CN103045760A - Primer set for detecting I-type herpes simplex virus and kit - Google Patents
Primer set for detecting I-type herpes simplex virus and kit Download PDFInfo
- Publication number
- CN103045760A CN103045760A CN2012105459842A CN201210545984A CN103045760A CN 103045760 A CN103045760 A CN 103045760A CN 2012105459842 A CN2012105459842 A CN 2012105459842A CN 201210545984 A CN201210545984 A CN 201210545984A CN 103045760 A CN103045760 A CN 103045760A
- Authority
- CN
- China
- Prior art keywords
- herpes simplex
- simplex virus
- virus type
- primer
- dna
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a primer set for detecting I-type herpes simplex virus and a kit. Primer sequences of the primer set respectively are SEQ ID NO:1 to 2. The kit disclosed by the invention consists of a DNA (Deoxyribose Nucleic Acid) amplification reagent, a forward and reverse primer combination, positive control nucleic acid and sterilized distilled water. According to the invention, viruses are detected by a method of designing and synthesizing forward and reverse primers capable of amplifying I-type herpes simplex virus specific DNA according to a conserved sequence of I-type herpes simplex virus genome DNA and utilizing a special PCR (Polymerase Chain Reaction) amplification reagent to direct perform PCR reaction without carrying out extraction on DNA of a sample. According to the invention, the step of extracting DNA of a template is omitted, one important link of losing the sample size is reduced and the highest utilization degree of limited clinical samples is ensured; and meanwhile, detection time is greatly shortened, a detection result can be obtained in 2 hours, and the primer set and the kit can take a good auxiliary effect on early diagnosis of clinical pathology.
Description
Technical field
The invention belongs to the rapid detection technical field of the common pathogenic microorganism of infectious eye disease, be specifically related to a kind of primer sets for detection of the I herpes simplex virus type and test kit.
Background technology
Simplexvirus is in the representative simplexvirus of herpetoviridae Alphaherpesvirinae, be divided into I type and two kinds of serotypes of II type according to antigenic difference, think that at present envelope glycoprotein G is a glycoprotein of difference maximum between I type and II type, can induce body to produce the type specificity antibody response.Hsv can cause human various diseases, and common viral keratitis is mainly by due to the I herpes simplex virus type infection cornea.The I herpes simplex virus type is by secretion body fluid and intimate contact mucomembranous surface or scratch mark and in interpersonal propagation, and primary infection is non-evident sympton normally, but from then on virus can hide in host.When body is subject to many factors such as ultraviolet ray (sun is exposed to the sun), heating, wound and nervous, bacterium or virus infection and after using the impact such as suprarenin, the virus of hiding is activated, the HSV-1 recurrent infection that activates can cause the eye pathology, recurrence can also cause that corneal scar forms, neovascularization repeatedly, even blind.The herpes simplex keratitis complicated clinical manifestation except typical branch, map and plate-like cornea focus form, also has some atypical Clinical changes, brings very large difficulty for diagnosis and treatment.Herpes simplex virus type keratitis has obvious rising and aggravation trend in recent years, because repeatedly outbreak, severe cases increases, the serious threat visual function.Therefore only have in time to make a definite diagnosis in early days be by due to which kind of virus infection, just can better instruct the rational use of drug in the successive treatment and select suitable opportunity of operation.
