme is a production technique
Through determining this test kit RT-PCR enzyme is that production technique is made up of preparation and packing two portions, and concrete preparation singly is:
Table 3
The M-MLV reversed transcriptive enzyme
Through supplier's audit, select the product of Promega company for use, specification is 200U/ μ L; The active disappearance of RNaseH, purity has the RNA reverse transcriptase activity greater than 90%; Do not contain RNase, free nucleic acid endonuclease activity or exonuclease activity do not freeze in-20 ± 5 ℃ of preservations.
RNase suppressor factor (RNasin)
Through supplier's audit, select the product of Promega company for use, specification is 40U/ μ L; Purity is greater than 90%; It is active to suppress RNaseA, RNaseB, RNaseC and people's placenta RNase, does not influence AMV reversed transcriptive enzyme, M-MLV reversed transcriptive enzyme, Taq dna polymerase activity, does not contain RNase; Free nucleic acid endonuclease activity or exonuclease activity do not freeze in-20 ± 5 ℃ of preservations.
The Taq archaeal dna polymerase
Through supplier's audit, select the product of KaTaRa company for use.Specification is 5U/ μ L, and purity＞95% has dna polymerase activity and 5 '-3 ' DNA 5 prime excision enzyme activity, the free nucleic acid endonuclease activity, and the tool thermostability, 94 ℃ still keep 50% activity after 1 hour, do not freeze in-20 ± 5 ℃ of preservations.
dNTPs
Through supplier's audit, select the product of Promega company for use.DATP, dCTP, dGTP, dTTP are clear solution, the sign value is 100mmol/L; With dATP: dCTP: dGTP: dTTP=1: be formulated as the mixed solution of 10mmol/L at 1: 1: 1.
Get a liquid mixing bottle, multiply by preparation people umber, mix by the amount of preparing single every kind of reagent.Using the single passage pipettor is by the packing of 60 μ L/ pipe with above-mentioned RT-PCR enzyme.Packing 1000 person-portion RT-PCR enzymes system needs 1 hour approximately.
3, nucleic acid extracting reagent production technique
Form by three parts through determining this test kit nucleic acid extracting reagent production technique, i.e. preparation before the preparation, preparation and packing.Confirm this test kit nucleic acid extracting reagent preparation Dan Rubiao 4 and table 5 according to production technique and reaction system result of study.
Table 4
Table 5
| Divide loading amount (mL/ pipe) | |
| RNA extracts 1 liquid | 6 |
| |
5 |
| |
5 |
| |
5 |
| |
5 |
| |
0.35 |
Get some liquid mixing bottles,, mix by the single formulated that various extracting solutions of preparation.Use the single passage pipettor that above-mentioned various extracting solutions are shown packing according to the packing list.Packing 1000 person-portion nucleic acid extracting reagents need 2 hours approximately.
Two, this test kit production technique major control point
1, gets the raw materials ready
Detection kit is used 1 reverse transcriptase primer, 2 hepatitis C virus specific amplimers and 5 probes; Be equipped with RT-PCR Buffer, archaeal dna polymerase (Taq), reversed transcriptive enzyme, dATP, dTTP, dCTP, four kinds of compositions such as mononucleotide monomer of dGTP, detect through reverse transcription reaction, PCR amplification in vitro and fluorescent signal and carry out the hepatitis C virus detection.
Above-mentioned primer and probe are special raw material, and the raw materials consumption amount is all less, and starting material sampling, check are all carried out at the quality inspection center, and cleaning shop carries out in the workshop for cleaning, drying, the sterilization of turnover container.
2, preparation
The preparation of HCV gene typing detecting reagent kit (PCR-fluorescent probe method) comprises that preparation, RNA that preparation, RNA that preparation, RNA that the preparation of nucleic acid extracting reagent: RNA extracts 1 liquid extract 3 liquid extract 4 liquid extract the preparation that preparation, the RNA of 5 liquid extract 6 liquid; RT-PCR reagent preparation: the preparation of the preparation of the preparation of enzyme system, the preparation of HCV 1b RT-PCR reaction solution, HCV 2a/6a RT-PCR reaction solution, the preparation of HCV 3a/3b RT-PCR reaction solution, HCV somatotype positive quality control product and the preparation of the negative quality control product of HCV somatotype; Wherein negative being prepared in the II level Biohazard Safety Equipment in 10,000 level work area (LWA)s of quality control product of HCV somatotype positive quality control product and HCV somatotype carried out; Sundry item be formulated between workshop preparation in carry out.
