CN108690884A - Hereby block viral primer and probe combinations and its kit for detecting - Google Patents
Hereby block viral primer and probe combinations and its kit for detecting Download PDFInfo
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- CN108690884A CN108690884A CN201710223281.0A CN201710223281A CN108690884A CN 108690884 A CN108690884 A CN 108690884A CN 201710223281 A CN201710223281 A CN 201710223281A CN 108690884 A CN108690884 A CN 108690884A
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Abstract
The present invention provides a kind of for detect the primer and probe combinations and its kit that hereby block virus, and the primer and probe combinations include a forward primer, a reverse primer and a probe, the DNA sequence dna such as SEQ ID NO of the forward primer:Shown in 1, the DNA sequence dna such as SEQ ID NO of the reverse primer:Shown in 2, the DNA sequence dna such as SEQ ID NO of the probe:Shown in 3.The present invention also provides a kind of kits for detecting hereby card viral diagnosis, and it includes the primer of the present invention and probe combinations.
Description
Technical field
The present invention relates to the nucleic acid detection methods for hereby blocking virus, are especially used to detect the primer and probe groups for hereby blocking virus
Conjunction and its kit.
Background technology
Hereby card viral infectious disease (Zika virus infection) is by hereby blocking caused by viral (Zika virus)
Acute infectious disease.Hereby card virus is one kind of flavivirus, is mainly propagated via mosquito bite.Common flavivirus includes western Buddhist nun
Sieve virus (West nile virus), yellow fever virus (Yellow fever virus), dengue fever virus (Dengue
Virus), chikungunya virus (Chikungunya virus) etc..Hereby card virus is most early in nineteen forty-seven in the hereby Carson of Uganda
It is separated in macaque body in woods, a small number of human cases also start to be reported in Africa and Asia in generation nineteen sixty;1].
It is divided into two kinds of types of Asian type and African type, the discipline that non-in, Southeast Asia and India etc. are found according to gene type at present
Record [2].
Hereby card viral transmission mode is mainly by mosquitoes spread to the mankind, Aedes Aegypti (Aedes aegypti) and white line spot
Two kinds of mosquitoes of mosquito (Aedes albopictus) are the main infection genesis in Taiwan.In addition, hereby card virus can also
Passability contacts and mother and baby's vertical transmission, America prevalence are to be aroused attention with mother and baby's vertical infection.Clinical manifestation range from
It is asymptomatic to similar cold symptoms, as grade fever, arthralgia, myalgia, weak sense, headache, maculopapule, conjunctivitis, eye socket pain,
Enlargement of lymph nodes and diarrhea etc..However, nonspecific clinical performance may obscure infection with other most of arboviruses, it is special
It is not the Gan Ran [ of dengue fever (Dengue) and chikungunya virus (Chikungunya virus);3].
Because currently without the instruments of inspection of " gold standard ", hereby the diagnosis of card virus infection is great challenging.Due to
If patient is infected by other flavivirus (including dengue fever), have antibody generation, so make hereby card viral diagnosis be positive result
(false positive) causes to be limited using serum.Therefore, hereby the confirmation method of card virus infection is mainly examined using RT-qPCR
The RNA for virus of breaking, it is a kind of quick, sensitive, and infects early stage i.e. workable diagnostic method.In order to effectively examine
It surveys, it is proposed that blood, serum or saliva sample can acquire after symptom appearance in 5 days.In addition, hereby blocking viral later stage rank in infection
Section can also be by taking urine come come [4].
Hereby card virus have passed through for halfth century after nineteen forty-seven is found, and only find the people distributed in Africa and Asia mostly
Class case is the whole world for the first time in Asia and non-until clustering epidemic situation is broken out on the islands Ya Pu in Micronesia Federated States in 2007
Area other than continent occurs, and just has more understanding to this disease.Nearest wave epidemic situation then starts from October, 2013
The South Pacific Oceans island such as polynesia area, thereafter in May, 2015;5], there is native country hereby in the Brazilian northeast of WHO confirmations
Card viral infectious disease confirmed cases are that America area is the first;Epidemic situation expands to dozens of country /region within 2016, main to concentrate
Yu Zhong, South America, Oceania, Asia and Africa also have Countries native country epidemic situation occur, including neighbouring Thailand, Philippine and
The ground Ma Er husband.Therefore, hereby card viral infectious disease is considered as the emerging Chuan Ranbing [ that will persistently expand to new area;6].