At present clinically main by typical cornea focus form (branch, map and plate-like), the medical history of multiple relapse, antibiotic therapy is invalid and cortin treatment deterioration, and corneal sensitivity is blunt or the clinical symptom such as disappearance is made a definite diagnosis herpes simplex virus type keratitis, but its clinical manifestation is complicated various after all, and this process has certain subjective dependency, so simple make a definite diagnosis or have certain difficulty by clinical manifestation.Laboratory diagnosis mainly contains viral isolated culture and FAST at present.The cultivation of carrying out virus from the pathology sample is the reliable basis of clarifying a diagnosis, but this cultural method length consuming time can not provide the early diagnosis result, delay treatment; Because the medication before the patient also may affect the positive of cultivation; And cultivation virus also needs the perfect of professional and technical personnel and hardware condition.Then the direct trace of diseased region cell is gone fluorescent antibody staining can judge within a short period of time, but the positive rate that this method detects is lower, also needs the fluorescent microscope of specialty.As seen the ocular infection that the I herpes simplex virus type is caused there is no fast and effectively method of early diagnosis at present, therefore needs clinically the diagnostic method of new fast and effectively viral keratitis badly.Utilize molecular biology method diagnosis pathogenic micro-organism to provide new thinking for the quick diagnosis of herpes simplex keratitis.Existing investigator is applied to the Infect And Diagnose that bacterium, fungi and virus cause with the method for regular-PCR, and the large and small subunit of the 16S rRNA of bacterium, fungi rRNA gene and viral species specificity sequence are increased and analyze, and can effectively identify to kind.Although regular-PCR method diagnosis cause of disease is efficiently special, carry out first the extraction of template DNA to sample, itself will cause the loss of sample this process; Add that Clinical Ophthalmology pathology sample size is original just seldom, even adopt efficiently easily test kit extracting method, the DNA yield still is very low.So consider and will carry out DNA extraction, regular-PCR is difficult to adapt to clinical needs so.The Direct PCR method just can be avoided the step of DNA extraction, clinical sample is directly carried out the amplification of I herpes simplex virus type specific DNA, both greatly shortened the time of detecting, efficiently special again, and can also further carry out genotypic evaluation if required.
Summary of the invention
The purpose of this invention is to provide a kind of primer sets for detection of the I herpes simplex virus type and test kit, can detect fast, efficiently and accurately the I herpes simplex virus type, save simultaneously clinical disease this DNA extraction step of changing so that detect more convenient, thereby remedy the deficiencies in the prior art.
The present invention designs and synthesizes the primer sets of type specificity according to the higher position of homology in the nucleotide sequence of the not homophyletic I herpes simplex virus type envelope glycoprotein G that announces among the GenBank.
Primer sets for detection of the I herpes simplex virus type of the present invention, its nucleotides sequence are classified as and are respectively SEQ ID NO:1～2, and the herpes simplex virus expanding fragment length is 346bp; Wherein:
Forward primer HSV-1F: its nucleotide sequence is shown in SEQ ID NO:1;
Reverse primer HSV-1R: its nucleotide sequence is shown in SEQ ID NO:2.
I herpes simplex virus type quick detection kit of the present invention comprises:
1) DNA cloning reagent:
The DNA cloning pack contains 2 * MightyAmp damping fluid, MightyAmp archaeal dna polymerase;
2) forward and reverse combination of primers:
The nucleotides sequence of forward and reverse primer is classified SEQ ID NO:1 as, and the sequence of reverse primer is SEQ ID NO:2.
3) positive control nucleic acid:
Positive control nucleic acid is the plasmid pUC-HSV-1 that contains I herpes simplex virus type sequence;
4) sterile purified water.
Utilize detection kit of the present invention the clinical sample of viral keratitis to be carried out the method for I herpes simplex virus type detection:
(1) processing of clinical sample: the pathological corneas scraping blade is resuspended in little centrifuge tube with the piping and druming of 5 ~ 10 μ L sterile purified waters; Aqueous humor or vitreum are inhaled behind high speed centrifugation and are abandoned supernatant, finally collect 5 ~ 10 μ L(and contain precipitation), then with pipettor piping and druming evenly; The conjunctival sac cotton swab is abandoned swab, remaining liquid the same branch of a family water treatment method after blowing and beating for several times with 500 μ L sterilization pure water.
(2) configuration of I herpes simplex virus type specific DNA amplification system:
In reaction tubes, add respectively following component:
(3) I herpes simplex virus type specific DNA amplification program (TouchDown method):
Step 1:98 ℃ 5 minutes,
Step 2:98 ℃ 30 seconds,
Step 3:65 ℃ 30 seconds, each circulating temperature reduces by 0.5 ℃,
Step 4:72 ℃ 30 seconds,
Step 5: repeat 2,3,4 step 9 circulation,
Step 6:98 ℃ 30 seconds,
Step 7:60 ℃ 30 seconds,
Step 8:72 ℃ 30 seconds,
Step 9: repeat 6,7,8 step 25 circulation,
Step 10:72 ℃ 10 minutes,
Step 11:4 ℃ of maintenance.
