CN101538619A - Kit for detecting RNA of hepatitis E virus - Google Patents
Kit for detecting RNA of hepatitis E virus Download PDFInfo
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Abstract
The invention relates to a kit for detecting RNA of a hepatitis E virus (HEV). The kit can carry out qualitative detection to an HEV RNA in blood serum sample and a fecal sample of man and animals by adopting fluorescent PCR technology, wherein the HEV nucleic acid is separated by adopting extracted nucleic acid in a silicone gel membrane method, and the detection of the HEV RNA is realized by adopting the fluorescent PCR technology.
Description
Technical field:
The present invention relates to test kit and detection method that a kind of nucleic acid (RNA) to hepatitis E virus (HEV) range gene type carries out external qualitative detection, can assist diagnosis, treatment and the animal HEV diagnosis of infection of human clinical hepatitis E.
Background technology
Hepatitis E (HE is called for short viral hepatitis type E) is similar to hepatitis A, is a kind of through intestinal transmitted self-limited disease.Up to 1980, the hepatitis A antibody detection reagent of special sensitivity is applied to the water source eruption and prevalence hepatitis that India takes place, people recognize that just a kind of and HAV have similar epidemiologic feature and clinical manifestation but be different from the virus of HAV, are called as through intestinal transmitted non A non B hepatitis virus.Nineteen eighty-three, USSR (Union of Soviet Socialist Republics) Balayan observes the non A non B hepatitis virus particle first under Electronic Speculum, and its infection is caused typical hepatitis symptom to a volunteer and macaque body.Subsequently, other scientists adopt molecular biological technology successfully to clone this virus, prove that it is the virus that is different from other hepatitis, are named as hepatitis E virus (HEV).HEV virus is mainly propagated by fecal oral route, has popular and distributes two kinds of forms.
HEV is that a kind of diameter is the no coating sub-thread positive chain RNA virus of 27nm ~ 34nm.Have at Nucleotide and amino acid on the basis of high homology regional distribution widely and certain characteristics such as genetic heterogeneity are arranged.The about 7.5kb of HEV genome total length comprises 3 ' poly (A) tail of growing 150 ~ 200 adenylic acid (AMP)s.With the strain of I type representative strains Burma is example, its 5 ' end contains the non-coding region (NCR) of 27 Nucleotide (nt), 3 ' end contains the non-coding region of 65 ~ 74 Nucleotide, between 5 ' and 3 ' non-coding region, contain three open reading frames (ORF), be followed successively by ORF1, ORF2 and ORF3, ORF1 (nt28 ~ 5107) Nonstructural Protein of encoding mainly comprises the needed enzyme of some virus replications; ORF2 (nt5147 ~ 7126) capsid protein of encoding is the structural protein of virus, can induce body to produce protective immunological reaction; ORF3 (nt5106 ~ 5474) function it be unclear that, can be by the epitope of patients serum's antibody recognition but contain.
Viral hepatitis type E mainly occurs in third world countries, and existing as Afghanistan, Mexico, China, Egypt, India, Mongolia, Burma, Thailand, Tunisia, the Sudan, Uzbekistan etc. reported the outburst of viral hepatitis type E in 29 countries and regions and confirmed through serology.The viral hepatitis type E eruption and prevalence of document reported first betides nineteen fifty-five India New Delhi.The viral hepatitis type E eruption and prevalence of maximum-norm has taken place in 1986-1988 China Xinjiang, it is reported that having 119,280 routine viral hepatitis type E patients during this period occurs.
Enzyme linked immune assay (EIA) be detect at present anti--HEV the most frequently used make things convenient for method.Generally speaking, the Case definition of HE is to detect the anti-HEV IgM of specificity antibody in patient's acute phase serum by ELISA, or the variation more than 4 times from low to high of anti-HEV IgG antibody.
Antigen used when being used among the EIA detecting anti-HEV IgG antibody and IgM is the generally acknowledged HEV structural area ORF2 and the recombinant protein or the synthetic peptide of ORF3 immunogenicity epi-position.Present used most of recombinant protein derives from a C-terminal or the part of ORF2, and total length or the C-terminal of ORF3.At present, have two kinds of EIAs comparatively extensive in whole world range of application: a kind of Genelabs-EIA of being, the antigen that it uses derive from ORF2 and 4 terminal short sequence recombinant proteins of ORF33 ' of the strain of prototype Burma and Mexico's strain; A kind of is Abbott-EIA, and the antigen that it uses is ORF2 partial sequence and two the total length expressed recombinant proteins of ORF3 that come from the strain of prototype Burma.
But under following factors: (1) blood sampling the time is in the window phase that HEV infects, anti-HEV as yet sun change (2) even morbidity early stage anti--HEV IgM positive rate only is 60% ~ 80%
[54](3) patient is to the HEV no response.The reaction that is negative of these situations all can cause hepatitis e virus infection person's serologic marker thing, thus the situation of failing to pinpoint a disease in diagnosis might be caused.Because used antigen difference also can cause the inconsistent of detected result.Discover, when the antigen of expressing with the HEV strain of different genotype and hypotype carries out the serology detection to HE patient, can show different susceptibility.Promptly use the strain of homologous genes type and hypotype, adopt different expression systems, different gene locuss, its antigenicity of resulting antigen also can be different.So also have to a certain degree uncertainty with the infection of ELISA method diagnosis hepatitis E virus.
Polymerase chain reaction (Polymerase chain reaction, PCR) technology is since 1989 begin to be applied to clinical diagnosis, advantages such as, sensitivity quick, easy with it become a hot technology of clinical experiment diagnostics very soon, at present, the diagnosis and the treatment monitoring of clinical disease have been widely used in.At first used in the macaque bile that RT-PCR method test experience infects since the HEV RNA widespread use in the research of viral hepatitis type E of RT-PCR method from nineteen ninety Reyes etc.Available RT-PCR detects HEV in acute phase serum or ight soil, and this method also can detect the water source of pollution and the HEV in other pollutents
[57]Consider in acute attack later stage HEV viremia and excrement and can weaken gradually, so PCR as far as possible early does.