It is TaiWan, China the second class Notifiable disease that hereby card viral infectious disease has been announced in January, 2016, and is promoted in 2 months
For the 5th class Notifiable disease, 2017 so far it has been found that 13 overseas move into confirmed cases.In TaiWan, China Aedes Aegypti and
White line spot mosquito is very common, and neighbouring country in Southeast Asia, also once detects hereby card virus, the hereby card virus of such as neighbouring country in Southeast Asia
Infectious disease epidemic situation does not obtain apparent control, hereby the relative risk of card poisoning intrusion TaiWan, China yet Ti Gao [ therewith;7][8].
It is therefore desirable to be able to exclusively detect the nucleic acid detection method for hereby blocking virus.
Invention content
On the one hand, the present invention provides a kind of for detecting the primer and probe combinations that hereby block virus, and it includes a forward directions
Primer, a reverse primer and a probe, the DNA sequence dna such as SEQ ID NO of the forward primer:Shown in 1, the reverse primer
DNA sequence dna such as SEQ ID NO:Shown in 2, the DNA sequence dna such as SEQ ID NO of the probe:Shown in 3.
On the other hand, the present invention also provides a kind of for detecting the kit for hereby blocking virus, and it includes primers as the aforementioned
And probe combinations.
The forward primer is an oligonucleotides, 5 '-ACCTCCGACTGA TGGCCAATGCC-3 ' (SEQ ID of sequence
NO:1);The reverse primer is an oligonucleotides, sequence 5 '-GGGTCTTGTCTTCCATGTGG-3 ' (SEQ ID NO:2);It should
Probe is an oligonucleotides, sequence 5 '-CCTGGTCAATCCATGGAAAGGGAGAATGG-3 ' (SEQ ID NO:3).
In the part preferred embodiment of the present invention, the 5 ' ends and 3 ' ends of the probe are respectively in connection with there is one first fluorescence
Group and one first fluorescent quenching group.
According to the present invention, the primer and probe combinations can be used for detecting the RNA samples for carrying out autoblood.
In the specific embodiment of the present invention, the primer and probe combinations are used for RT-qPCR.
Part preferred embodiment according to the present invention, it is described hereby to block viral kit for detect and further wrap
The primer and probe combinations for being used for internal control containing one.It is described for detecting hereby in the preferred embodiment of the present invention
The kit for blocking virus also includes a positive quality control product and a negative quality-control product.
It should be appreciated that both previous general description and detailed description below are all merely illustrative and explanatory and be not intended to limit this
Invention.
Cooperation attached drawing reading can more preferably understand the present invention such as preceding general introduction and detailed description below.Mesh is shown in attached drawing
Preceding preferred embodiment.
Description of the drawings
Fig. 1 display present invention's hereby blocks viral specific primers and probe to the detection situation of hereby card viral RNA, Yi Jiyong
In the primer of detection internal control gene M S2 and the fluorophor (HEX) of probe to hereby blocking viral specific primers and probe
The interference cases of fluorophor (FAM)."MS2":The hereby card virus specific primers of MS2RNA+MS2 primers and probe+present invention
And probe;" hereby blocking ":Hereby card viral RNA+MS2 primers and the hereby card virus specific primers and probe of probe+present invention;And
" mixing ":MS2RNA+ hereby card viral RNA+MS2 primers and the hereby card virus specific primers and probe of probe+present invention.
Specific implementation mode
It is described in detail below to be all merely illustrative and explanatory rather than limitation of the present invention.
On the one hand, the present invention provides a kind of for detecting the primer and probe combinations that hereby block virus, and it includes a forward directions
Primer, a reverse primer and a probe, the DNA sequence dna such as SEQ ID NO of the forward primer:Shown in 1, the reverse primer
DNA sequence dna such as SEQ ID NO:Shown in 2, the DNA sequence dna such as SEQ ID NO of the probe (probe):Shown in 3.