(4) agarose gel electrophoresis of I herpes simplex virus type specific DNA detects:
Conventional agarose gel electrophoresis detects I herpes simplex virus type specific DNA, whether define without positive amplification, thereby determine to cause the Causative virus type of viral keratitis if conforming to I herpes simplex virus type specific DNA predetermined molecular weight according to the amplified production molecular weight.
The present invention is by the conserved sequence according to I herpes simplex virus type genomic dna, design and synthesize forward and reverse primer of the I herpes simplex virus type type specificity DNA that to increase, utilize special pcr amplification reagent to need not to carry out that sample DNA extracts and the method for directly carrying out the PCR reaction detects virus.The present invention has saved the template DNA extraction step, has reduced an important step of sample size loss, has guaranteed the maximum availability of limited clinical sample; Also so that the time of detecting shortens greatly, detected result can be gone out in 2 hours simultaneously, good booster action can be played to the early diagnosis of clinical pathology.The Direct PCR amplifing reagent amplification efficiency that the present invention adopts is very high, although amplification is the study that extracts without template DNA, special polysaccharase still has very high amplification efficiency.The amplification program of the TouchDown that the present invention adopts had both been avoided the study non-specific amplification that the complicacy of template may be brought in this, had guaranteed again the efficient amplification of distinguished sequence.
Embodiment
The below carries out concrete description to test kit of the present invention and detection method.
One, the acquisition of amplimer group
According to the corresponding US4 zone design of envelope glycoprotein G pcr amplification primer relatively conservative in the I herpes simplex virus type genome sequence, from the GenBank database, download 9 not nucleotide sequences of homophyletic I herpes simplex virus type coding glycoprotein G, corresponding A ccession Number is as follows: AB297670.1, AY240761.1, JX142173.1, HQ833203.1, GQ898900.1, JN420337.1, HM585514.1, JQ780693.1, HM585511.1.Utilize sequence analysis software DNAMAN version 6 to carry out homology relatively, it is 100% position in homology, design pcr amplification primer, utilize the primer analytic function in the DNAMAN software that designed primer is carried out the concrete analysis of parameters, finally determine forward and reverse primer sets of the present invention.
Two, the preparation of test kit
Direct PCR detects the preparation (5 samples) of I herpes simplex virus type test kit:
(1) DNA cloning reagent, 1 pipe, (every sample need be done 1 positive reaction to in-built 15 parts of amplified reactions, 1 laboratory sample reaction, 1 negative reaction) required reagent, 10.5 μ L/ parts, every part contains 10 μ L, 2 * MightyAmp damping fluid, 0.5 μ L MightyAmp archaeal dna polymerase; (MightyAmp damping fluid and corresponding DNA polysaccharase are a kind of PCR reaction systems that TaKaRa company produces, and article No. is DR071)
(2) forward and reverse combination of primers, 1 pipe, in-built 15 parts, 2 μ L/ parts, every part of concentration that contains I herpes simplex virus type forward primer and reverse primer is 5 μ M, the concrete sequence of primer such as SEQ ID NO:1～2.
(3) positive control nucleic acid, 1 pipe, in-built 5 μ L comprise the plasmid of I herpes simplex virus type target DNA, called after pUC-HSV-1.The concrete operations of plasmid preparation are to utilize above-mentioned forward and reverse primer that I herpes simplex virus type genomic dna is carried out pcr amplification, the fragment of the purpose size of gained is reclaimed test kit and reclaims with cutting glue, then be connected on the cloned plasmids pMD-18T, change in the bacillus coli DH 5 alpha, through ammonia benzyl culture medium flat plate screening and culturing, the picking positive colony carries out sequence verification, after being defined as the interior conserved sequence of I herpes simplex virus type envelope glycoprotein G type, the plasmid that extracts this positive colony namely can be used as positive nucleus acid.
(4) sterile purified water, 1 pipe, in-built 1mL.