Chaukan in 1993 etc. use the RT-PCR method and detect HEV RNA among the hepatitis E patients serum.Jameel in 1992 etc. use RT-nPCR and detect HEV RNA in the hepatitis E patient ight soil.Because this method adopts two pairs of primers, carry out the twice PCR amplification, be equivalent to 4 specific probes and carry out several times liquid phase molecular hybridization, therefore, sensitivity and the specific degree of RT-nPCR detection HEV RNA are all higher than RT-PCR.China has successively set up RT-PCR and RT-nPCR method respectively at 1991 with nineteen ninety-five.With the macaque bile of 2 experimental infections of RT-PCR method detection, the HEV RNA in serum and the ight soil.These two macaques are all positive in infecting back the 7th day HEV RNA in the bile, and wherein a macaque is the of short duration positive, turn out cloudy in back 12 days in infecting; Another macaque lasting masculin, HEV RNA is still positive in infecting the 14th day bile in back.Serum HEV RNA promptly change at the unusual preceding sun of ALT, but the time length is shorter, is about for 1～2 week in infecting the back 7 ~ 20 days.Generally before anti-HEV sun changes or sun change disappearance simultaneously.HEV RNA almost occurs simultaneously with viremia in the ight soil, but the time length is long than the viremia time, is about for 2～3 weeks, resists-HEV sun commentaries on classics 1～2 week of back in serum, still can detect HEV RNA in the ight soil.Use ORF1 RT-nPCR and detect 41 fens sporadic hepatitis E patients' acute phase serum (average course of disease is 10.1 days) and the serum of macaque of 11 parts of experimental infection HEV, the positive rate of HEV RNA is respectively 68.3% and 81.8% as a result.To 15 parts of serum of ORF1 RT-nPCR male (wherein 1 part is serum of macaque), carry out ORF2 RT-nPCR again and detect, HEV RNA is also positive.
The primer of HEV genome different zones is used for the report that PCR all has success, and the primer that generally is used for the Mexico strain is unsuitable for the Asia strain, has the HEV RNA detection of conservative ORF23 ' primer 6227nt ~ 6603nt applicable to source, different region only.
Detect the indication that HEV RNA is acute HIV infection in the clinical sample, yet do not find the possibility that HEV does not get rid of acute HIV infection, whether the PCR positive must be noted that because due to the experimental pollution.Because PCR is a kind of highstrung detection method, so in that to be used for clinical diagnosis very careful.
The real-time fluorescence PCR technology was released by U.S. Applied Biosystems company in 1996.Compare with conventional PCR, it is stronger that it has specificity, can effectively solve PCR and easily pollute and avoid problems such as general pcr amplification time length, complicated operation, level of automation height, can carry out characteristics such as accurately quantitative to starting template simultaneously, be used widely clinically at present.Detect kind and comprise communicable disease such as hepatitis B virus, hepatitis C virus, human immunodeficiency virus, venereal disease pathogenic agent etc.; Genetic diseases diagnosis such as thalassemia etc.; The detection of transgenic plant etc.
The real-time fluorescence quantitative PCR reaction is outside a pair of primer of conventional PCR, adds two ends and has fluorescently-labeled oligonucleotide probe.Under the intact state of probe, the exciting light of 5 ' end report fluorophor is suppressed by the cancellation fluorophor of 3 ' end.In the PCR reaction process, extension along with chain, the Taq enzyme moves to the binding site of fluorescence labeling probe along dna profiling, bring into play its 5 ' → 3 ' exonuclease activity, fluorescent probe is cut off, discharge the fluorescent signal of report fluorophor, whenever synthetic template, the signal of a reporter group discharges, and d/d number and the PCR product that swashs from the report fluorophor is man-to-man relation.By regularly each circulation of dynamic monitoring of quantitative PCR instrument, can obtain the actual amplification curve of sample, find the logarithmic phase of pcr amplification.Software compares by the logarithmic phase of the product to be tested of standard substance, obtains the initial content of each sample template DNA.
The characteristics that fluorescent PCR detects mainly contain: (1) has the dual specificity of primer and probe, and CR compares with conventional P, and specificity greatly improves.(2) susceptibility reaches 10 usually
2IU/ml, and linearity range is very wide, is 0-10
11IU/ml.The number of pathogenic agent is 0-10 in the clinical samples in general
10IU/ml, sample need not dilute.(3) FQ-FCR quite stable as a result, because thresholding is arranged on the index amplification phase, this moment, each reactive component concentration was relatively stable, the logarithm of CT value and fluorescent signal is linear.And after PCR reaction enters plateau, the exhausting of each component of reaction system, the reduction of enzymic activity and the reasons such as feedback inhibition of product cause product no longer to increase, and reach plateau.Comparing the CT value with end-point method can be more stable, reflects the content of starting template more accurately.
Fluorescent PCR detects employed fluorescence chemical method and mainly contains: SYBR Green I, Taqman probe, molecular beacon, double cross probe, Scorpion primers etc.What be used for that HEV detects mainly contains SYBR GreenI and two kinds of methods of Taqman probe.
Germano Orr ù etc. sets up a kind of high sensitivity, and (the single stage method RT-PCR method of 10cDNA molecule/PCR) detects faecal samples.This method utilizes SYBR Green I to carry out quantitatively.Experiment shows because the concentration of HEV in serum and ight soil is often lower, and the hepatitis E patient just began toxin expelling in 7 ~ 16 days before clinical symptom occurring, so select this sensitivity, timesaving nucleic acid quantification method highly beneficial to clinical diagnosis.But the investigator does not have the detection case of the different genotype of present HEV existence is done report, and the primer that they use in SYBRGreen RT-PCR test is weak point (15～16mer), its Tm value is lower, may increase the possibility of the non-purpose segment amplification of potential.
Detect the HEV nucleic acid relevant report of existing several examples abroad with the real-time fluorescence RT-PCR method.Utilizations such as Jothikumar detect the HEV sample based on the real time fluorescent quantitative method of TaqMan probe.
We at first set up and have developed the fluorescence PCR detecting method at HEV nucleic acid at home in 2007, and the pattern detection that is used for humans and animals of success.And through continuous follow-up optimization production go out can commercial kit for detecting RNA of hepatitis E virus (fluorescent PCR method).
Hepatitis E virus has been the arch-criminal who causes the intestinal transmitted non-A non-B hepatitis in the torrid zone, subtropics now, continuous discovery along with the animal host, HEV is also increasing to the threat that public health causes, therefore, detection method is the important step of hepatitis E prevention, control and treatment with setting up efficiently to improve reliable diagnostic reagent.
Summary of the invention:
The invention provides a kind of adopt the fluorescent PCR technology can carry out the qualitative detection test kit to the HEVRNA in humans and animals serum and the fecal sample, wherein adopt silica gel embrane method nucleic acid extracting reagent to separate hepatitis E virus nucleic acid, and the employing fluorescence PCR detection reagent is realized the detection to RNA of hepatitis E virus.
The present invention adopts pellosil specific adsorption principle to realize separating of HEV nucleic acid in serum and the ight soil.Cardinal principle and operating process comprise several sections: 1 nucleic acid dispose procedure: the effect by lysate, settling agent and dehydrated alcohol realizes that nucleic acid separates and the concentrated of nucleic acid separated out with proteinic; Wherein the similar reagent with other of this test kit is compared, and has increased the process of nucleic acid settling agent, has improved the separating power of product to trace dna.2. nucleic acid adsorption process: adopt the pellosil that nucleic acid (RNA) is had a specific adsorption effect to adsorb cleaved spissated nucleic acid molecule; 3. nucleic acid purification process: use washing lotion A and washing lotion B that foreign protein and inorganic ion are had a cleaning function that the nucleic acid (RNA) that is incorporated on the pellosil is carried out purifying respectively.4. nucleic acid elution process: use elutriant with the nucleic acid (RNA) of purifying wash-out from the film.The template that the nucleic acid molecule that wash-out obtains can directly be tested as follow-up foranalysis of nucleic acids.