Connect get the raw materials ready between and supplied materials between purifying, behind electronic balance weighing, prepare, the quality inspection check, salable product are delivered to each post packing of packing.
3, packing
The packing of HCV gene typing detecting reagent kit (PCR-fluorescent probe method) comprises the packing of nucleic acid extracting reagent: packing, RNA that packing, the RNA that packing, the RNA that packing, the RNA that packing, the RNA that the packing of RNA purification column, RNA extract 1 liquid extracts 2 liquid extracts 3 liquid extracts 4 liquid extracts 5 liquid extract the packing of 6 liquid, the packing of RNA collection tube, the packing of eight PCR pipes (pipe arrangement lid); RT-PCR reagent packing: the packing of the packing of the packing of RT-PCR enzyme system, the packing of HCV 1b RT-PCR reaction solution, the packing of HCV 2a/6a RT-PCR reaction solution, the packing of HCV 3a/3b RT-PCR reaction solution, HCV somatotype positive quality control product and the negative quality control product of HCV somatotype.Wherein negative being divided in the II level Biohazard Safety Equipment in 10,000 level work area (LWA)s of quality control product of HCV somatotype positive quality control product and HCV somatotype carried out; Other project be divided between workshop preparation in carry out.
Connect get the raw materials ready between and supplied materials between preparation, carry out packing by manual work, salable product are delivered to outer packaging post, workshop assembling.
4, packing
Connect the work in-process of sending here between packing, manual work by the following packing box that is assembled into precooling, is assembled into detection kit with the work in-process that are up to the standards on wrapping table.
Test kit comprises nucleic acid extracting reagent and RT-PCR reagent two portions; Every box specification is 10 person-portions; Wherein nucleic acid extracting reagent contains RNA purification column, RNA collection tube (RNase free), each 1 bag of eight PCR pipes (pipe arrangement lid), and RNA extracts 1 liquid, RNA and extracts 2 liquid, RNA and extract 3 liquid, RNA and extract that 4 liquid, RNA extract respectively one bottle of 5 liquid, RNA extracts 6 liquid, one pipe; RT-PCR reagent contains each 1 pipe of the negative quality control product of RT-PCR enzyme system, HCV 1b RT-PCR reaction solution, HCV 2a/6a RT-PCR reaction solution, HCV 3a/3b RT-PCR reaction solution, HCV somatotype positive quality control product and HCV somatotype.
After the assembling of this test kit, nucleic acid extracting reagent 2-8 ℃ preservation, RT-PCR reagent is stored in-20 ± 5 ℃, should avoid multigelation.Validity period is 6 months.The sampling censorship can be put in storage after the assay was approved after the finished product assembling was accomplished.
5, personnel purify
The operator of 100,000 grades of clean areas need after changing footwear, unrobing, wear clean garment, handwashing disinfection, to get into the operational zone; The staff of 10,000 grades of clean areas needs through changing footwear, wearing the aseptic working suit of disjunctor and get into clean operational zone again.
Three, the preparation method of standard substance (calibration object), quality control product
Enterprise work is with reference to the preparation method of article and the situation of tracing to the source
Work is with reference to the composition of article: positive with reference to article (7 parts, CP1-CP7), feminine gender reference article (3 parts, CN1-CN3), the lowest detection amount with reference to article (1 part, CS1), precision with reference to article (1 part, CP1).
1, positive in article
Collecting the clinical detection result is the serum or the plasma sample of the deactivation of hepatitis c virus-positive; HCV gene typing detecting reagent kit (PCR-fluorescent probe method) with our company carries out the fluorescent PCR detection; Therefrom choose the sample of 1b list hypotype, 2a list hypotype, the single hypotype of 3a, the single hypotype of 3b, the single hypotype of 6a, 1b&2a mixing hypotype, seven kinds of common types of 1b&6a mixing hypotype, the PCR product of these samples is checked order by Shanghai Ying Jun Bioisystech Co., Ltd with primer L-pri-C1&C2 or L-pri-C1&C3.The sequence that records is through website ht tp: //www.ncbi.nlm.nih.gov/BLAST carries out sequence relatively; Clinical sample serum or blood plasma that comparative result is consistent with the fluorescent PCR detected result; Adopt " hepatitis C virus nucleic acid detection by quantitative test kit (PCR-fluorescent probe method) " (No. the 3400677th, state's food medicine prison tool (standard) word 2009; Section China biotechnology limited-liability company) it is carried out detection by quantitative, choosing detected result concentration is 5 * 10
4IU/ml～5 * 10
6The serum of IU/mL or plasma sample.The serum or the blood plasma of homologous genes hypotype are mixed, by the packing of 1000 μ L/ pipe, 7 parts in 1 cover, numbering CP1-CP7 ,-20 ± 5 ℃ of preservations.