Detection method is carried out according to following (1)-(4) program:
(1) processing of clinical sample: pathological corneas scraping thing is resuspended in little centrifuge tube with the piping and druming of 5 ~ 10 μ L sterile purified waters; Aqueous humor or vitreum are inhaled behind high speed centrifugation and are abandoned supernatant, finally collect 5 ~ 10 μ L(and contain precipitation), then with pipettor piping and druming evenly; The conjunctival sac cotton swab is abandoned swab, remaining liquid the same branch of a family water treatment method after blowing and beating for several times with 500 μ L sterilization pure water.
(2) configuration of I herpes simplex virus type specific DNA amplification system:
In reaction tubes, add following component:
Simultaneously respectively do the positive and negative control, namely template respectively adds corresponding positive nucleic acid in the positive reaction, and negative reaction does not then add template.
(3) I herpes simplex virus type specific DNA amplification program (TouchDown method):
98 ℃ 5 minutes/98 ℃ 30 seconds, 65 ℃-60 ℃ 30 seconds (each circulating temperature reduce by 0.5 ℃), 72 ℃ 30 seconds; (9 circulations)/98 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 30 seconds; (25 circulations)/72 ℃ 10 minutes, 4 ℃ of maintenances.
(4) agarose gel electrophoresis of I herpes simplex virus type specific DNA detects:
Conventional agarose gel electrophoresis detects I herpes simplex virus type specific DNA, whether define without positive amplification, thereby determine to cause the Virus Type of viral keratitis if conforming to I herpes simplex virus type specific DNA predetermined molecular weight according to the amplified production molecular weight.
Primer and test kit that direct PCR method of the present invention detects the I herpes simplex virus type not only can carry out the detection of I herpes simplex virus type to focus scraping blade or the conjunctival sac swab sample of epidemic keratoconjunctivitis, can also carry out the corresponding former detection of causing a disease to aqueous humor and the vitreum sample of viral endophthalmitis.Following specific embodiment further specifies the present invention, but the restriction of the present invention of should not opposing.
The sensitivity Detection of embodiment 1 test kit of the present invention
Be 10 with known titre I herpes simplex virus type suspension gradient dilution
4, 10
3, 10
2, 10
1, 10
0PFU/ μ L adopts DNA cloning reagent and combination of primers in the test kit of the present invention to increase, and estimates the susceptibility that Direct PCR detects the I herpes simplex virus type, and concrete grammar is as follows:
1) DNA cloning reagent 10.5 μ L, each gradient I herpes simplex virus type suspension 1 μ L, forward and reverse combination of primers 2 μ L mend sterile purified water to 20 μ L;
2) 98 ℃ 5 minutes/98 ℃ 30 seconds, 65 ℃-60 ℃ 30 seconds (each circulating temperature reduce by 0.5 ℃), 72 ℃ 30 seconds; (9 circulations)/98 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 30 seconds; (25 circulations)/72 ℃ 10 minutes, 4 ℃ of maintenances.
3) after the PCR reaction finishes, in each concentration gradient reaction tubes, add sample-loading buffer, carry out agarose gel electrophoresis and detect.
The result shows that test kit of the present invention just can amplify positive findings to the I herpes simplex virus type of trace, and detection sensitivity can reach 10 copy numbers, and sensitivity is very high, false negative can not occur when use primer of the present invention detects in actually operating.
Embodiment 2: the specific detection of test kit of the present invention
Utilize other common viruses of ophthalmology such as adenovirus, varicella zoster virus, cytomegalovirus; another serotype of herpes simplex virus II herpes simplex virus type; staphylococcus epidermidis in common other cause of diseases of ophthalmology such as the bacterium, the corneal epithelial cell that often is mixed with in the Fusarinm solani in the fungi and thorn ameba protozoon and the cornea scraping blade has carried out specific detection to the primer of I herpes simplex virus type.
Concrete operations are with the concrete grammar of embodiment 1, the result shows, an I herpes simplex virus type combination of primers specific amplified I herpes simplex virus type does not produce amplification to other pathogenic micro-organisms in the test kit of the present invention, illustrates that primer of the present invention false positive can not occur when detecting.
Embodiment 3: test kit of the present invention is to the detection of animal pathology tissue sample
Utilize and to suspect highly that clinically the viral keratitis pathological corneas organizes the scraping thing that test kit of the present invention is carried out validation verification, concrete grammar is as follows:
(1) pathological corneas scraping thing adds the piping and druming of 5 ~ 10 μ L sterile purified waters and scatters to evenly.