The present invention uses the fluorescent PCR method to detect hepatitis E virus nucleic acid.The fluorescent PCR method adopts the fluorescent PCR technology based on the Taqman fluorescent probe, be meant and in the PCR reaction system, add HEV specificity fluorescent probe, fluorescent probe is taken turns in the specific pcr amplification process at each and is degraded by the Taq polysaccharase, and discharges fluorescent signal.Test kit can utilize the fluorescent probe integration of signals whole PCR process of monitoring in real time on the fluorescent PCR analyser, and unknown template is analyzed.The employed fluorophor of this product is the specific Taqman fluorescent probe of HEV.
The principle of work of TaqMan fluorescent probe is: add a specific fluorescent probe during pcr amplification when adding a pair of primer, this probe is an oligonucleotide, and two ends are mark report fluorophor and a cancellation fluorophor respectively.When probe was complete, the reporter group fluorescent signal emitted was absorbed by quenching group; During pcr amplification, 5 '-3 ' 5 prime excision enzyme activity of Taq enzyme is cut degraded with the probe enzyme, the report fluorophor is separated with the cancellation fluorophor, thereby the fluorescence monitoring system can receive fluorescent signal, it is DNA chain of every amplification, just there is a fluorescence molecule to form, realized that the accumulation of fluorescent signal and PCR product form fully synchronously.
Employed HEV PCR Auele Specific Primer and fluorescent probe are the different HEV genotype designs at humans and animals among the present invention, and selected pcr amplification position is positioned at the common conserved regions of all known humans and animals HEV genotype and strain at present.Can detect four genotype of all known HEV.
According to above principle, the invention provides a kind of test kit and detection method of different genotype hepatitis E virus nucleic acid (HEV RNA) being carried out external qualitative detection.
The invention provides a kind of kit for detecting RNA of hepatitis E virus at different genotype, described test kit is made up of separate nucleic acid reagent and augmentation detection reagent, wherein,
Consisting of of separate nucleic acid reagent:
Nucleic acid adsorption column: pellosil adsorption column;
Settling agent: material with precipitate nucleic acids; As: Acryl Carrier, glycogen (glycogen), yeast tRNA etc.;
Washing lotion A: guanidinium isothiocyanate liquid and Tris/HCI liquid;
Elutriant: Tris/HCI damping fluid;
Sleeve pipe: nuclease free sleeve pipe;
Lysate: guanidinium isothiocyanate liquid, Triton X-100 liquid, Tris/HCI liquid;
Washing lotion B:Tris/HCI damping fluid;
Consisting of of augmentation detection reagent:
HEV reaction solution: form by HEV specific PCR primer, HEV specificity T aqman fluorescent probe, 1 * PCR damping fluid, four kinds of deoxyribonucleotide mixtures and depc water mixed preparing;
Reversed transcriptive enzyme: have the active reversed transcriptive enzyme of RNA reverse transcription; As: AMV reversed transcriptive enzyme, M-MLV reversed transcriptive enzyme etc.;
Archaeal dna polymerase: have the active polysaccharase of dna replication dna; As: Taq polysaccharase, HotStart Taq polysaccharase, pfu archaeal dna polymerase etc.;
HEV negative control: new-born calf serum solution, perhaps negative serum;
The critical positive control of HEV: contain the new-born calf serum of HEV artificial pseudovirus or the matrix of other no HEV RNA; As: negative serum, depc water, PBS damping fluid etc.;
HEV strong positive contrast: contain the new-born calf serum of HEV artificial pseudovirus or the matrix of other no HEV RNA; As: negative serum, depc water, PBS damping fluid etc.;
Preferred test kit of the present invention, wherein,
1. separate nucleic acid reagent:
The nucleic acid adsorption column: 2.0ml nuclease free sleeve pipe includes a dismountable pellosil adsorption column.
Settling agent: the Acryl polymer, perhaps other settling agents such as glycogen etc. have the material of precipitate nucleic acids.
Washing lotion A:5M guanidinium isothiocyanate, 10mM Tris/HCI.
Elutriant: 10mM Tris/HCI damping fluid.
Sleeve pipe: be 2.0ml nuclease free sleeve pipe.
Lysate: 5M guanidinium isothiocyanate, 10%Triton X-100,10mM Tris/HCI.
Washing lotion B:10mM Tris/HCI damping fluid.
2. augmentation detection reagent
HEV reaction solution: by HEV specific PCR primer (0.10-0.20mmol/L), HEV specificity T aqman fluorescent probe (0.10-0.20mmol/L), 1 * PCR damping fluid (100mmol/LTris-HCl, pH9.2,500mmol/L KCl, 10mmol/L MgCl2), four kinds of deoxyribonucleotide mixtures (each 0.10-0.20mmol/L of dATP/dTTP/dGTP/dCTP) and depc water mixed preparing form.
Reversed transcriptive enzyme: the AMV reversed transcriptive enzyme, or other have the active reversed transcriptive enzyme of RNA reverse transcription.
Archaeal dna polymerase: the Taq polysaccharase, or other have the active polysaccharase of dna replication dna.
The HEV negative control: new-born calf serum solution, perhaps negative serum.
The critical positive control of HEV: contain the new-born calf serum of HEV artificial pseudovirus, perhaps the matrix of negative serum, other no HEVRNA.
HEV strong positive contrast: contain the new-born calf serum of HEV artificial pseudovirus, perhaps the matrix of negative serum, other no HEV RNA.
[packing specifications] 48 person-portions/box
[purposes] this product is used for the RNA of hepatitis E virus of the different genotype of humans and animals serum and fecal sample is carried out external qualitative detection, the diagnosis and the treatment of auxiliary clinical hepatitis E.
[inspection principle] this product detects principle according to fluorescent PCR, adopts HEV specificity T aqman probe that the ORF3 gene conserved regions of hepatitis E virus different genotype is carried out specific recognition, thereby realizes the qualitative detection to RNA of hepatitis E virus.
[condition of storage and validity period] separate nucleic acid reagent is in 2-8 ℃ of preservation; Augmentation detection reagent should be avoided multigelation in-20 ℃ of preservations.
[being suitable for instrument] full-automatic fluorescent PCR amplification instrument.
Fresh serum, plasma sample and the fecal sample of [sample requirement] humans and animals.
Preferred test kit of the present invention, reagent concentration is wherein formed, and packing and reaction conditions are preferably, and effect is better.