On the other hand, the present invention also provides a kind of for detecting the kit for hereby blocking virus, and it includes primers as the aforementioned
And probe combinations.
The forward primer is an oligonucleotides, 5 '-ACCTCCGACTGA TGGCCAATGCC-3 ' (SEQ ID of sequence
NO:1);The reverse primer is an oligonucleotides, sequence 5 '-GGGTCTTGTCTTCCATGTGG-3 ' (SEQ ID NO:2);It should
Probe (probe) is an oligonucleotides, sequence 5 '-CCTGGTCAATCCATGGAAAGGGAGAATGG-3 ' (SEQ ID NO:
3)。
In the part preferred embodiment of the present invention, the 5 ' ends and 3 ' ends of the probe are respectively in connection with there is one first fluorescence
Group and one first fluorescent quenching group.
In the specific embodiment of the present invention, which is FAM, which is
TAMRA, but not limited to this.
According to the present invention, the primer and probe combinations can be used for detecting the RNA samples for carrying out autoblood.The present invention's
In one specific embodiment, the blood sample of individual to be detected is extracted via conventional method after obtaining RNA samples, then with described
Primer and probe combinations be detected.
In the specific embodiment of the present invention, the primer and probe combinations are used for RT-qPCR.
Part preferred embodiment according to the present invention, it is described hereby to block viral kit for detect and further wrap
The primer and probe combinations for being used for internal control containing one.
It is described for detecting the kit for hereby blocking virus also comprising a positive in the preferred embodiment of the present invention
Quality-control product and a negative quality-control product.For example, the positive quality control product can be to contain artificial synthesized hereby card conserved viral section NS5 bases
The in-vitro transcription RNA of cause, and the feminine gender quality-control product can be secondary water.
In some embodiments of the present invention, this be used for the primer of internal control and probe combinations can specificity distinguish
Recognize MS2 bacteriophages, it includes following primer and probes:DNA sequence dna such as SEQ ID NO:Forward primer shown in 4, DNA sequence dna is such as
SEQ ID NO:Such as SEQ ID NO of reverse primer and DNA sequence dna shown in 5:Probe shown in 6.
A specific embodiment according to the present invention, the DNA sequence dna such as SEQ ID NO:5 ' the ends and 3 ' ends of probe shown in 6
Respectively in connection with having one second fluorophor and one second fluorescent quenching group.For example, second fluorophor is HEX, this second
Fluorescent quenching group is TAMRA.
The present invention for detect hereby block virus primer and probe combinations or for detect hereby block viral kit can
It is used in one-step method (one step) real-time and quantification polymerase chain reaction (RT-qPCR), to detect hereby card virus.One-step method
RT-qPCR mainly carries out at-once monitor reaction product amount using fluorescein stain and polymeric enzyme reaction, and polymerase chain reaction is with DNA
Polymerase (for example, Taq polymerase) extends the region that specific primers are bonded and synthesizes new double-stranded DNA.One-step method RT-
RNA can be inverted to cDNA by qPCR directly by hereby card viral nucleic acid progress RT-qPCR in blood without additionally reverse transcription is first carried out
The step of, there is advantage quick, easy to operate and good susceptibility compared to general RT-qPCR the method.
The present invention is further illustrated by following instance, is provided and is not intended to limit the present invention as the purposes illustrated.
Specific experiment condition and method, usually according to normal condition, such as Sambrook etc. are not specified in the following example
The molecular cloning of people chief editor:Laboratory manual (New York:Cold Spring Harbor Laboratory Press,
1989) condition described in, or the condition with reference to proposed by reagent manufacturer and step.