(2) look sample and measure the above-mentioned sample suspension of 1 ~ 5 μ L and add respectively in 1 little PCR reaction tubes, add 10.5 μ L DNA cloning reagent, and then add I herpes simplex virus type combination of primers 2 μ L, add sterile purified water and supply 20 μ L.Simultaneously to doing the positive control that adds positive nucleic acid and the negative control that does not add template.
(3) above-mentioned reaction tubes is put into the PCR instrument and is carried out TouchDown PCR reaction: 98 ℃ 5 minutes/98 ℃ 30 seconds, 65 ℃-60 ℃ 30 seconds (each circulating temperature reduces by 0.5 ℃), 72 ℃ 30 seconds; (9 circulations)/98 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 30 seconds; (25 circulations)/72 ℃ 10 minutes, 4 ℃ of maintenances.
(4) after the PCR reaction finishes, carry out conventional agarose gel electrophoresis and detect.
Viral keratitis pathological corneas tissue sample amplifies the I herpes simplex virus type specific DNA band identical with positive reaction pipe size as a result, and the negative reaction pipe is without amplification.
Embodiment 4: test kit of the present invention involves the detection of humoral sample to animal tissues's pathology
Utilize and suspect highly that clinically the aqueous humor sample of viral endophthalmitis carries out validation verification to test kit of the present invention, concrete grammar is as follows:
Get the centrifugal 10min of aqueous humor sample 10000r/min of collection, inhale and abandon supernatant, stay about 20 μ L samples, piping and druming evenly; Remaining step is with (2) among the embodiment 3～(4), and the result shows that the aqueous humor example reaction pipe of viral endophthalmitis amplifies the I herpes simplex virus type specific DNA band identical with positive reaction pipe size, and the negative reaction pipe increases without the positive.
Claims (4)
1. for detection of the primer sets of I herpes simplex virus type, it is characterized in that the nucleotide sequence of described primer sets is respectively SEQ ID NO:1 and SEQ ID NO:2.
2. primer sets claimed in claim 1 is used for the detection of I herpes simplex virus type.
3. I herpes simplex virus type quick detection kit comprises following component:
1) DNA cloning reagent:
Every part of DNA cloning reagent contains 10 μ L, 2 * MightyAmp damping fluid, 0.5 μ L MightyAmp archaeal dna polymerase;
2) forward and reverse combination of primers:
Forward and reverse combination of primers is forward and the reverse primer equivalent mixed liquor that designs and synthesizes for I herpes simplex virus type conserved sequence; Wherein forward and reverse primer is primer sets claimed in claim 1, and its forward primer nucleotides sequence is classified SEQ ID NO:1 as, and the sequence of reverse primer is SEQ ID NO:2;
3) positive control nucleic acid:
Positive control nucleic acid is the plasmid pUC-HSV-1 that contains I herpes simplex virus type sequence
4) sterile purified water.