The main production process of test kit of the present invention:
PCR primer and fluorescent probe
Use multiple molecular biology software design,, entrusts domestic professional DNA Synesis Company to prepare the pure or HPLC purifying of PAGE through the checking of repeatedly primer probe shaker test.
Upstream primer: 5`CGGTGGTTTCTGGGGTGA 3`
Downstream primer: 5`GCGAAGGGGTTGGTTGGA 3`
Taqman fluorescent probe: 5`TGATTC TCA GCC CTT CGC 3`
Taq polysaccharase and AMV reversed transcriptive enzyme
Taq polysaccharase and AMV reversed transcriptive enzyme are outsourcing.
Be used to preserve the wrapping material that primer, probe, reaction solution and other reagent place use
Use low absorption, no RNase and the residual disposable laboratory consumptive material of DNase, the reaction tubes that carries out the fluorescent PCR amplified reaction is selected the special-purpose consumptive material of the full-automatic quantitative fluorescent PCR analyser of different model respectively for use.
Separate nucleic acid reagent
Optimize repeatedly through a large amount of tests, adopt the separate nucleic acid reagent of pellosil absorption method principle, main raw material(s) is imported raw material.
The HEV artificial pseudovirus
The HEV artificial pseudovirus entrusts specialized laboratory to make up, and is that the external source produced in fragments forms with HEV I hypotype ORF3 gene conserved regions.
Other chemical feedstockss: all from homemade or import reagent, for analytical pure or top grade pure.Molecular biosciences class reagent is the molecular biosciences special agent of nuclease free.
Below illustrate application of the present invention by the detection of introducing hepatitis E virus (HEV) RNA sample.
Get the clinical viral hepatitis type E serum sample of 100ul, extract HEV RNA sample according to this product description method.Experimental technique is seen sample pre-treatment and the nucleic acid extraction step in [method of inspection].
The preparation of separate nucleic acid reagent: with RL, RW1, RW2 and the components such as adsorption column, sleeve pipe in the viral nucleic acid extraction test kit (QIAamp Viral RNAMini Kit) of QIAGEN company, use settling agent that other suppliers provide and elutriant etc. simultaneously, above component is assembled into the separate nucleic acid test kit by production processes such as packing, labeling, assemblings.
Should use operation instructions after the optimization that this product provides to obtain the best organic efficiency to micro-viral RNA when clinical serum and fecal sample are handled operation, the operating process of product is:
5 μ l settling agents, 200 μ l lysates and 100 μ l serum samples are mixed, concuss 2 minutes, room temperature left standstill 10 minutes.Add 240 μ l dehydrated alcohols, put upside down mixing, cleavage mixture is drawn in the adsorption column.Centrifugal 2 minutes of 3500g takes off sleeve pipe, outwells the liquid in the sleeve pipe, and centrifugal 1 minute of 6000g outwells the liquid in the sleeve pipe, adds 500 μ l washing lotion A, and centrifugal 1 minute of 6000g changes a new casing.Add 500 μ l washing lotion B, left standstill 1 minute, centrifugal 1 minute of 6000g outwells the liquid in the sleeve pipe, adds 500 μ l washing lotion B, and centrifugal 1 minute of 6000g changes a new casing, centrifugal 3 minutes of 13000rpm.With the adsorption column nuclease free 1.5ml centrifuge tube of packing into, carefully add 50 μ l preheating elutriants in adsorption film central authorities, room temperature left standstill 4 minutes, centrifugal 2 minutes of 10000rpm, liquid is purifying RNA to be checked in the centrifuge tube.
The preparation of HEV specific PCR primer and fluorescent probe: the synthetic of HEV specific PCR primer and fluorescent probe synthesized by the precious biotech firm of living worker in Shanghai or Dalian respectively, and the Taqman fluorescent probe requires 5` end mark reporter group (FAM fluorescein or other luminous fluorescent elements), 3` to hold mark quenching group (TAMRA, Dabcyl or other have the group of cancellation effect); Require the HPLC purifying.The TE (pH8.0) that synthetic primer that obtains or probe lyophilized powder use depc water or go nuclease to handle is diluted to 40mmol/L concentration.HEV reaction solution preparation: with HEV specific PCR upstream and downstream primer (0.10-0.20mmol/L), HEV specificity T aqman fluorescent probe (0.10-0.20mmol/L), 1 * PCR damping fluid (100mmol/LTris-HCl, pH9.2,500mmol/L KCl, 10mmol/L MgCl2), it is formulated that four kinds of deoxyribonucleotide mixtures (each 0.10-0.20mmol/L of dATP/dTTP/dGTP/dCTP) and depc water mix the back.
The detection of the reaction system of HEV RNA:
Successively with 20 μ l HEV reaction solutions, 3U Taq polysaccharase, the 2U reversed transcriptive enzyme, and 10 μ l purifying RNA to be checked softly mixes.Carry out the fluorescent PCR amplified reaction by following condition: 50 ℃ 30 minutes, 95 ℃ 3 minutes; 95 ℃ 15 seconds, 50 ℃ 30 seconds, 72 ℃ 1 minute, totally 5 circulations; 95 ℃ 10 seconds, 55 ℃ 40 seconds, totally 40 circulations are collected the FAM fluorescent signals at 55 ℃.After reaction finished, if can detect amplification curve, promptly illustrating had HEV RNA to exist in the sample.
RNA of hepatitis E virus detection method (FQ-PCR method) is according to the polymerase chain reaction principle, and employing Taqman fluorescent probe technique realizes the detection to HEV nucleic acid.This product can effectively detect the nucleic acid of human and animal's different genotype hepatitis E virus, and its detectivity is better than shell type RT-PCR laboratory detection technology commonly used at present.The development of this reagent provides a kind of possibility for the early diagnosis of hepatitis E.Immunology detection technology to routine in clinical HEV diagnosis has stronger complementary action.
Determining of test kit linearity range and sensitivity
1.HEV the preparation of artificial pseudovirus:
The HEV artificial pseudovirus is to adopt genetic engineering technique expressed proteins-RNA complex body, and it is by being wrapped to form the protein coat of target RNA sequence with phage, and its similar is in natural viral.Have following characteristics: 1) stable, not by the RNA enzyme liberating; 2) lifeless matter safety problem does not have the danger of infection; 3) natural structure of simulated virus can carry out true Quality Control to cracking, the sepn process of virus; 4) can prepare in a large number.Used the HEV artificial pseudovirus in this test kit, its target RNA is a HEV virus O RF3 conserved regions.This test kit is with the index of HEV artificial pseudovirus as this product positive reference substance component and assessment test kit performance.
