CN103725800A - Human adenovirus detection kit - Google Patents
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Abstract
The invention provides a human adenovirus (HAdV) detection kit. The kit comprises a nucleic releaser and PCR (polymerase chain reaction) liquid, wherein the nucleic releaser contains 0.01mM/L to 0.5mM/L of surfactin, 20mM/L to 300mM/L of potassium chloride, 0.01 to 2 percent of sodium dodecyl sulfate and 0.05 to 1 percent of ethanol; the PCR reaction liquid contains primers for HAdV detection and a probe sequence, wherein the HAdV upstream primer is AGTGTAACATGACCAAAGACTGGTTC, the HAdV downstream primer is AAGAAGGAGTACATGCGRTCCTT, and the HAdV probe is ACTACAACATTGGCTACCAGGGCTTCTA. The sensitivity of the kit for detecting the HAdV can reach 400copies/ml, and the quantitative linear range is 400 to 4.00 E+09copies/ml; by utilizing the kit, the HAdV-DNA (deoxyribonucleic acid) in unknown samples such as sputum and throat swab can be rapidly and accurately detected, and reliable experimental reference can be provided for the early diagnosis of the infection of the HAdV.
Description
Technical field
The invention provides a kind of adenovirus hominis detection kit, specifically a kind of adenovirus detection kit based on fluorescent PCR.
Background technology
((Human adenoviruses, HAdV) belongs to Adenoviridae to adenovirus hominis, is the widely distributed DNA virus of a group, can be divided into six subspecies of A～F, totally 51 serotypes.Adenovirus is nonenveloped virus, can stable existence under low ph environment, there is the ability of the effect of the very strong reagent of resistance to physics and chemistry, adenovirus can produce tolerance to gastrointestinal secretion thing and bile, therefore adenovirus can copy in stomach and intestine, produces very high virus load.The adenovirus breeding in pharynx, conjunctiva, enteron aisle and Lymphoid tissue of being everlasting, and cause various clinical symptom, such as the infection of respiratory system, conjunctivitis, gastro-enteritis, hepatitis, hemorrhagic cystitis, neural disorder etc.Different adenovirus subspecies can cause different diseases, as adenovirus hominis C subspecies mainly cause paediatric upper respiratory tract infection, adenovirus hominis B subspecies be everlasting cause in grownup popular, adenovirus hominis B and E subspecies are Etiologicals that military camp personnel infect, and adenovirus hominis F subspecies can cause infectious diarrhea.Adenovirus is gentle at immune host's In vivo infection, and has self limiting.Adenovirus can be passed through people, water, vehicle and apparatus propagation, and under room temperature condition, adenovirus exists the cycle can extend to 3 weeks in dirt.Adenovirus more easily occurs to infect and is popular on a large scale in children and military camp personnel, and most of infants at least infected a kind of adenopathy strain in postnatal 5 years.In the past few years, adenovirus often causes high incidence and mortality ratio immunologic hypofunction host in as the patient of aids patient, immune genetic defect, marrow recipient, solid organs and hematopoietic stem cell transplantation person as main pathogenic agent; Paediatrics stands the patient of isotype stem cell transplantation, and after it infects adenovirus, mortality ratio can be up to 60%.Some fatal infection are often caused by adenoviral serotype 1,2,3, often there will be the situation of the microorganism coinfections such as bacterium, fungi in these patient bodies.Aids patient infects adenovirus can produce the complication such as pneumonia, hepatitis, meningomalacia, ephritis, gastro-enteritis.Therefore can make a definite diagnosis in time which kind of cause of disease and cause a disease, accomplish early to find early treatment, sb.'s illness took a turn for the worse in minimizing, therefore early diagnosis is very important.