4. I herpes simplex virus type quick detection kit claimed in claim 3 is used for viral keratitis or endophthalmitis clinical disease changed and originally carries out the I herpes simplex virus type and detect.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012105459842A CN103045760A (en) | 2012-12-14 | 2012-12-14 | Primer set for detecting I-type herpes simplex virus and kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012105459842A CN103045760A (en) | 2012-12-14 | 2012-12-14 | Primer set for detecting I-type herpes simplex virus and kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103045760A true CN103045760A (en) | 2013-04-17 |
Family
ID=48058619
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2012105459842A Pending CN103045760A (en) | 2012-12-14 | 2012-12-14 | Primer set for detecting I-type herpes simplex virus and kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103045760A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109576396A (en) * | 2018-12-12 | 2019-04-05 | 上海博威生物医药有限公司 | Method and kit for the detection of HSV1 virion number |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002061390A2 (en) * | 2001-01-31 | 2002-08-08 | Mayo Foundation For Medical Education And Research | Detection of herpes simplex virus |
| CN101824409A (en) * | 2003-04-25 | 2010-09-08 | 贝克顿·迪金森公司 | Detect 1 type and herpes simplex types 2 virus by nucleic acid amplification |
| CN101979668A (en) * | 2010-11-05 | 2011-02-23 | 武汉百泰基因工程有限公司 | Fluorescence quantitative polymerase chain reaction (PCR) kit for quickly detecting herpes simplex viruses 1 |
-
2012
- 2012-12-14 CN CN2012105459842A patent/CN103045760A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002061390A2 (en) * | 2001-01-31 | 2002-08-08 | Mayo Foundation For Medical Education And Research | Detection of herpes simplex virus |
| CN101824409A (en) * | 2003-04-25 | 2010-09-08 | 贝克顿·迪金森公司 | Detect 1 type and herpes simplex types 2 virus by nucleic acid amplification |
| CN101979668A (en) * | 2010-11-05 | 2011-02-23 | 武汉百泰基因工程有限公司 | Fluorescence quantitative polymerase chain reaction (PCR) kit for quickly detecting herpes simplex viruses 1 |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109576396A (en) * | 2018-12-12 | 2019-04-05 | 上海博威生物医药有限公司 | Method and kit for the detection of HSV1 virion number |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103966358B (en) | A kind of mandarin fish infectious spleen and kidney necrosis virus fluorescent quantificationally PCR detecting kit and detection method | |
| CN103045761B (en) | For detecting test kit and the detection method of infectious eye disease pathogenic microorganism | |
| CN111321248B (en) | African swine fever virus MGF-505R gene fluorescence PCR detection reagent, kit and application thereof | |
| CN108384899B (en) | Fluorescent quantitative PCR kit for detecting novel goose astrovirus and application thereof | |
| CN103131798A (en) | Norovirus real-time fluorescent RT-PCR detection kit and application thereof | |
| CN104152584B (en) | Capripoxvirus virus Taqman-MGB probe multiple real-time fluorescence quantitative PCR detection primer, method and test kit | |
| CN103757139A (en) | Canine distemper virus and canine parvovirus duplex TaqMan-MGB fluorescent quantitative PCR (polymerase chain reaction) detection kit and detection method thereof | |
| CN104195266A (en) | Quadruple fluorogenic quantitative PCR kit for detecting three pathogens of infantile pneumonia | |
| CN103484565A (en) | Kit for detecting coronavirus through real-time fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) and application thereof | |
| CN105463132A (en) | Genetic marker of canine parvovirus and specific primers as well as probe thereof | |
| CN108048603A (en) | A kind of K subgroup avian leucosis virus detection kit | |
| CN104745689A (en) | Primers, probe and kit used for detecting bordetella pertussis | |
| CN102643931B (en) | Reverse transcription-polymerase chain reaction (RT-PCR) method for verifying avian infectious bronchitis virus (IBV) epidemic strains and vaccine strains | |
| CN104232789A (en) | RT-PCR (reverse transcription-polymerase chain reaction) technique capable of simultaneously identifying porcine reproductive and respiratory syndrome virus (PRRSV) | |
| CN102634605A (en) | Method for detecting egg drop syndrome viruses and kit for method | |
| CN103820574B (en) | Human herpes virus type 6 real-time fluorescence quantitative PCR parting detecting reagent | |
| CN101376909B (en) | A segment of sequence and a restriction enzyme site for differentiating canine distemper viral vaccine strain and wild strain | |
| CN104946753A (en) | Specificity primer pair for cow mycoplasma detection, detection kit, as well as using method and application of detection kit | |
| CN104593486A (en) | Primer, probe and kit all used for detecting blood fluke | |
| CN103045760A (en) | Primer set for detecting I-type herpes simplex virus and kit | |
| CN101178350A (en) | Method and reagent box for testing rabies viruses | |
| CN104745724A (en) | Primers, probe and kit used for detecting JC viruses (JCVs) | |
| CN104745722A (en) | Primers, probe and kit used for detecting varicella zoster viruses (VZVs) | |
| CN103131797B (en) | A kind of bocavirus real-time fluorescence PCR detection reagent kit and application thereof | |
| CN101985664A (en) | Nucleotide sequence and kit for detecting type-A influenza virus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20130417 |