With enterprise positive with reference to article as sample to be checked; HCV gene typing detecting reagent kit (PCR-fluorescent probe method) with our company; Detect according to the test kit specification sheets, the somatotype result should be consistent with sequencing result, and promptly CP1-CP7 is the corresponding type positive.
2, negative with reference to article
Collect the serum or the plasma sample of deactivation; Adopt " hepatitis C virus nucleic acid detection by quantitative test kit (PCR-fluorescent probe method) " (No. the 3400677th, state's food medicine prison tool (standard) word 2009; Section China biotechnology limited-liability company) (state's food medicine is supervised tool (standard) word 2008 No. 3400932 with " hbv nucleic acid detection by quantitative test kit (PCR-fluorescent probe method) "; Section China biotechnology limited-liability company) it is carried out detection by quantitative, select the sample that hepatitis C virus is negative and hepatitis B virus is negative, mix; By the packing of 1000 μ L/ pipe, numbering CN1.-20 ± 5 ℃ of preservations.
Collect the serum or the plasma sample of deactivation; Adopt " hepatitis C virus nucleic acid detection by quantitative test kit (PCR-fluorescent probe method) " (No. the 3400677th, state's food medicine prison tool (standard) word 2009; Section China biotechnology limited-liability company) (state's food medicine is supervised tool (standard) word 2008 No. 3400932 with " hbv nucleic acid detection by quantitative test kit (PCR-fluorescent probe method) "; Section China biotechnology limited-liability company) it is carried out detection by quantitative, select the negative and hepatitis B virus male sample of hepatitis C virus, mix; By the packing of 1000 μ L/ pipe, numbering CN2-CN3.-20 ± 5 ℃ of preservations.
As sample to be checked, the HCV gene typing detecting reagent kit (PCR-fluorescent probe method) with our company detects according to the test kit specification sheets with enterprise's negative reference article, and it is all negative that detected result should be CN1-CN3.
3, lower limit of detectability is with reference to article
Collecting the clinical detection result be the serum or the plasma sample of the deactivation of hepatitis c virus-positive, carry out fluorescent PCR with the HCV gene typing detecting reagent kit (PCR-fluorescent probe method) of our company and detect, and warp checks order and turns out to be HCV 1b type; Filter out HCV 1b gene hypotype positive sample; Adopt " hepatitis C virus nucleic acid detection by quantitative test kit (PCR-fluorescent probe method) " (No. the 3400677th, state's food medicine prison tool (standard) word 2009 again; Section China biotechnology limited-liability company) virus concentration of lower limit of detectability with reference to the article mother liquor carried out quantitatively, get 10 quantitative MVs greater than 1.0 * 10
7The sample of IU/mL; Adopt the negative serum or the blood plasma of deactivation then, with quantitative values greater than 1.0 * 10
7The diluted sample to 1.0 of IU/mL * 10
7IU/mL, the sample mix after the dilution is even, and by the packing of 1000 μ L/ pipe, 1 cover, 1 pipe is numbered CS1.-20 ± 5 ℃ of preservations.
With lower limit of detectability with reference to article as sample to be checked, the HCV gene typing detecting reagent kit (PCR-fluorescent probe method) with our company detects according to the test kit specification sheets, it is positive that the result should be the single hypotype of 1b.
4, precision is with reference to article
With reference to article, by the packing of 1000 μ L/ pipe, 1 overlaps 1 pipe to the positive reference of employing article CP1, is numbered CP1 behind the mixing as precision.-20 ± 5 ℃ of preservations.
Detect as sample to be checked with reference to article CP1 with 1 part of precision, the HCV gene typing detecting reagent kit (PCR-fluorescent probe method) with our company detects according to the test kit specification sheets, repeats 10 times.10 detected results all should be that the single hypotype of HCV 1b is positive.