The detection of adenovirus is depended on to the type of disease and the sample obtaining, and the existence of making a definite diagnosis adenovirus can adopt the technology such as viral cell cultures, antigen measuring and genome detection.Although cell cultures remains the gold standard of HAdV, it is insensitive to clinical samples, and slow, is subject to the pollution of bacterium and fungi.Detection of antigen is commonly used to direct-detection adenovirus in respiratory tract and GI infection, faster and sensitivity higher.Immunofluorescence (especially to respiratory tract specimens, throat swab and living tissue specimen) and enzyme immunoassay (especially for stool sample) are conventional methods, and compared with cell cultures, the sensitive performance that immunofluorescence detects adenovirus improves 40～60%.Other method of directly measuring antigen comprises immunochromatographic method and latex agglutination.Studies confirm that, compared with cell cultures detection method, the sensitivity of using immune chromatography reagent kit to measure can reach 90%.
In recent years, poly-ribozyme chain reaction (PCR) method has manifested its advantage on detecting gradually, and Fluorescence PCR assay is sensitiveer, more special based on normal PCR technology the one that grows up in conjunction with spectroscopic techniques, more accurate nucleic acid detection technique.Detected result is accurate, and repeatability is high, can the forward and backward pathogenic agent dynamic change of dynamic response patient treatment and with clinical relation, and in whole process, avoided normal PCR to need the problem of aftertreatment, reduced pollution.
The detection that round pcr is adenovirus hominis provides another new approach.The appearance of quantitative PCR, break PCR situation qualitatively, wherein quantitative fluorescent PCR is with features such as its highly sensitive, high specific, low pollution rate, Real-Time Monitorings, can be clinically provide more accurately, objective detected result, and understand in time the state of an illness and prognosis.At present domestic also do not have fluorescent PCR diagnostic kit to use in clinical HAdV Infect And Diagnose, and the HAdV nucleic acid fluorescent PCR diagnostic kit tool of therefore designing and developing highly sensitive, high specific has very great significance.
Use round pcr to detect and be mainly concerned with two aspects, the extraction of nucleic acid and the augmentation detection of nucleic acid.
The direct boiling method of at present domestic main employing clinically extracts the HAdV nucleic acid in sputum, throat swab equal samples, specifically, to adding certain nucleic acid lysate through in pretreated sample, directly boils, and high speed centrifugation, supernatant is template; The method nucleic acid extraction process is more complicated, sample process length consuming time, and when processing sample, pass through multiple steps such as boiling lysis, high speed centrifugation enrichment DNA, there is loss in the DNA in sample, especially insufficient to the sample cracking of high density, enrichment is incomplete, can cause a large amount of loss of DNA to cause sample quantitatively on the low side, simultaneously owing to having adopted the heat step of water-bath or metal bath, easily cause Aerosol Pollution.
The detection method of HAdV-DNA is mainly the technology based on real-time fluorescence quantitative PCR at present, Real-Time Fluorescent Quantitative PCR Technique is development in recent years a kind of nucleic acid detection technique rapidly, use a kind of pcr amplification instrument with fluorescence detection device, fluorescence detection device can according to certain property program loop send the exciting light of specific wavelength, collect and detect fluorescent signal, by detecting the dynamic change of fluorescent signal, reflect in real time the level of amplification of each circulation of PCR, after off-test, can obtain amplification curve by software automatic analysis, according to the shape of the intersection point of amplification curve and fluorescence threshold line (being Ct value) and amplification curve, can judge yin and yang attribute result, if have quantitative reference material or the standard substance of concentration known in same reaction, can obtain typical curve by software automatic analysis, realize thus the definite value (being detection by quantitative) to unknown sample.Compare with traditional PCR, it has increased the two ends probe of mark fluorescent reporter group and quenching group respectively in reaction system.When probe structure is complete, the fluorescent energy that fluorescence report group sends is quenched group and absorbs, and presents quenching effect; If there is the existence of target sequence in amplification procedure, along with the extension of target fragment, probe molecule is cut off by Taq enzymic hydrolysis gradually, fluorescence report group and quenching group dissociate mutually, blocked the two fluorescence energy transfer effect, the fluorescent signal that fluorescence report group sends is collected by fluorescence detection device.Along with the carrying out of amplification, fluorescent signal presents linear enhancing along with the amplification of object fragment.After off-test, the software automatic analysis data that can carry by fluorescent PCR instrument, can obtain the definite value result of yin and yang attribute result and concentration of specimens, therefore, this technology is in the detection and quantitative analysis of target polynucleotide sample, replace gradually traditional PCR method, obtain applying very widely.