2 linearity range and sensitivity
Use negative serum that the HEV artificial pseudovirus is carried out 10 times of serial gradient dilutions, use this test kit to detect.The result shows that this test kit can detect 1: 1 * 10
8The HEV artificial pseudovirus of dilution doubly, sensing range is crossed over 8 dilution orders of magnitude, the dependency R of formed typical curve
2=0.9968.The wherein minimum HEV artificial pseudovirus that detects 50 copy/ml.
The test kit reference substance
1. strong positive reference substance and critical positive reference substance
This product is provided with 1 strong positive contrast and 1 critical positive control, so that product situation in operation is carried out Quality Control.
2. negative control product
This product is provided with 1 negative control, to prevent to occur in operation pollution condition.
Product uses the assessment of instrument
Different full-automatic fluorescent PCR instruments mainly concentrate on two aspects, the i.e. difference of the difference of PCR thermal cycle conditions and reaction system to the influence that product detects.
The PCR reaction conditions that this product adopted is a relative standardization and open working conditions, use this condition can guarantee that all instrument normally finishes pcr amplification circulation and fluorescent signal collection process for various fluorescent PCR instrument, (ABI7000, ABI7300, RG3000, rich day Linegene series, SLAN etc.) test on the fluorescent PCR instrument of various models, and the result does not find differences.For Luo Shi Lightcycler series product, descend for avoiding the Taq polymerase activity in addition, therefore its denaturation temperature is adjusted into 93 ℃ by 95 ℃ when using these series product, and all the other conditions are constant.
For realizing the versatility of reagent on different fluorescent PCR analytical instrument, this product adopts the pcr amplification reaction system of 30ul.The HEV reaction solution of 30ul volume of preparation is packed into and all can be kept suitable liquid height in the PCR optical tube on Lightcycler series, RG3000, SLAN, ABI7000, the rich day full-automatic fluorescent PCR amplification instrument such as Linegene series, satisfies the requirement of collecting fluorescent signal fully.
The advantage of test kit of the present invention
1. test kit specificity
This test kit detects HAV, HBV, HCV and HIV viral nucleic acid positive sample, and is all negative, and showing does not have non-specific amplification.Adopt the product (20061101 batches) of trial production, to detecting from 309 routine clinical samples (containing 135 routine chronic hepatitis patients, 50 routine patient with liver cirrhosis, 124 routine medical examiners and general out patient service patient), specificity reaches 100%.The specificity that shows test kit has reached design requirements.
2. test kit sensing range
This test kit PCR primer and probe are for to design at all hypotype ORF3 conservative fragments, this test kit in product design and product system are optimized repeatedly the HEV serum of end user's different subtype sample and pig and fecal sample as primer, probe screening and optimization and template, by using the product (20060814 batches) of laboratory lab scale, the product of the product of follow-up trial production (20061101) and three batches of trial productions, serum and fecal sample to humans and animals detect, the result shows that reagent can effectively detect at human and animal's HEV viral nucleic acid.
3. the clinical use of product
Use the import MP HEV IgM of company antibody test reagent reagent in contrast, 725 routine clinical serum samples are carried out HEV RNA detect, the result shows that the coincidence rate of this product and the HEV IgM of MP company antibody test reagent is 81.8%.
Use this product that the acute hepatitis serum sample of 249 routine IgM antibody positives is detected, the positive rate of this product is 70.6%, far above 30 ~ 60% HEV RNA positive rate of reported in literature.
Use this product to detect the HEV RNA positive to 12 examples, and other detect the trace detection that all negative patient of index carries out continuous 4 weeks, as a result 1-2 after week other indexs detect the positive successively as (HEV IgM and IgG antibody), show that this product is the early diagnosis reagent that a kind of HEV infects, and has shortened the window phase of clinical viral hepatitis type E greatly.
In the at present clinical HEV diagnosis, still needleless uses this product that 94 routine acute viral hepatitis type E patients' fecal sample is detected to the detection means of viral hepatitis type E patient fecal sample, and confirms that the 53 routine HEV positives are arranged, and shows that patient is in the ight soil toxin expelling phase.Therefore this product has been filled up the technology and the product blank in this field.
Embodiment:
Further specify the present invention by the following examples, but not as limitation of the present invention.
The embodiment 1 test kit method of inspection
1. sample pre-treatment
Serum and plasma sample prepare according to a conventional method, and fresh sample detects immediately, or in-70 ℃ frozen, avoid multigelation.
Fecal sample: get 1.5 ~ 2.0 gram ight soil and place the 50ml centrifuge tube, add 30ml PBS solution (containing 2%BSA), shook 15 minutes, during left standstill 2-3 minute, centrifugal 30 minutes of 3000rpm gets that suspension detects or frozen in-20 ℃.
2. nucleic acid extraction: (please in strict accordance with requiring operation)
Attention: a) press the label indication at washing lotion A and washing lotion B bottle adding dehydrated alcohol, fully mixing with preceding.B) with elutriant in 70 ℃ of preheatings.C) reagent reference substance and freezing sample melt with preceding room temperature, the concussion mixing.D) the centrifugal setting (rpm and g) in the differentiation operation, it is centrifugal that this test is room temperature.E) settling agent low temperature may be gluey, and the piping and druming mixing can use.
1) in the 1.5ml centrifuge tube, successively adds 5 μ l settling agents and 200 μ l lysates, adding 100 μ l serum samples (is provided with negative control, critical positive control and strong positive and contrasts each 1, operate identical with sample), concuss 2 minutes, room temperature left standstill 10 minutes.
2) add 240 μ l dehydrated alcohols, put upside down mixing, cleavage mixture is drawn in the adsorption column.
3) 3500g is centrifugal 2 minutes, takes off sleeve pipe, outwells the liquid in the sleeve pipe, and adsorption column is reloaded sleeve pipe.
4) 6000g is centrifugal 1 minute, outwells the liquid in the sleeve pipe, and adsorption column is reloaded sleeve pipe.
5) add 500 μ l washing lotion A, centrifugal 1 minute of 6000g changes a new casing with adsorption column.
6) add 500 μ l washing lotion B, left standstill 1 minute, centrifugal 1 minute of 6000g outwells the liquid in the sleeve pipe, and adsorption column is reloaded sleeve pipe.
7) add 500 μ l washing lotion B, centrifugal 1 minute of 6000g changes a new casing with adsorption column, centrifugal 3 minutes of 13000rpm.
8) adsorption column is packed into a nuclease free 1.5ml centrifuge tube carefully adds 50 μ l preheating elutriants in adsorption film central authorities, and room temperature left standstill 4 minutes, centrifugal 2 minutes of 10000rpm, and liquid is purifying RNA to be checked in the centrifuge tube.
3. fluorescent PCR amplification:
Please take out the HEV reaction solution in advance and melt, use behind the mixing that slightly vibrates in room temperature.