5, HCV somatotype positive quality control product
Adopt positive reference article CP1 as HCV somatotype positive quality control product, manage packing ,-20 ± 5 ℃ of preservations by 100 μ L/ behind the mixing.
,, detect by the test kit specification sheets with the qualified HCV gene typing detecting reagent kit of quality inspection (PCR-fluorescent probe method) as sample to be checked with HCV somatotype positive quality control product, it is positive that detected result should be the single hypotype of HCV 1b.
6, the negative quality control product of HCV somatotype
Adopt negative reference article CN1 as the negative quality control product of HCV somatotype, press the packing of 100 μ L/ pipe behind the mixing ,-20 ± 5 ℃ of preservations.
With the negative quality control product of HCV somatotype is sample to be checked, with the qualified HCV gene typing detecting reagent kit of quality inspection (PCR-fluorescent probe method), detects by the test kit specification sheets, and detected result should be negative.
Four, test kit staple of the present invention
These article are chosen HCV gene 5 '-UTR zone; Design specific primers and fluorescent probe; Adopt PCR-fluorescent probe method, HCV 1b, 2a, 3a, 3b and 6a subtype virus nucleic acid in the test sample can detect HCV 1b, 2a, 3a, 3b and 6a hypotype and infect separately and polyinfection.
1, main moity of this test kit such as table 6.Explain: 1, the component of different lot numbers can be exchanged use in the test kit.2, HCV somatotype positive quality control product derives from positive the infected's of HCV 1b of clinical collection plasma sample, suitably dilutes the liquid of back gained with saline water; The negative quality control product of HCV somatotype derives from the negative plasma sample of HCV of clinical collection.Above quality control product all passes through inactivation treatment.
Table 6
2, condition of storage:
Test kit I (including RT-PCR reagent and quality control product) is stored in-20 ± 5 ℃, should avoid multigelation.Test kit II (including nucleic acid extracting reagent) is stored in 2-8 ℃.The validity period of this product under the above corresponding condition of storage is 6 months.
3, be suitable for instrument
Comprise instruments such as ABI series such as Agilent Stratagene Mx3000P, ABI Prism 7500,7300,7000.
Five, sample preparation
This test kit sample source is serum or blood plasma.
1, serum pref: extract person under inspection's venous blood 2mL with disposable sterilized injector; Inject aseptic dry glass tube; Room temperature was placed 30-60 minute, but was no more than 4 hours, but the blood specimen self-solidifying is separated out serum; Or the centrifugal 5-10 of 1500rpm minute, the absorption upper serum also is transferred in the aseptic centrifuge tube;
2, blood plasma preparation: extract person under inspection's venous blood 2mL with disposable sterilized injector; Injection contains the Glass tubing of EDTA Disodium (EDTA) or sodium citrate anticoagulant; Put upside down Glass tubing immediately gently and make venous blood and the abundant mixing of antithrombotics; Room temperature was placed 5-10 minute, and the centrifugal 5-10 of 1500rpm minute, the absorption upper plasma also was transferred in the aseptic centrifuge tube.
3, serum for preparing or blood plasma room temperature are placed and are no more than 2 hours, and 4 ℃ of preservations are no more than 48 hours, and-20 ℃ of preservations are no more than half a year, should avoid multigelation.Need during transportation to add the ice bag sealing with 0 ℃ of curling stone or bubble chamber, the time limit in transit should not be above 48 hours.
The use of 2 test kits of embodiment
One, HCV RNA extracts
The parallel RNA of positive, negative quality control product and clinical sample extracts
Annotate: RNA extracts 1 liquid cryopreservation crystalline deposit possibly occur, and 37 ℃ are incubated to deposition and dissolve fully and can use.
1, draw 600 μ l RNA and extract 1 liquid and change clean 1.5ml centrifuge tube over to, add 100 μ l clinical samples (or positive quality control product, or negative quality control product) again, the mixing that promptly vibrates, room temperature left standstill 5 minutes.
2, add 500 μ l RNA and extract 2 liquid, mixing promptly vibrates.
3, draw 600 μ l cracking mixed solutions and be transferred in the adsorption column of RNA purification column centrifugal 1 minute of room temperature, 8000rpm.