Example
Example 1:With fluorescence detection method one-step method real-time and quantification is carried out to hereby blocking virus
1.1 material
Specific primers and probe solution (every reaction tube (if any addition), 10 μM of SEQ ID NO:1 forward primer
1.0 μ L, 10 μM of SEQ ID NO:2 1.0 μ L of reverse primer, 10 μM of SEQ ID NO:3 0.5 μ L of probe, 10 μM of SEQ
ID NO:4 1.0 μ L of forward primer, 10 μM of SEQ ID NO:5 1.0 μ L of forward primer and 10 μM of SEQ ID NO:6
0.5 μ L of probe),RNA-to-CtTM10.0 μ L of 1-Step Mix reaction solutions, reverse transcriptase (Reverse
Transcriptase) 0.5uL and ddH26.0 μ L of O, positive quality control product are to contain artificial synthesized hereby card conserved viral section
The in-vitro transcription RNA of NS5 genes, negative quality-control product (non-template control, NTC) are secondary water, for internal control
The gene of system is MS2 bacteriophages.SEQ ID NO:5 ' the ends and 3 ' ends of 3 probe are respectively in connection with there is FAM and TAMRA.SEQ ID
NO:5 ' the ends and 3 ' ends of 6 probe are respectively in connection with there is HEX and TAMRA.
1.2 method
Hereby the preparation of card viral RNA genes positive quality control product is summarized as follows:It is prepared and is purified into containing NS5 bases by authorized company
The hereby card viral RNA of cause measures its light absorption value (A260) to detect RNA concentration in wavelength 260nm using spectrophotometer, and counts
Its RNA copy number is calculated, dilutes RNA concentration to 10 using sterile water8Replisome/mL-105Replisome/mL is as standard items.It is negative
Quality-control product and hereby card virus-positive quality-control product take 5.0 μ L respectively per concentration, and 10.0 μ L of polymeric enzyme reaction liquid and each single-minded are added
Property primer and probe solution, then with distilled water (ddH2O it) will often react tube reaction total volume to adjust to 20 μ L, same concentration carries out
The repetition of three reaction tubes is tested.Fluoroscopic examination is carried out in instant quantitative poly reaction instrument, instrument condition is set as:In 48 DEG C,
15 minutes and 95 DEG C, the preliminary program of 1 cycle is carried out under 10 minutes, then with 95 DEG C, 15 seconds and 60 DEG C, 1 minute condition
Carry out the amplified reactions of 45 cycles, it is finally for 15 seconds at 95 DEG C and 60 DEG C at continue 1 minute, and with 0.15 DEG C per second
Speed rises to 95 DEG C and carries out melting curve (melting curve) test.
1.3 result
Repeat experiment three times, the results are shown in Figure 1.The recurring number (Ct values) of obtained each group carries out statistical analysis, group
Between difference (inter-assay) CV%<4 and group difference (intra-assay) CV%<2, the smaller then accuracy of numerical value is higher,
Inspection result is stablized between indicating different batches, and repeatability is high.In Fig. 1, the sample of " MS2 " group is MS2RNA+MS2 primers and spy
The hereby card virus specific primers and probe of needle+present invention;The sample of " hereby blocking " group is hereby card viral RNA+MS2 primers and probe
The hereby card virus specific primers and probe of+the present invention;And the sample of " mixing " group is that hereby card viral RNA+MS2 draws MS2RNA+
The hereby card virus specific primers and probe of object and probe+present invention." MS2 " group the results show that the present invention hereby card virus
Specific primers and probe non-will not exclusively recognize MS2RNA." hereby block " group the results show that MS2 primers and probe will not shadows
The hereby card virus specific primers and probe of the present invention are rung, and non-will not exclusively recognize hereby card viral RNA.The knot of " mixing " group
Fruit shows that the fluorescence (HEX) of MS2 probes does not interfere the fluorescence (FAM) of hereby card Viral Probe.
Example 2:To hereby blocking the computer for analysis of viral specificity
It by the primer and probe of the present invention, is analyzed with Clustal Omega softwares, is hereby blocked with known in the mankind and mosquito
Viral pnca gene is compared, and as a result display is all 100% pairing (result is not shown), indicates primer and the spy of the present invention
All there is high specificity for hereby card Strain not of the same race.In addition, by the primer of the present invention and probe and the viral phase of hereby card
As 8 strain virus strains (including west nile virus, yellow fever virus, dengue fever virus, chikungunya virus) gene with BLASTN
Be compared, as a result show the present invention primer and probe not with its specific pairs (result is not shown).
