CN107858404A - It is a kind of for the neck ring primer of multiplexed PCR amplification and its application - Google Patents
It is a kind of for the neck ring primer of multiplexed PCR amplification and its application Download PDFInfo
- Publication number
- CN107858404A CN107858404A CN201711402111.5A CN201711402111A CN107858404A CN 107858404 A CN107858404 A CN 107858404A CN 201711402111 A CN201711402111 A CN 201711402111A CN 107858404 A CN107858404 A CN 107858404A
- Authority
- CN
- China
- Prior art keywords
- primer
- amplification
- neck ring
- sequence
- pcr amplification
- 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
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
Abstract
Present invention offer is a kind of for the neck ring primer of multiplexed PCR amplification and its application, and the primer is the oligonucleotides that a segment length is 35 ~ 50bp；3 ' ends of the oligonucleotides are the sequence complementary with target gene, and 5 ' ends are one section of sequence artificially edited.The present invention is applied to any multiplexed PCR amplification, and target gene is more, and effect is better；The present invention is applicable not only to sonde method fluorescent PCR, is also applied for dye method PCR；Suitable for various clinical purposes, particularly solution can be provided for clinical microorganism identification.
Description
Technical field
The invention belongs to biological technical field, and in particular to a kind of for the neck ring primer of multiplexed PCR amplification and its application.
Background technology
PCR (PCR) technology is a kind of simple and fast, highly sensitive, special gene amplification method, its
Process generally includes the 20-50 circulations formed by being denatured, annealing and extending three-step reaction.Can be special within 1.5-3 hours
Ground amplifying target nucleic acid sequence is to millions of times.In recent years, because the appearance of real time PCR instrument makes regular-PCR no longer need gel electric
Swimming analysis, operates after without PCR, has prevented PCR pollutions.Therefore real time pcr is clinically using more and more extensive.
In real-time PCR detection technology it is most widely used be mark fluorescent nucleic acid probe, such as in 5'-Exonuclease technology
The Taqman probes that use, molecular beacon etc..
With the progress of fluorescence probe labelling technique and exploitation and the vitro detection of multiple real time fluorescence quantifying PCR instrument
Market increasingly increases the demand of multi-target joint-detection, and multi-fluorescence Real-time quantitative PCR is using more and more extensive.Just
The multiple PCR technique of phase, the two-wheeled amplification technique mainly using nest-type PRC as representative, and due to the primed probe number in system
Amount is excessive, causes primer dimer and the increase of non-specific amplification probability, and then influence sensitivity and the specificity of amplification.
So in order to meet clinical demand, need that invention is a kind of can to improve the more of detection sensitivity and specific amplification badly
Weight fluorescence PCR method.
The content of the invention
It is an object of the invention to provide a kind of for the neck ring primer of multiplexed PCR amplification and its application.
To achieve the above object, the present invention adopts the following technical scheme that：
A kind of primer design method of multiplex PCR is included in the present invention, primer dimer is avoided with the design method of uniqueness
Occur, and Reverse transcriptase caused by the difference between different target gene template concentrations is eliminated after being expanded by the first round, fit
Detect and analyze simultaneously for more target genes.
The primer that the present invention designs includes two kinds：A kind of is the specificity amplification primer of tape label sequence, for the first step
Specific amplification；One kind is Tag primer, for the universal amplification of second；Specific primer includes upstream and downstream primer, its
Sequence label is consistent, and Tag primer only has one, i.e., also serves as anti-sense primer as sense primer.Described label sequence
It is classified as CAAGAAGGTGGTGAA.
The specificity amplification primer of tape label sequence, total length are 35 ~ 50bp, wherein 15 ~ 20bp of sequence label, specificity
20 ~ 30bp of sequence.As shown in Figure 1,3 ' ends are the specific primer for the design of not target-gene sequence to its structure；5 ' ends are label
Sequence, the sequence label of all primers are consistent.Because 5 ' terminal sequences of all primers are all the same, greatly reduce
Probability that primer dimer is formed, the stability for reducing dimer formation.
When the first step expands, specific sequence and the complementary combination of target gene of specific primer.After completing amplification, template sequence
Row both ends are added to sequence label, as shown in Figure 2.After next round denaturation, these products are single-stranded itself to form a ring
Shape structure, structure is as shown in figure 3, these cyclic products will not participate in ensuing amplification.So as to the different target bases in system
In the case of because of initial concentration difference, the Competitive assays between different templates are reduced as far as possible.
After first step amplification terminates, different target gene template numbers expand, second close to unanimously followed by second step
Step amplification will be completed by Tag primer.Tag primer sequence is one section of sequence with high amplification efficiency of artificial Screening Treatment
Row, and all templates are all expanded by this primer, the template for efficiently obtaining the first step in the amplification of this step passes through
Amplification is converted to fluorescence signal, reaches the purpose of more target genes while amplification.