4, the liquid in the collection tube of RNA purification column is outwelled repeating step (3).
5, the liquid in the collection tube of RNA purification column is outwelled, added 500 μ lRNA in the adsorption tube and extract 3 liquid, centrifugal 1 minute of room temperature, 8000rpm.
6, the liquid in the collection tube of RNA purification column is outwelled, added 500 μ lRNA in the adsorption tube and extract 4 liquid, centrifugal 1 minute of room temperature, 8000rpm.
7, the liquid in the collection tube of RNA purification column is outwelled, added 500 μ lRNA in the adsorption tube and extract 5 liquid, centrifugal 1 minute of room temperature, 8000rpm.
8, the liquid in the collection tube of RNA purification column is outwelled, adsorption tube is reinstalled in the collection tube.Centrifugal 1 minute of room temperature, 12000rpm abandon the collection tube of RNA purification column, and the adsorption tube of RNA purification column is put in the clean RNA collection tube.
9, extract 6 liquid toward the Dropwise 35 μ l RNA of adsorption tube central authorities, after room temperature leaves standstill 2 minutes, centrifugal 2 minutes of 12000rpm room temperature.Collect stream and wear liquid ,-20 ℃ of preservations are subsequent use.
Two, RT-PCR amplification
Get each hypotype HCV RT-PCR reaction solution; Get three centrifuge tubes, adding each hypotype HCV RT-PCR reaction solution 38 * (n+2) μ l and enzyme in each centrifuge tube respectively is 2 * (n+2) μ l, (n=examined samples number); Mixing, each pipe install in the PCR reaction tubes by 40ul/ pipe branch respectively.The RNA that adds sample or quality control product again in each hypotype HCV RT-PCR reaction solution respectively extracts each 10 μ l of product.
Flick the tube wall mixing, should avoid bubble to produce, 6000rpm instantaneous centrifugal after, put into the fluorescent PCR appearance, press tabulation 7 said procedure conditions amplifications.
Table 7
Resorcinolphthalein is provided with: all detect Kong Jun and are set to FAM-TAMRA and HEX-TAMRA (no HEX can be set to JOE);
Fluorescent signal is collected: Stage4:60 ℃---45sec;
Reaction volume: 50ul.
Interpretation of result: after reaction finishes; (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 2～3, the End value can be located at 5～10; The amplification curve of adjustment negative control is straight or be lower than threshold line), click Analysis and obtain analytical results automatically, watch the result at the Report interface)
Quality control:
Negative quality control product: each reaction tubes does not all have typical S type amplification curve or Ct value＞25.1;
Positive quality control product: FAM mark curve is typical S type amplification curve and Ct value≤25.1 in the HCV 1b RT-PCR reaction tubes; Remaining reaction Guan Zhongjun does not have typical S type amplification curve or Ct value＞25.1;
Need more than to require in once testing, satisfying simultaneously, otherwise this experiment is invalid, need carry out again.
Three, the judgement of test-results
Do not have typical S type amplification curve or Ct value＞25.1 if detect sample, then declaring sample is that HCV1b, 2a, 3a, 3b and 6a are negative.
Be typical S type amplification curve and Ct value≤25.1 if detect sample, then according to the hypotype of table 8 judgement sample.
Table 8
Reference value (term of reference)
According to the test-results of clinical sample, behind statistical analysis, the Cutoff value of confirming this test kit is 25.1.
The explanation of assay
When detecting sample, if the FAM resorcinolphthalein is typical S type amplification curve and Ct value≤25.1 in the HCV 1b RT-PCR reaction tubes, possible situation is that HCV 1b hypotype infects or MOI; If the FAM resorcinolphthalein is typical S type amplification curve and Ct value≤25.1 in the HCV 2a/6a RT-PCR reaction tubes, possible situation is that HCV 2a hypotype infects or MOI; If the HEX resorcinolphthalein is typical S type amplification curve and Ct value≤25.1 in the HCV 2a/6a RT-PCR reaction tubes, possible situation is that HCV 6a hypotype infects or MOI; If the FAM resorcinolphthalein is typical S type amplification curve and Ct value≤25.1 in the HCV 3a/3b RT-PCR reaction tubes, possible situation is that HCV 3a hypotype infects or MOI; If the HEX resorcinolphthalein is typical S type amplification curve and Ct value≤25.1 in the HCV 3a/3b RT-PCR reaction tubes, possible situation is that HCV 3b hypotype infects or MOI;
Product performance index
1, lower limit of detectability: this test kit is 1.0 * 10 to the lower limit of detectability of sample
3IU/ml.