The external existing scientific research test kit that detects HAdV-DNA based on Real-Time Fluorescent Quantitative PCR Technique at present, but these test kits extract nucleic acid with boiling method mostly, and its detection sensitivity is not high, about 500～1000copies/ml left and right; In addition, these test kits lack perfect system of quality control mostly, also need further improve and improve technical level, and make this series products more meet the needs of clinical Accurate Diagnosis.
Summary of the invention
The invention provides the application of a kind of nucleic acid releasing agent in adenovirus hominis detects, described nucleic acid releasing agent comprises Buddhist Sha graceful (surfactin) 0.01～0.5mM/L, Repone K 20～300mM/L, sodium laurylsulfonate 0.01～2% and ethanol 0.05～1%.
In nucleic acid releasing agent of the present invention, its solvent can be conventional for this area, for example, be sterilized water or TE damping fluid.The present invention is disclosed in first in adenovirus detection and uses the nucleic acid releasing agent containing strong protein denaturant, simplified to a great extent the step of this detection of nucleic acids, and detection sensitivity is greatly improved.
The invention provides a kind of adenovirus hominis HAdV detection kit, described test kit comprises nucleic acid releasing agent and PCR reaction solution, described nucleic acid releasing agent comprises Buddhist Sha graceful (surfactin) 0.01～0.5mM/L, Repone K 20～300mM/L, sodium laurylsulfonate 0.01～2% and ethanol 0.05～1%; In described PCR reaction solution, comprise the primer and the probe sequence that for HAdV, detect as follows,
HAdV upstream primer: AGTGTAACATGACCAAAGACTGGTTC,
HAdV downstream primer: AAGAAGGAGTACATGCGRTCCTT,
HAdV probe: ACTACAACATTGGCTACCAGGGCTTCTA.
Use the method for the nucleic acid releasing agent release nucleic acid in test kit of the present invention and the detected result of boiling method extraction nucleic acid there is no notable difference, and adopt strong protein denaturant while extracting nucleic acid in the present invention, rapid damage pathogenic agent coat protein structure, discharge pathogen nucleic acid, without heating, can complete release and the extraction of DNA; The sensitivity that test kit of the present invention detects adenovirus can reach 400copies/ml, and quantitative linearity scope is 400～4.00E+09copies/ml.In addition, test kit of the present invention, because adopting above-mentioned primer and the probe sequence for detection of target polynucleotide, has good specificity.
In the present invention, in preferred described test kit, also comprise interior mark, and in described PCR reaction solution, also comprise for detection of interior target primer and probe sequence,
Interior mark: CACCACTTAAATCCTAAGGTTCCAGCTCTGTCATCCAGTTTTGCTGACTCA CGTATTCGTAGCCAATCTTCTGGAGGTGCAATCTCAATTATGTCATCAG;
Interior mark upstream primer: CACCACTTAAATCCTAAGGTTCCAG,
Interior mark downstream primer: CTGATGACATAATTGAGATTGCACC,
Interior mark probe: TTTTGCTGACTCACGTATTCGTAGCCAA.
A segment length who is designated as insertion pUC18T carrier in described in the present invention is the recombinant chou of the DNA artificial sequence synthetic of 100 base pairs, it is plasmid, it prevents as the positive internal reference in pcr amplification system the false negative causing due to the PCR interfering substance that may exist in sample.
In preferred test kit of the present invention, also comprise enzyme mixation, in described enzyme mixation, comprise the uracil dna glycosylase (UNG enzyme) of hot resistant DNA polymerase (Taq enzyme) and 0.5～2U/ μ l, in described PCR reaction solution, also comprise dUTP simultaneously.Wherein the function of UNG enzyme is the PCR product that degraded contains dU, utilizes the dUTP in UNG enzyme and PCR reaction solution can play the effect of prevention PCR product pollution, thereby prevents pattern detection false positive.
In an embodiment, described test kit comprises nucleic acid releasing agent, interior mark, PCR reaction solution, enzyme mixation, the quantitative reference material of adenovirus hominis, adenovirus hominis positive control, adenovirus hominis negative control and adenovirus hominis concentrated solution.