1) prepares: in the 1.5ml centrifuge tube, successively add 20 μ l * n HEV reaction solution, 0.6 μ l * n Taq archaeal dna polymerase, 0.2 μ l * n reversed transcriptive enzyme (n is a reaction tubes number to be amplified), concussion mixing, the centrifugal liquid that makes is poly-to managing at the end, divides with 20 μ l/ pipe to be filled to fluorescent PCR amplification pipe.
2) application of sample: in fluorescent PCR amplification pipe, add 10 μ l purifying RNA to be checked respectively, the tight pipe lid of lid.2000rpm made liquid poly-to managing the end in centrifugal 20 seconds.The pipe that will increase is put into fluorescent PCR amplification instrument, carries out the fluorescent PCR amplified reaction.
3) fluorescent PCR amplified reaction:
The fluorescent PCR amplification program is: reverse transcription and sex change: 50 ℃ 30 minutes, 95 ℃ 3 minutes; Pre-amplification: 95 ℃ 15 seconds, 50 ℃ 30 seconds, 72 ℃ 1 minute, totally 5 circulations.Amplification and phosphor collection: 95 ℃ 10 seconds, 55 ℃ 40 seconds, totally 40 circulations are collected the FAM fluorescent signals at 55 ℃.(for Lightcycler fluorescent PCR amplification instrument, change denaturation temperature into 93 ℃ by 95 ℃, all the other conditions are constant.)
[reference range]
Each test should be provided with strong positive contrast, critical positive control and negative control, and should meet following requirement, otherwise test-results is false.
1. negative control does not have the Ct value or the Ct value is 0.
2. strong positive contrast Ct value is less than 23, and critical positive control Ct value is less than 30.
[explanation of assay]
1. the fluorescein of fluorescent probe mark is 5`FAM and 3`TAMRA in this product, should select F1/F2 (FAM/TAMRA) or F1 (FAM) passage analytical test result.
2. the baseline scope requires to set according to instrument, and purpose is the correcting background fluorescence interference.The threshold line height does not have Ct value or 0 with negative control, and strong positive contrast Ct value is less than 23, and critical positive control Ct value is as the criterion less than 30.
3.Ct value reports that less than 32.0 o'clock this test HEV RNA detects positive; The Ct value equals 0 or to report that this test HEV RNA detects when not having the Ct value negative, shows that the sample rna level is lower than sensing range; The Ct value should if still positive report is positive suspicious, need to judge in conjunction with other indexs as suspicious sample duplicate detection greater than 32.0 o'clock.
[product performance index]
1. this product can effectively detect the RNA of people source and each hypotype HEV virus of pig source.
2. this product is fit to the HEV virus in human or animal's ight soil is detected.
3. the artificial constructed negative serum dilution that contains the pseudovirus gradient of HEV gene fragment is detected, this product can detect the HEV artificial pseudovirus of 50 copy/ml.
This product has been finished the clinical study test of product, in the development status on-site verification by State Food and Drug Administration's tissue in 2007, and obtain the external diagnosis reagent manufacturing enterprise quality management system examination return on qualification that State Food and Drug Administration issues at this product.Three batches of products of quantity-produced in 2008 obtain the registration qualification test report of Nat'l Pharmaceutical ﹠ Biological Products Control Institute.
The preparation method of embodiment 2 test kits (please provide detailed preparation and wrapping process)
The preparation of test kit component:
The nucleic acid adsorption column: outsourcing, the qualified back of quality inspection is as the test kit component.
Sleeve pipe: be 2.0ml nuclease free sleeve pipe.
Settling agent: outsourcing, the qualified back of quality inspection is as the test kit component.
The preparation of lysate: get guanidinium isothiocyanate 590.8 grams respectively, 100ml Triton X-100 joins among the 10mMTris/HCI (pH8.0), is settled to 1000 milliliters after the dissolving.Autoclaving, the qualified back packing of quality inspection is as the test kit component.
The preparation of washing lotion A: get guanidinium isothiocyanate 590.8 grams respectively, join among the 10mM Tris/HCI (pH8.0), be settled to 1000 milliliters after the dissolving.Autoclaving, the qualified back packing of quality inspection is as the test kit component.
The preparation of washing lotion B: take by weighing 1.57 gram Tris.base and be dissolved in the depc water, regulate pH value to 7.0, be settled to 1000 milliliters with dense HCI, autoclaving, the qualified back packing of quality inspection is as the test kit component.
The preparation of elutriant: take by weighing 1.57 gram Tris.base and be dissolved in the depc water, regulate pH value to 8.0, be settled to 1000 milliliters with dense HCI, autoclaving, the qualified back packing of quality inspection is as the test kit component.
The preparation of HEV reaction solution:
In the container of a nuclease free, successively add 300 μ l, 10 * PCR damping fluid, 120 μ l MgCl2,21 μ l 25mM dNTPs, 2 μ l upstream primer stock solutions, 4 μ l downstream primer stock solutions, 4 μ l fluorescent probe stock solutions, add depc water to 1920 μ l, the lucifuge mixing is in 2-8 ℃ of preservation.The qualified back packing of quality inspection is as the test kit component.
The AMV reversed transcriptive enzyme, outsourcing, the qualified back packing of quality inspection is as the test kit component.
The Taq archaeal dna polymerase: outsourcing, the qualified back packing of quality inspection is as the test kit component.
HEV negative control: new-born calf serum solution.
The critical positive control of HEV: the HEV artificial pseudovirus is diluted with new-born calf serum with 1: 1000000 times, as the test kit component.
HEV strong positive contrast: the HEV artificial pseudovirus is diluted with new-born calf serum with 1: 10000 times, as the test kit component.
The packing of test kit component
This product comprises two packing boxes altogether, be respectively separate nucleic acid test kit and detection kit, wherein contain 7 kinds of components in the separate nucleic acid test kit altogether, comprise 1 bottle of nucleic acid adsorption column 1 bag (48), sleeve pipe (96), settling agent 1 pipe, 1 bottle of lysate, washing lotion A1 bottle, washing lotion B1 bottle and elutriant respectively.
Contain 6 kinds of components in the detection kit altogether, comprise HEV reaction solution 1 pipe, AMV reversed transcriptive enzyme 1 pipe, TaqDNA polysaccharase 1 pipe, HEV negative control 1 pipe, critical positive control 1 pipe of HEV and HEV strong positive contrast 1 pipe respectively.
The calibrating of finished product test kit:
This product need adopt enterprise's quality control product to examine and determine by the factory inspection of finished product test kit, and use qualified back.
1. physical examination: outward appearance is neat, and component is complete, the liquid components ne-leakage.
2. specificity Quality Control product coincidence rate: false positive must not appear in 7 parts of specificity Quality Control product.
3. positive quality control product coincidence rate: false negative must not appear in 6 parts of positive quality control product.
4. limit of identification: 4 parts of sensitivity quality control products must detect 3 parts.