2, cross reaction: test kit is for the HCV negatives, and detected result is negative, no cross reaction.
Four, advantage and effect:
The technological method that like product is ratified the listing situation at home and abroad and adopted:
In SFDA DB " medicine registration approval in-formation ", be keyword with " HCV "; Retrieve 3 information (see table 9) relevant with the hepatitis C virus nucleic acid detection reagent; Be the qualitative or detection by quantitative reagent of HCV; The technological method that is adopted is all fluorescent PCR, does not have the HCV gene type reagent of approval listing.
Table 9
Through consulting document and related web site, find abroad have following several kinds of approveds listing HCV gene parting detecting reagent (seeing table 9):
1) INNO-LiPA HCV II: be to carry out the test kit that the HCV somatotype detects by the reverse linear hybridizing method that utilizes that Innogenetics company (Belgium) develops; Amplification 5 ' UTR district; Can detect HCV 1-6 hypotypes such as totally 6 kinds of different gene types and 1a, 1b, 2a/c, 2b, 3a-c, 4a-h, 5a, 6a, through the CE authentication.
2) Versant HCV Genotype Assay (LiPA) 2.0: the new edition HCV gene type reagent by French Bayer associating Innogenetics company of company (Belgium) releases, passed through the CE authentication.Principle is linear probe hybridization equally, but more accurate to the differentiation of 1 type and 6 types and 1a and 1b, because except that 5 ' UTR, has increased C region sequence information.
3) Abbott HCV ASR: the product of U.S. Abbott company.Target region is 5 ' UTR and NS5B, adopts polychrome three pipe Real-time RT-PCR technology, can detect HCV oligogene type 1-6, can also distinguish 1a and 1b.Through the FDA authentication.
4) Trugene 5 ' NC Genotyping Kit:Bayer Company products; It is commercial HCV sequencing reagent; It has special test kit, sequencing system and analysis software; Surveyed area is at 5 ' UTR, and distinguishing oligogene type and LiPA has good consistence, but can not accurately detect hypotype.The HCV gene type test kit of external approval listing is seen table 10.
Table 10
Application for registration product and the comparison of like product abroad
From the method that detects principle, detect the HCV gene typing detecting reagent kit (PCR-fluorescent probe method) of our company being applied for the registration of several aspects such as gene type, reagent expense with obtained card abroad and used the comparison that more general test kit carries out, see table 11.
Table 11
Can find out that from last table in four kinds of test kits, it is basic identical that application for registration product and Abbott HCVASR detect principle, also be the method that adopts real-time fluorescence PCR to detect.Aspect the detection type; Abbott HCV ASR test kit can detect the 1-6 type simultaneously, and the type that the test kit of our company's research and development detects is 1b, 2a, 3a, 3b and 6a type, is less than Abbott HCV ASR test kit; But according to bibliographical information and the test of our clinical examination; The oligogene hypotype of domestic HCV virus is 1b, 2a, 3a, 3b and 6a type, does not find 4,5 types as yet.Thereby the design of test kit can be satisfied domestic HCV genotype detection demand.In addition, Abbott HCV ASR detection reagent price is high, is not easy to carry out at home.
INNO-LiPA HCV II (VERSANT HCV Genotype 2.0 Assay) and Trugene 5 ' NC Genotyping reagent are for application for registration reagent; Detecting instrument or reagent cost an arm and a leg; Be inappropriate for and clinically generally promote the use of; And exist not highly to some oligogene type detection specificity, can not accurately distinguish hypotype, to shortcomings such as the detectivity of polyinfection are low.
The application registers 1 test of reagent and in 3 pipe PCR, detects the several genes sequence simultaneously, and it is higher to detect flux; Sensitivity, specificity height can be distinguished common hypotype, and detected result can be judged by analysis software automatically; Cost is low, and is simple to operate, and detection time is short, is suitable for promoting the use of in common Molecular Biology Lab and clinical labororatory, has the good clinical application prospect.
Quality control system is reliable and stable; Product stability can be good, and detected result is reliably accurate, has high clinical value.Once experiment can detect domestic common 5 the oligogene hypotypes (1b, 2a, 3a, 3b, 6a) of HCV simultaneously; Test set is required simple.