The present invention also provides a kind of adenovirus hominis HAdV detection kit, and described test kit comprises PCR reaction solution, comprises the primer and the probe sequence that for HAdV, detect as follows in described PCR reaction solution,
HAdV upstream primer: AGTGTAACATGACCAAAGACTGGTTC,
HAdV downstream primer: AAGAAGGAGTACATGCGRTCCTT,
HAdV probe: ACTACAACATTGGCTACCAGGGCTTCTA.
Adenovirus hominis fluorescent quantificationally PCR detecting kit operation provided by the invention fast, method is easy, detection sensitivity is high, sensing range is wide, applying this test kit can detect fast and accurately to the HAdV-DNA in the unknown sample such as sputum, throat swab, for diagnosis adenovirus infection provides reliable experimental basis.
Embodiment
Below be only the preferred embodiment of the present invention, protection scope of the present invention is not limited to this, and any those skilled in the art is in technical scope disclosed by the invention, within can being easy to the change carried out or changing and be encompassed in protection scope of the present invention.Therefore, protection scope of the present invention should be as the criterion with the protection domain of claims.
In addition, as do not do special explaining, the percentage composition in the present invention refers to quality percentage composition.
Embodiment 1
The present embodiment provides a kind of concrete adenovirus detection kit, and it comprises following component:
1. nucleic acid releasing agent: comprise Buddhist Sha graceful (surfactin) 0.1mmol/L, Repone K 100mmol/L, the second alcohol and solvent TE damping fluid of sodium laurylsulfonate (SDS) 0.1%, 0.1%.
2. mark (positive internal reference) in: for a segment length who inserts pUC18T carrier is the recombinant chou of the DNA artificial sequence synthetic of 100 base pairs, it is plasmid, concentration is 2.00E+04copies/ml, and the sequence of 100 base pairs is: 5 '-CACCACTTAAATCCTAAGGTTCCAGCTCTGTCATCCAGTTTTGCTGACTCACGTAT TCGTAGCCAATCTTCTGGAGGTGCAATCTCAATTATGTCATCAG-3 '.
3. PCR reaction solution: comprise 10 × PCR reaction buffer, 5 μ l, the dNTP of 0.2mmol/L, upstream and downstream primer for target polynucleotide amplification is 0.3 μ mol/L, the probe detecting for target polynucleotide is 0.3 μ mol/L, upstream and downstream primer for interior mark fragment amplification is 0.3 μ mol/L, for detection of interior target probe, is 0.1 μ mol/L.Wherein, described 10 × PCR reaction buffer is the 200mmol/L Tri(Hydroxymethyl) Amino Methane Hydrochloride solution that comprises pH7.5,30mmol/L magnesium chloride solution, 500mmol/L Klorvess Liquid, 0.2% Triton solution and 10% formamide soln; Described dNTP comprises dATP, dCTP, dUTP, dGTP and dTTP; The described upstream and downstream primer increasing for target polynucleotide and the probe detecting for target polynucleotide are primer and the probes that comes from the conservative region of adenoviral nucleic acid, and its base sequence is respectively:
HAdV upstream primer: 5 '-AGTGTAACATGACCAAAGACTGGTTC-3 ';
HAdV downstream primer: 5 '-AAGAAGGAGTACATGCGRTCCTT-3 ';
HAdV probe: 5 '-ACTACAACATTGGCTACCAGGGCTTCTA-3 ';
Described is respectively for detection of interior target primer probe sequence:
Interior mark upstream primer: 5 '-CACCACTTAAATCCTAAGGTTCCAG-3 ';
Interior mark downstream primer: 5 '-CTGATGACATAATTGAGATTGCACC-3 ';
Interior mark probe: 5 '-TTTTGCTGACTCACGTATTCGTAGCCAA – 3 '.
4. enzyme mixation: the hot resistant DNA polymerase (Taq enzyme) that comprises 5U/ μ l and the uracil dna glycosylase (UNG enzyme) of 1U/ μ l.