5. accuracy: accuracy Quality Control product are repeated to examine and determine 5 times, calculate the variation coefficient of Ct value, CV (%)＜10%.
Claims (10)
1. the kit for detecting RNA of hepatitis E virus at different genotype is characterized in that, described test kit is made up of separate nucleic acid reagent and augmentation detection reagent, wherein,
Consisting of of separate nucleic acid reagent:
Nucleic acid adsorption column: pellosil adsorption column;
Settling agent: material with precipitate nucleic acids;
Washing lotion A: guanidinium isothiocyanate liquid and Tris/HCI liquid;
Elutriant: Tris/HCI damping fluid;
Sleeve pipe: nuclease free sleeve pipe;
Lysate: guanidinium isothiocyanate liquid, Triton X-100 liquid, Tris/HCI liquid;
Washing lotion B:Tris/HCI damping fluid;
Consisting of of augmentation detection reagent:
HEV reaction solution: form by HEV specific PCR primer, HEV specificity T aqman fluorescent probe, 1 * PCR damping fluid, four kinds of deoxyribonucleotide mixtures and depc water mixed preparing;
Reversed transcriptive enzyme: have the active reversed transcriptive enzyme of RNA reverse transcription;
Archaeal dna polymerase: have the active polysaccharase of dna replication dna;
HEV negative control: new-born calf serum solution, perhaps negative serum;
The critical positive control of HEV: the matrix that contains new-born calf serum, negative serum or other no HEVRNA of HEV artificial pseudovirus;
HEV strong positive contrast: the matrix that contains new-born calf serum, negative serum or other no HEV RNA of HEV artificial pseudovirus.
2, the detection kit of claim 1 is characterized in that, wherein,
Consisting of of separate nucleic acid reagent:
The nucleic acid adsorption column: 2.0ml nuclease free sleeve pipe includes a dismountable pellosil adsorption column;
Settling agent: Acryl polymer;
Washing lotion A:5M guanidinium isothiocyanate liquid, 10mM Tris/HCI liquid;
Elutriant: 10mM Tris/HCI damping fluid;
Sleeve pipe: be 2.0ml nuclease free sleeve pipe;
Lysate: 5M guanidinium isothiocyanate liquid, 10%Triton X-100 liquid, 10mM Tris/HCI liquid;
Washing lotion B:10mM Tris/HCI damping fluid;
Consisting of of augmentation detection reagent:
HEV reaction solution: by 0.10-0.20mmol/L HEV specific PCR primer, 0.10-0.20mmol/L HEV specificity T aqman fluorescent probe, 100mmol/L Tris-HCl, pH9.2,500mmol/L KCl, 10mmol/LMgCl21 * PCR damping fluid, four kinds of deoxyribonucleotide mixtures and the depc water mixed preparing of each 0.10-0.20mmol/L of dATP/dTTP/dGTP/dCTP form;
Reversed transcriptive enzyme: AMV reversed transcriptive enzyme;
Archaeal dna polymerase: Taq polysaccharase;
HEV negative control: new-born calf serum solution, perhaps negative serum;
The critical positive control of HEV: the matrix that contains new-born calf serum, negative serum or other no HEVRNA of HEV artificial pseudovirus;
HEV strong positive contrast: the matrix that contains new-born calf serum, negative serum or other no HEV RNA of HEV artificial pseudovirus.
3, the detection kit of claim 1 is characterized in that, is to realize detection to hepatitis E virus nucleic acid according to Taqman fluorescent probe principle.
4, the detection kit of claim 1 is characterized in that, the RNA of hepatitis E virus in detected object behaviour and animal serum, blood plasma and the ight soil.
5, the detection kit of claim 1 is characterized in that, uses strong positive contrast and the critical positive control of the HEV artificial pseudovirus of genetically engineered structure as kit for detecting RNA of hepatitis E virus, in addition as the Quality Control thing that product is carried out quality control.
6, the detection kit of claim 1 is characterized in that, uses the annealing temperature that improves to realize improving the target of reverse transcription PCR amplified reaction efficient.
7, the detection kit of claim 1 is characterized in that, adds settling agent to improve the sensitivity and the rate of recovery of separate nucleic acid in separate nucleic acid reagent.
8, the detection kit of claim 1 is characterized in that, test kit is applicable to various full-automatic fluorescent PCR amplification instrument.
9, the detection kit of claim 1 is characterized in that, test kit can be with clinical hepatitis E is carried out early diagnosis.
10, the preparation method of the detection kit of claim 1 is characterized in that,
The preparation of test kit component:
The nucleic acid adsorption column: outsourcing, quality inspection is qualified back as the test kit component,
Sleeve pipe: be 2.0ml nuclease free sleeve pipe,
Settling agent: outsourcing, quality inspection is qualified back as the test kit component,
The preparation of lysate: get guanidinium isothiocyanate 590.8 gram respectively, 100ml Triton X-100 joins among the 10mMTris/HCI (pH8.0), is settled to 1000 milliliters after the dissolving, autoclaving, and the qualified back packing of quality inspection is as the test kit component,
The preparation of washing lotion A: get guanidinium isothiocyanate 590.8 grams respectively, join among the 10mM Tris/HCI (pH8.0), be settled to 1000 milliliters after the dissolving, autoclaving, the qualified back packing of quality inspection is as the test kit component, the preparation of washing lotion B: take by weighing 1.57 gram Tris.base and be dissolved in the depc water, regulate pH value to 7.0 with dense HCI, be settled to 1000 milliliters, autoclaving, the qualified back packing of quality inspection is as the test kit component
The preparation of elutriant: take by weighing 1.57 gram Tris.base and be dissolved in the depc water, regulate pH value to 8.0, be settled to 1000 milliliters with dense HCI, autoclaving, the qualified packing afterwards of quality inspection is as the test kit component,
The preparation of HEV reaction solution:
In the container of a nuclease free, successively add 300 μ l, 10 * PCR damping fluid, 120 μ l MgCl2,21 μ l 25mM dNTPs, 2 μ l upstream primer stock solutions, 4 μ l downstream primer stock solutions, 4 μ l fluorescent probe stock solutions are added depc water to 1920 μ l, the lucifuge mixing, in 2-8 ℃ of preservation, the qualified back packing of quality inspection is as the test kit component
The AMV reversed transcriptive enzyme, outsourcing, the qualified back packing of quality inspection, as the test kit component,
The Taq archaeal dna polymerase: outsourcing, the qualified back packing of quality inspection, as the test kit component,
The HEV negative control: new-born calf serum solution,