In addition, being analyzed by Clustal Omega softwares, primer more of the invention and probe and relevant previous disclosure
Primer and probe, to the single-minded identification situation of different hereby card Strain, as a result as shown in table 1 below, primer of the invention and spy
It is widest in area that needle can recognize hereby card Strain.
Table 1. compares primer of the invention and previously disclosed primer
F:Forward primer;R:Reverse primer;P:Probe.
Person skilled in the art scholar, which should be understood that, can be changed the above embodiments without departing from its extensive invention generally
It reads.It is therefore to be understood that the present invention is not only restricted to revealed specific embodiment, but it is intended to cover determine in such as claims
Modification in the spirit and scope of the present invention of justice.
Bibliography:
[1]Fang Y et al.Comparative thermostability of West Nile,St.Louis
encephalitis,and western equine encephalomyelitis viruses during heat
inactivation for serologic diagnostics.Am J Trop Med Hyg,80(5):862–3;2008
[2]Lanciotti et al.Genetic and serologic properties of Zika virus
associated with an epidemic,Yap state,Micronesia,Emerg Infect Dis,14(8):1232–
9;2008
[3]Lyle et al,Zika Virus.N Engl J Med,374:1552-1563;2016
[4]CDC.Trioplex Real-time RT-PCR Assay.Atlanta,GA:US Department of
Health and Human Services;2016
[5]Duffy et al,Zika virus outbreak on Yap Island,Federated States of
Micronesia.N Engl J Med,360:2536-43;2009
[6]Zanluca et al,First report of autochthonous transmission of Zika
virus in Brazil.Mem Inst Oswaldo Cruz,110:569-72;2015
[7]Angela et al,A new reportable disease is born:Taiwan Centers for
Disease Control's response to emerging Zika virus infection.Journal of the
Form Med Assoc,115,223e225;2016
[8]http://www.cdc.gov.tw/home/Zika_virus
[9]Tappe Det al.First case of laboratory-confirmed Zika virus
infection imported into Europe.Euro Surveill,19(4):20685;2014
[10]Pyke AT et al.Imported Zika virus infection from the Cook Islands
into Australia.PLoS Curr,6:6;2014
[11]Waggoner et al.Zika Virus:Diagnostics for an emerging pandemic
threat.J Clinic Microbio,54(4);2016
[12]Goebel et al.A sensitive virus yield assay for evaluation of
Antivirals against Zika Virus.J Viro Met,238(13-20);2016
[13]Corman et al.Assay optimization for molecular detection of Zika
virus.Bull World Health Organ,94:879–892;2016
Sequence table
<110>Declare victory stem cell Sheng Ji limited liability companies
<120>Hereby block viral primer and probe combinations and its kit for detecting
<130> MRD0007TW
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 23
<212> DNA
<213>Artificial sequence
<220>
<223>Forward primer
<400> 1
acctccgact gatggccaat gcc 23
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>Reverse primer
<400> 2
gggtcttgtc ttccatgtgg 20
<210> 3
<211> 29
<212> DNA
<213>Artificial sequence
<220>
<223>Primer/probe
<400> 3
cctggtcaat ccatggaaag ggagaatgg 29
<210> 4
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>Forward primer
<400> 4
ccctcagcaa tcgcagcaaa 20
<210> 5
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>Reverse primer
<400> 5
ccgcttccag tagcgacaga 20
<210> 6
<211> 23
<212> DNA
<213>Artificial sequence
<220>
<223>Primer/probe
<400> 6
acgccggcca ttcaaacatg agg 23
Claims (13)
1. a kind of for detecting the primer and probe combinations that hereby block virus, it includes a forward primer, a reverse primer and one to visit
Needle, the DNA sequence dna such as SEQ ID NO of the forward primer:Shown in 1, the DNA sequence dna such as SEQ ID NO of the reverse primer:Shown in 2,
The DNA sequence dna of the probe such as SEQ ID NO:Shown in 3.
2. it is according to claim 1 for detect hereby block virus primer and probe combinations, wherein the probe 5 ' hold and
3 ' ends are respectively in connection with having one first fluorophor and one first fluorescent quenching group.