5. the quantitative reference material of adenovirus hominis: derive from the HAdV strong positive plasmid after the quantitative linearity reference material L1～L5 of end user's adenovirus enterprise definite value, the quantitative reference material of this adenovirus comprises the gradient reference material of A, B, C, tetra-concentration compositions of D, and its concentration is respectively 4.00E+07copies/ml(A), 4.00E+06copies/ml(B), 4.00E+05copies/ml(C), 4.00E+04copies/ml(D).
6. adenovirus hominis positive control: the HAdV strong positive throat swab sample of deactivation of collecting for clinical hospitals, after concentrated centrifugal, add sterile saline dissolution precipitation, and after the qualified detection of adenovirus hominis test kit definite value, with sterile saline, being diluted to concentration is 4.00E+05copies/ml.
7. adenovirus hominis negative control: the negative throat swab sample of HAdV of the deactivation that clinical hospitals is collected, with after 100 times of sterile saline dilutions, detects negative through qualified HAdV test kit.
8. adenovirus hominis concentrated solution: polyethylene glycol 6000 (PEG-6000) 10～100mmol/L(mass/volume), sodium-chlor 50～500mmol/L(mass/volume).
Embodiment 2
The present embodiment provides described in above-described embodiment 1 test kit for detection of the operation steps of HAdV-DNA in the unknown sample such as sputum, throat swab:
One, reagent is prepared
According to the quantity of sample to be tested, adenovirus negative control, adenovirus positive control and the quantitative reference material A～D of adenovirus, PCR reaction solution (38 μ l/ person-portion), enzyme mixation (2 μ l/ person-portion) and the interior mark 0.5 μ l/ person-portion of getting in proportion respective amount, be fully mixed into PCR-mix; Instantaneous centrifugal rear standby.
Two, nucleic acid extraction
1. sputum sample: the physiological saline that adds 2～3 times of volumes in sputum sample, fully concussion mixes latter standing one hour and makes sputum liquefaction, the sample 1000 μ l that get after liquefaction (avoid the obvious solid impurity of sucking-off) in 1.5ml centrifuge tube, in centrifuge tube, add adenovirus hominis concentrated solution 100 μ l, the centrifugal 5min of 12,000rpm after vibration mixes, abandon supernatant, in precipitation, add 50 μ l nucleic acid releasing agents, concussion or liquid-transfering gun are inhaled to beat and are mixed, standby as sample to be tested.
2. throat swab sample: add 1ml stroke-physiological saline solution toward collecting in the sample collection tube of throat swab, fully vibration mixes, then whole liquid is poured into (cotton swab abandons after extracting by centrifugal tube wall) in 1.5ml sterilizing centrifuge tube, in centrifuge tube, add adenovirus hominis concentrated solution 100 μ l, the centrifugal 5min of 12,000rpm after vibration mixes, abandon supernatant, in precipitation, add 50 μ l nucleic acid releasing agents, concussion or liquid-transfering gun are inhaled to beat and are mixed, standby as sample to be tested.
3. adenovirus negative control, adenovirus positive control, the quantitative reference material of adenovirus: adenovirus negative control, adenovirus positive control, the quantitative reference material A～D of adenovirus get respectively 10 μ l and 10 μ l nucleic acid releasing agents mix stand-by.
Three, to application of sample in PCR reaction tubes
In each PCR reaction tubes, add the each 10 μ l of sample to be tested after treatment in above-mentioned second step, adenovirus negative control, adenovirus positive control and the quantitative reference material A～D of adenovirus; After standing 10 minutes, every pipe adds the PCR-mix40 μ l in step 1, inhales to beat to mix 2-3 time, removes bubble bonnet upper tube cap, centrifugal 30 seconds of 2000rpm.
Four, Fluorescence PCR and interpretation of result (carrying out on fluorescent quantitative PCR instrument)
1) PCR reaction tubes is put into amplification instrument sample cell, by correspondence order, sample to be tested title and quantitative reference material concentration are set.
2) fluorescence detection channel is selected: select FAM passage (Reporter:FAM, Quencher:None) to detect adenovirus; Select HEX or VIC passage (Reporter:VIC, Quencher:None) to detect interior mark; Reference fluorescence (Passive Reference) is set to none.