This test kit is at the performance study that has mainly carried out comprising the following aspects aspect the performance obtained in laboratory evaluation: lower limit of detectability, accuracy, specificity, precision, repeatability and polyinfection etc.
See also Fig. 1 to Figure 11:
The negative reference of Fig. 1-3 article CN1-CN3 is the pcr amplification curve in HCV 1b RT-PCR reaction solution, 2a/6aRT-PCR reaction solution and 3a/3b RT-PCR reaction solution respectively;
The positive reference of Fig. 4-10 article CP1-CP7 is the pcr amplification curve in HCV 1b RT-PCR reaction solution, 2a/6aRT-PCR reaction solution and 3a/3b RT-PCR reaction solution respectively;
Figure 11 is that the lowest detection amount is with reference to the pcr amplification curve of article CS1 in HCV 1b RT-PCR reaction solution, 2a/6aRT-PCR reaction solution and 3a/3b RT-PCR reaction solution.
Analyze the lower limit of detectability experimental result and show that it is 1 * 10 that this test kit can stably detect Cmin
3IU/m L HCV serum sample;
The precision of analysis experimental result shows that for dissimilar positive sample, this test kit detected result all fits like a glove with sequencing result;
Analyze the specificity experimental result and show that the clinical negatives of HCV do not disturb the detected result of this test kit, do not have the non-specific amplification reaction;
The precision result of study show this test kit criticize in batch between high conformity, withinrun precision and betweenrun precision be equal≤10%.
Repetitive research result is illustrated under the various experiment conditions and can repeated multiple times is stable detects five kinds of clinical common HCV gene hypotypes, and repeatability is 100%.
The polyinfection test-results shows that this test kit mixes the hypotype infection to domestic common HCV 1b&2a and the infection of HCV 1b&6a mixing hypotype all can detect exactly.
The above is merely embodiments of the invention; Be not so limit claim of the present invention; Every equivalent structure or equivalent flow process conversion that utilizes specification sheets of the present invention and accompanying drawing content to be done; Or directly or indirectly be used in other relevant technical fields, all in like manner be included in the scope of patent protection of the present invention.
Claims (6)
1. the RT-PCR detection kit of five kinds of hypotypes of hepatitis C virus is characterized in that, comprising: the RNA extracting solution, and the PCR reaction solution, RT-PCR enzyme system, positive quality control product, negative quality control product, said PCR reaction solution comprises:
Primer L-pri-C1:5 '-ACTGCCTGATAGGGTGCTTGCGAG-3 ';
Primer L-pri-C2:5 '-AATTGCCAGGACGACCGGGTCCTT-3 ';
Primer L-pri-C3:5 '-GAAAGCGTCTAGCCATGGCGTTAGT-3 ';
Probe L-pro-C1:5 '-CCCGCTCAATGCCTGGAGATTTGGGC-3 ';
Probe L-pro-C2:5 '-GGAATTGCCGGGAAGACTGGGTCCTTTC-3 ';
Probe L-pro-C3:5 '-ACCGGAATCGCTGGGGTGACCGGGTCC-3 ';
Probe L-pro-C4:5 '-AACCCGCTCAATGCCCGGAAATTTGG-3 ';
Probe L-pro-C5:5 '-CCCGCTCAATGCCTGGAGATTTGGGCGT-3 ';
Said probe 5 ' end is used fluorochrome label, and 3 ' end is used the cancellation fluorochrome label.
2. the RT-PCR detection kit of five kinds of hypotypes of hepatitis C virus according to claim 1 is characterized in that, said fluorescent probe 5 ' end flag F AM or HEX, 3 ' end mark TAMARA or BHQ1.
3. the RT-PCR detection kit of five kinds of hypotypes of hepatitis C virus according to claim 1 is characterized in that, said RT-PCR enzyme is to comprise M-MLV reversed transcriptive enzyme, RNase suppressor factor, Taq archaeal dna polymerase.
4. the RT-PCR detection kit of five kinds of hypotypes of hepatitis C virus according to claim 1 is characterized in that said PCR reaction solution also comprises pure water, RT-PCR buffer, MgCl
2, dATP, dTTP, dGTP, dCTP.