The critical positive control of HEV: the HEV artificial pseudovirus is diluted with new-born calf serum with 1: 1000000 times, as the test kit component,
The contrast of HEV strong positive: the HEV artificial pseudovirus is diluted with new-born calf serum with 1: 10000 times, as the test kit component,
The packing of test kit component
This product comprises two packing boxes altogether, be respectively separate nucleic acid test kit and detection kit, wherein contain 7 kinds of components in the separate nucleic acid test kit altogether, comprise nucleic acid adsorption column 1 bag (48) respectively, sleeve pipe (96), settling agent 1 pipe, 1 bottle of lysate, washing lotion A1 bottle, 1 bottle of washing lotion B1 bottle and elutriant, contain 6 kinds of components in the detection kit altogether, comprise HEV reaction solution 1 pipe respectively, AMV reversed transcriptive enzyme 1 pipe, TaqDNA polysaccharase 1 pipe, HEV negative control 1 pipe, critical positive control 1 pipe of HEV and HEV strong positive contrast 1 pipe
The calibrating of finished product test kit:
This product need adopt enterprise's quality control product to examine and determine by the factory inspection of finished product test kit, and use qualified back,
1). physical examination: outward appearance is neat, and component is complete, the liquid components ne-leakage,
2). specificity Quality Control product coincidence rate: false positive must not appear in 7 parts of specificity Quality Control product,
3). the positive quality control product coincidence rate: false negative must not appear in 6 parts of positive quality control product,
4). limit of identification: 4 parts of sensitivity quality control products must detect 3 parts,
5). accuracy: accuracy Quality Control product are repeated to examine and determine 5 times, calculate the variation coefficient of Ct value, CV (%)＜10%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910136169A CN101538619A (en) | 2009-04-30 | 2009-04-30 | Kit for detecting RNA of hepatitis E virus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910136169A CN101538619A (en) | 2009-04-30 | 2009-04-30 | Kit for detecting RNA of hepatitis E virus |
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| CN101538619A true CN101538619A (en) | 2009-09-23 |
Family
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200910136169A Pending CN101538619A (en) | 2009-04-30 | 2009-04-30 | Kit for detecting RNA of hepatitis E virus |
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| CN (1) | CN101538619A (en) |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101962689A (en) * | 2010-10-28 | 2011-02-02 | 浙江出入境检验检疫局检验检疫技术中心 | Identification and detection method for hepatitis E virus by utilizing quadruple fluorescence quantitative PCR (Polymerase Chain Reaction) |
| CN102559935A (en) * | 2012-02-23 | 2012-07-11 | 天津出入境检验检疫局动植物与食品检测中心 | M-gene based fluorescent RT-PCR (Reverse Transcription Polymerase Chain Reaction) detection method of Nipah virus |
| CN102827947A (en) * | 2011-06-17 | 2012-12-19 | 东北制药集团辽宁生物医药有限公司 | Kit and detection method for rapid quantitative detection of hepatitis virus nucleic acid |
| WO2014144578A1 (en) * | 2013-03-15 | 2014-09-18 | The Usa, As Represented By The Secretary, Department Of Health And Human Services | Selective detection of hepatitis a, b, c, d, or e viruses or combinations thereof |
| CN105176969A (en) * | 2015-07-13 | 2015-12-23 | 杭州优思达生物技术有限公司 | Nucleic acid extraction method and nucleic acid extraction reagent for biological samples |
| CN107287347A (en) * | 2017-05-09 | 2017-10-24 | 广州机场出入境检验检疫局综合技术服务中心 | HEV real-time fluorescence reverse transcription PCR detection primer, probe, detection kit and detection method |
| US10047406B2 (en) | 2013-08-14 | 2018-08-14 | Gen-Probe Incorporated | Compositions and methods for detecting HEV nucleic acid |
| CN110878378A (en) * | 2019-12-02 | 2020-03-13 | 昆明理工大学 | Primer combination for detecting hepatitis E virus in body fluid and application thereof |
| CN111235315A (en) * | 2020-03-13 | 2020-06-05 | 苏州智享众创孵化管理有限公司 | Method for simultaneously detecting multiple genotypes of hepatitis E virus |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101962689A (en) * | 2010-10-28 | 2011-02-02 | 浙江出入境检验检疫局检验检疫技术中心 | Identification and detection method for hepatitis E virus by utilizing quadruple fluorescence quantitative PCR (Polymerase Chain Reaction) |
| CN101962689B (en) * | 2010-10-28 | 2013-07-03 | 浙江出入境检验检疫局检验检疫技术中心 | Identification and detection method for hepatitis E virus by utilizing quadruple fluorescence quantitative PCR (Polymerase Chain Reaction) |
| CN102827947A (en) * | 2011-06-17 | 2012-12-19 | 东北制药集团辽宁生物医药有限公司 | Kit and detection method for rapid quantitative detection of hepatitis virus nucleic acid |
| CN102827947B (en) * | 2011-06-17 | 2014-09-24 | 东北制药集团辽宁生物医药有限公司 | Kit and detection method for rapid quantitative detection of hepatitis virus nucleic acid |
| CN102559935A (en) * | 2012-02-23 | 2012-07-11 | 天津出入境检验检疫局动植物与食品检测中心 | M-gene based fluorescent RT-PCR (Reverse Transcription Polymerase Chain Reaction) detection method of Nipah virus |
| WO2014144578A1 (en) * | 2013-03-15 | 2014-09-18 | The Usa, As Represented By The Secretary, Department Of Health And Human Services | Selective detection of hepatitis a, b, c, d, or e viruses or combinations thereof |
| US10047406B2 (en) | 2013-08-14 | 2018-08-14 | Gen-Probe Incorporated | Compositions and methods for detecting HEV nucleic acid |
| CN105176969A (en) * | 2015-07-13 | 2015-12-23 | 杭州优思达生物技术有限公司 | Nucleic acid extraction method and nucleic acid extraction reagent for biological samples |
| CN107287347A (en) * | 2017-05-09 | 2017-10-24 | 广州机场出入境检验检疫局综合技术服务中心 | HEV real-time fluorescence reverse transcription PCR detection primer, probe, detection kit and detection method |
| CN107287347B (en) * | 2017-05-09 | 2020-12-25 | 广州机场出入境检验检疫局综合技术服务中心 | Real-time fluorescence reverse transcription PCR (polymerase chain reaction) detection primer, probe, detection kit and detection method for hepatitis E virus |
| CN110878378A (en) * | 2019-12-02 | 2020-03-13 | 昆明理工大学 | Primer combination for detecting hepatitis E virus in body fluid and application thereof |
| CN111235315A (en) * | 2020-03-13 | 2020-06-05 | 苏州智享众创孵化管理有限公司 | Method for simultaneously detecting multiple genotypes of hepatitis E virus |
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Inventor after: Cao Jianrong Inventor after: Zhang Zha Inventor after: Yan Baoshan Inventor after: Zhang Luming Inventor after: Li Lin Inventor before: Cao Jianrong Inventor before: Zhang Zha Inventor before: Yan Baoshan |
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Application publication date: 20090923 |