3) quantitative fluorescent PCR reaction conditions is in Table 1:
Table 1
4) interpretation of result
After reaction finishes, the automatic saving result of instrument, can utilize the software that instrument carries to carry out automatic analysis, and starting value, end value and threshold value that also can manual regulation baseline be analyzed, and then record sample Ct value and result.The intersection point of amplification curve and threshold line, is called Ct value (be cycle threshold, refer to the cycling numerical value that fluorescent signal in PCR reaction tubes experiences while reaching the threshold value of setting); Instrument software, according to each sample Ct value size, by the typical curve of 4 quantitative reference materials draftings of concentration gradient, can be tried to achieve the detection by quantitative result of each sample automatically.For measuring Ct value≤39(Ct value > 0) sample, be reported as the HAdV-DNA positive, now the amplification curve of sample to be tested is S-type; For measuring the sample showing without Ct value, interior mark test positive (Ct value≤39), is reported as HAdV-DNA feminine gender simultaneously, and now sample to be tested amplification curve is straight; For the sample of measuring Ct value >39, interior mark test positive (Ct value≤39) simultaneously, is reported as lower than detecting lower limit.If interior mark Ct value >39 or interior mark show without Ct value, the detected result of this sample is invalid, should search and get rid of reason, and this sample is carried out to revision test.
First the present invention finds out the conservative section of homology of 51 serotypes of HAdV-DNA, then designs its primer and probe at its conservative region, can detect HAdV and infect, but can not detect non-HAdV pathogenic agent, illustrates that test kit of the present invention has good specificity.Its specific test is shown: with common disease substance no cross reaction as equal in UU, MP, CP, TB, EBV and influenza virus etc.
The detection sensitivity of test kit of the present invention can reach 400copies/ml, and quantitative linearity scope is 400copies/ml～4.00E+09copies/ml.Use test kit of the present invention to detect enterprise work reference material, its yin and yang attribute reference material coincidence rate is 100%, and the detected result of sensitivity reference material meets quality standard.Use the precision test of test kit of the present invention to show, batch in and batch between reproducible, the variation coefficient <10% of detected result Ct value, concentration variation coefficient <50%.
Claims (6)
1. the application of nucleic acid releasing agent in adenovirus hominis detects, described nucleic acid releasing agent comprises Buddhist Sha graceful (surfactin) 0.01～0.5mM/L, Repone K 20～300mM/L, sodium laurylsulfonate 0.01～2% and ethanol 0.05～1%.
2. an adenovirus hominis HAdV detection kit, described test kit comprises nucleic acid releasing agent and PCR reaction solution, described nucleic acid releasing agent comprises Buddhist Sha graceful (surfactin) 0.01～0.5mM/L, Repone K 20～300mM/L, sodium laurylsulfonate 0.01～2% and ethanol 0.05～1%; In described PCR reaction solution, comprise the primer and the probe sequence that for HAdV, detect as follows,
HAdV upstream primer: AGTGTAACATGACCAAAGACTGGTTC,
HAdV downstream primer: AAGAAGGAGTACATGCGRTCCTT,
HAdV probe: ACTACAACATTGGCTACCAGGGCTTCTA.
3. detection kit according to claim 2, is characterized in that, also comprises interior mark in described test kit, and in described PCR reaction solution, also comprises for detection of interior target primer and probe sequence,
Interior mark: CACCACTTAAATCCTAAGGTTCCAGCTCTGTCATCCAGTTTTGCTGACTCA CGTATTCGTAGCCAATCTTCTGGAGGTGCAATCTCAATTATGTCATCAG;
Interior mark upstream primer: CACCACTTAAATCCTAAGGTTCCAG,
Interior mark downstream primer: CTGATGACATAATTGAGATTGCACC,
Interior mark probe: TTTTGCTGACTCACGTATTCGTAGCCAA.
4. detection kit according to claim 2, it is characterized in that, in described test kit, also comprise enzyme mixation, in described enzyme mixation, comprise the uracil dna glycosylase (UNG enzyme) of hot resistant DNA polymerase (Taq enzyme) and 0.5～2U/ μ l, in described PCR reaction solution, also comprise dUTP simultaneously.