3. according to claim 2 for detecting the primer and probe combinations that hereby block virus, wherein first fluorophor
For FAM.
4. according to claim 2 for detecting the primer and probe combinations that hereby block virus, wherein first fluorescent quenching
Group is TAMRA.
5. it is according to claim 1 for detecting the primer and probe combinations that hereby block virus, it is used to detect and carrys out autoblood
RNA samples.
6. it is according to claim 1 for detecting the primer and probe combinations that hereby block virus, it is used for RT-qPCR.
7. a kind of for detecting the kit for hereby blocking virus, it includes as claimed in any one of claims 1 to 6 for detecting hereby
Block the primer and probe combinations of virus.
8. it is according to claim 7 for detecting the kit for hereby blocking virus, also include drawing for internal control
Object and probe combinations.
9. according to claim 8 hereby block viral kit for detect, wherein this be used for internal control primer and
Probe combinations include:DNA sequence dna such as SEQ ID NO:Forward primer shown in 4, DNA sequence dna such as SEQ ID NO:It is anti-shown in 5
To primer and DNA sequence dna such as SEQ ID NO:Probe shown in 6.
10. according to claim 9 for detecting the kit for hereby blocking virus, the wherein DNA sequence dna such as SEQ ID NO:6
Shown in probe 5 ' ends and 3 ' ends respectively in connection with having one second fluorophor and one second fluorescent quenching group.
11. according to claim 10 for detecting the kit for hereby blocking virus, wherein second fluorophor is HEX.
12. according to claim 10 for detecting the kit for hereby blocking virus, wherein second fluorescent quenching group is
TAMRA。
13. it is according to claim 7 for detecting the kit for hereby blocking virus, also include a positive quality control product and one
Negative quality-control product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201710223281.0A CN108690884A (en) | 2017-04-07 | 2017-04-07 | Hereby block viral primer and probe combinations and its kit for detecting |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710223281.0A CN108690884A (en) | 2017-04-07 | 2017-04-07 | Hereby block viral primer and probe combinations and its kit for detecting |
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| CN108690884A true CN108690884A (en) | 2018-10-23 |
Family
ID=63842169
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103602760A (en) * | 2013-11-21 | 2014-02-26 | 安徽华卫集团禽业有限公司 | Method for detecting Newcastle disease viruses through real-time fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) by using virus-like particle interior label |
| CN105734171A (en) * | 2016-03-10 | 2016-07-06 | 中山大学达安基因股份有限公司 | Zika virus fluorescent PCR detecting kit |
| CN105936946A (en) * | 2016-06-27 | 2016-09-14 | 扬州大学 | One step method inverse transcription PCR kit for detecting and differentiating Zika viruses and detection method thereof |
-
2017
- 2017-04-07 CN CN201710223281.0A patent/CN108690884A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103602760A (en) * | 2013-11-21 | 2014-02-26 | 安徽华卫集团禽业有限公司 | Method for detecting Newcastle disease viruses through real-time fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) by using virus-like particle interior label |
| CN105734171A (en) * | 2016-03-10 | 2016-07-06 | 中山大学达安基因股份有限公司 | Zika virus fluorescent PCR detecting kit |
| CN105936946A (en) * | 2016-06-27 | 2016-09-14 | 扬州大学 | One step method inverse transcription PCR kit for detecting and differentiating Zika viruses and detection method thereof |
Non-Patent Citations (4)
| Title |
|---|
| J. DREIER等: "Use of Bacteriophage MS2 as an Internal Control in Viral Reverse Transcription-PCR Assays", 《JOURNAL OF CLINICAL MICROBIOLOGY》 * |
| M. R. ZAMBENEDETTI等: "Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics", 《MEM INST OSWALDO CRUZ》 * |
| NCBI: "Zika virus polyprotein gene, complete cds-EU545988.1", 《GENBANK》 * |
| O. FAYE等: "Quantitative real-time PCR detection of Zika virus and evaluation with field-caught Mosquitoes", 《VIROLOGY JOURNAL》 * |
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Application publication date: 20181023 |