5. the RT-PCR detection method of five kinds of hypotypes of hepatitis C virus is characterized in that, comprises following concrete steps:
The RNA of a, extraction testing sample;
B, the RNA that extracts with step a are template, adopt single stage method RT-PCR method to carry out the PCR fluoroscopic examination with said primer of claim 1 and probe, judge the sample detection result according to amplification curve.
6. the RT-PCR detection method of five kinds of hypotypes of hepatitis C virus according to claim 5 is characterized in that said step a is specially:
1) draw 600 μ l RNA and extract the 1.5ml centrifuge tube that 1 liquid changes cleaning over to, add 100 μ l testing samples again, the mixing that promptly vibrates left standstill 5 minutes;
2) add 500 μ l RNA and extract 2 liquid, mixing promptly vibrates;
3) the cracking mixed solution that absorption 600 μ l steps 2) obtains is transferred in the adsorption column of RNA purification column centrifugal 1 minute of 8000rpm;
4) liquid in the collection tube of RNA purification column is outwelled repeating step 3);
5) liquid in the collection tube of RNA purification column is outwelled, added 500 μ lRNA in the adsorption tube and extract 3 liquid, centrifugal 1 minute of 8000rpm;
6) liquid in the collection tube of RNA purification column is outwelled, added 500 μ lRNA in the adsorption tube and extract 4 liquid, centrifugal 1 minute of 8000rpm;
7) liquid in the collection tube of RNA purification column is outwelled, added 500 μ lRNA in the adsorption tube and extract 5 liquid, centrifugal 1 minute of 8000rpm;
8) liquid in the collection tube of RNA purification column is outwelled, adsorption tube is reinstalled in the collection tube, centrifugal 1 minute of 12000rpm abandons the collection tube of RNA purification column, and the adsorption tube of RNA purification column is put in the clean RNA collection tube;
9) extract 6 liquid toward the Dropwise 35 μ l RNA of adsorption tube central authorities, leave standstill 2 minutes after, centrifugal 2 minutes of 12000rpm collects stream and wears liquid ,-20 ℃ of preservations are subsequent use.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103773897A (en) * | 2014-01-16 | 2014-05-07 | 江苏硕世生物科技有限公司 | Multiplex fluorescence PCR detection kit for hepatitis C virus nucleic acid detection and genotyping and application thereof |
| CN109593887A (en) * | 2018-12-29 | 2019-04-09 | 中山大学达安基因股份有限公司 | A kind of kit of hepatitis C virus nucleic acid quantitative detection |
| CN111893216A (en) * | 2020-08-11 | 2020-11-06 | 北京毅新博创生物科技有限公司 | Product for detecting DNA/RNA by nucleic acid mass spectrum and detection method |
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| CN101250592A (en) * | 2007-11-22 | 2008-08-27 | 扬子江药业集团北京海燕药业有限公司 | One-step fluorescence quantitative RT-PCR detecting method for hepatitis C virus(HCV) and reagent case thereof |
| CN101921871A (en) * | 2010-01-27 | 2010-12-22 | 南京迪安医学检测中心有限公司 | Hepatitis C sequencing and typing kit and detection method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101250592A (en) * | 2007-11-22 | 2008-08-27 | 扬子江药业集团北京海燕药业有限公司 | One-step fluorescence quantitative RT-PCR detecting method for hepatitis C virus(HCV) and reagent case thereof |
| CN101921871A (en) * | 2010-01-27 | 2010-12-22 | 南京迪安医学检测中心有限公司 | Hepatitis C sequencing and typing kit and detection method thereof |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103773897A (en) * | 2014-01-16 | 2014-05-07 | 江苏硕世生物科技有限公司 | Multiplex fluorescence PCR detection kit for hepatitis C virus nucleic acid detection and genotyping and application thereof |
| CN103773897B (en) * | 2014-01-16 | 2015-07-01 | 江苏硕世生物科技有限公司 | Multiplex fluorescence PCR detection kit for hepatitis C virus nucleic acid detection and genotyping and application thereof |
| CN109593887A (en) * | 2018-12-29 | 2019-04-09 | 中山大学达安基因股份有限公司 | A kind of kit of hepatitis C virus nucleic acid quantitative detection |
| CN111893216A (en) * | 2020-08-11 | 2020-11-06 | 北京毅新博创生物科技有限公司 | Product for detecting DNA/RNA by nucleic acid mass spectrum and detection method |
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