5. according to the test kit described in any one in claim 2～4, it is characterized in that, described test kit comprises nucleic acid releasing agent, interior mark, PCR reaction solution, enzyme mixation, the quantitative reference material of adenovirus hominis, adenovirus hominis positive control, adenovirus hominis negative control and adenovirus hominis concentrated solution.
6. an adenovirus hominis HAdV detection kit, described test kit comprises PCR reaction solution, comprises the primer and the probe sequence that for HAdV, detect as follows in described PCR reaction solution,
HAdV upstream primer: AGTGTAACATGACCAAAGACTGGTTC,
HAdV downstream primer: AAGAAGGAGTACATGCGRTCCTT,
HAdV probe: ACTACAACATTGGCTACCAGGGCTTCTA.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105586439A (en) * | 2016-01-27 | 2016-05-18 | 中国人民解放军军事医学科学院基础医学研究所 | Kit for detecting B and E type human adenovirus nucleic acid and detection method thereof |
| CN109504804A (en) * | 2018-11-22 | 2019-03-22 | 李越希 | A kind of RPA method, its primer special and probe and purposes detecting 3 type adenovirus hominis |
| CN110387439A (en) * | 2019-07-12 | 2019-10-29 | 湖南圣湘生物科技有限公司 | Primer and probe, kit and method for adenovirus detection and parting |
| CN111549178A (en) * | 2020-04-30 | 2020-08-18 | 嘉兴实践医学科技有限公司 | Universal nucleic acid detection kit and method for human adenovirus |
| CN113227363A (en) * | 2018-12-21 | 2021-08-06 | 卢森堡科学技术研究院 | DNA aptamers specific for adenovirus types |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1406285A (en) * | 2000-02-08 | 2003-03-26 | 阿文蒂斯药物股份有限公司 | Method for detecting and quantifying adenovirus |
| CN101107366A (en) * | 2004-12-24 | 2008-01-16 | 卡洛斯Ⅲ世健康研究所 | Probes and methods for the simultaneous detection and identification of multiple viruses that cause respiratory infections in humans |
| CN103122396A (en) * | 2013-01-10 | 2013-05-29 | 湖南圣湘生物科技有限公司 | Kit for detecting human cytomegalovirus (HCMV) |
-
2014
- 2014-01-15 CN CN201410017219.2A patent/CN103725800B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1406285A (en) * | 2000-02-08 | 2003-03-26 | 阿文蒂斯药物股份有限公司 | Method for detecting and quantifying adenovirus |
| CN101107366A (en) * | 2004-12-24 | 2008-01-16 | 卡洛斯Ⅲ世健康研究所 | Probes and methods for the simultaneous detection and identification of multiple viruses that cause respiratory infections in humans |
| CN103122396A (en) * | 2013-01-10 | 2013-05-29 | 湖南圣湘生物科技有限公司 | Kit for detecting human cytomegalovirus (HCMV) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105586439A (en) * | 2016-01-27 | 2016-05-18 | 中国人民解放军军事医学科学院基础医学研究所 | Kit for detecting B and E type human adenovirus nucleic acid and detection method thereof |
| CN105586439B (en) * | 2016-01-27 | 2020-12-18 | 中国人民解放军军事医学科学院基础医学研究所 | Kit for detecting B, E type human adenovirus nucleic acid and detection method thereof |
| CN109504804A (en) * | 2018-11-22 | 2019-03-22 | 李越希 | A kind of RPA method, its primer special and probe and purposes detecting 3 type adenovirus hominis |
| CN113227363A (en) * | 2018-12-21 | 2021-08-06 | 卢森堡科学技术研究院 | DNA aptamers specific for adenovirus types |
| CN110387439A (en) * | 2019-07-12 | 2019-10-29 | 湖南圣湘生物科技有限公司 | Primer and probe, kit and method for adenovirus detection and parting |
| CN110387439B (en) * | 2019-07-12 | 2023-04-25 | 圣湘生物科技股份有限公司 | Primers, probes, kit and method for adenovirus detection and typing |
| CN111549178A (en) * | 2020-04-30 | 2020-08-18 | 嘉兴实践医学科技有限公司 | Universal nucleic acid detection kit and method for human adenovirus |
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