The advantages of invention：The advantage of the present invention is 1, improves multiplexed PCR amplification sensitivity；2nd, the hair of primer dimer is reduced
It is raw, improve the specificity of multiplexed PCR amplification；3rd, interference caused by different templates initial concentration difference in same system is eliminated.
The beneficial effect of invention：1st, the present invention is applied to any multiplexed PCR amplification, and target gene is more, and effect is better；
2nd, the present invention is applicable not only to sonde method fluorescent PCR, is also applied for dye method PCR；3rd, suitable for various clinical purposes, it is particularly
Solution can be provided for clinical microorganism identification.
Brief description of the drawings
Specific primer structure charts of the Fig. 1 containing sequence label.
Fig. 2 first steps expanded after template sequence structure chart.
After the denaturation of Fig. 3 first steps product, the cyclic structure figure of single-stranded formation.
Fig. 4 second steps expand schematic diagram.
Fig. 5 samples 01（HBV is 1 with internal control concentration：1）Amplification.
Fig. 6 samples 02（HBV is 100 with internal control concentration：1）Amplification.
Fig. 7 samples 03（HBV is 1000 with internal control concentration：1）Amplification.
Fig. 8 samples 04（HBV is 10000 with internal control concentration：1）Amplification.
Fig. 9 samples 05（HBV is 100000 with internal control concentration：1）Amplification.
Figure 10 samples 06（Concentration is 5.0 × 102copies/mL）Amplification.
Figure 11 samples 07（Concentration is 5.0 × 104copies/mL）Amplification.
Figure 12 samples 08（Concentration is 5.0 × 105copies/mL）Amplification.
Figure 13 samples 09（Concentration is 5.0 × 106copies/mL）Amplification.
Embodiment
Embodiment one：Neck ring Tag primer system quantitatively detects for hbv nucleic acid
The immunology detection of hepatitis type B virus (Hepatitis B Virus, HBV) is to diagnose the tradition side of HBV infection
Method.It is generally believed that in serum or blood plasma hepatitis B virus surface antigen (HBsAg) presence, indicate that and infected B-type hepatitis
Poison, but HBsAg appearance can not provide the information of viral duplication activity in vivo.Single e antigens (HBeAg) conduct
Judge whether hepatitis B virus duplication is active, infectiousness is strong and weak immune indexes are not definite enough.With gene diagnosis technology
Continue to develop and clinical practice, serum HBV-DNA level are considered as both can prove that the presence of hepatitis B, and can reflection
The activity of virus, its confidence values is higher than immunization, significant to the clinical conditions rational use of medicines and judging prognosis.
HBV-DNA detection generally use is fluorescence quantitative PCR method at this stage, and the testing process of the method is：Sample
Machine testing → interpretation of result is gone up in acquisition → nucleic acid extraction → PCR reaction systems are prepared →.In order to ensure the accuracy of result,
HBV-DNA can add internal control to monitor the homogeneity of nucleic acid extraction process, the validity of system and instrument while detection.But
It is due to the amplification expanded with HBV-DNA of internal control in same system while carries out, both has the relation of Competitive assays.
When HBV-DNA concentration is higher, the amplification of internal control will be suppressed, so as to situation of the internal control without amplification occur.And neck
Ring Tag primer system can be good at avoiding this situation of the target without amplification a certain caused by Competitive assays.
In order to verify the validity of neck ring Tag primer system, the HBV of various concentrations and the aggregate sample of internal control is respectively adopted
Originally verified.Sample is as follows：
The primer probe sequence that neck ring Tag primer system includes is as follows：
HBV sense primers：5'-CAAGAAGGTGGTGAATCCTGTCCTCCAATTTGTCC-3'；
HBV anti-sense primers：5'-CAAGAAGGTGGTGAAGGTCCCGTACTGGTTGTTGT-3'；
HBV probes：FAM-5'-TTCCTCTTCATCCTGCTGCT-3'-BHQ1；
Internal control sense primer：5'-CAAGAAGGTGGTGAAACCGGACTGAAGGAGCTG-3'；
Internal control anti-sense primer：5'-CAAGAAGGTGGTGAAACTGCTGACTATGTCCCGC-3'；
Internal control probe：HEX-5'-CCTGCCCTGTGCAACGTGGA-3'-BHQ1；
Tag primer：5'-CAAGAAGGTGGTGAA-3'；
Reaction system formula is as follows：
Reaction condition is：95 DEG C of pre-degeneration 10min；95 DEG C of 15s, 62 DEG C of 40s, 15 circulations；95 DEG C of 15s, 58 DEG C of 40s, 35
Individual circulation.
Interpretation of result：After employing neck ring Tag primer system, no matter HBV-DNA concentration level, internal control can be normal
Amplification, illustrate that neck ring Tag primer system can be good at eliminating the competition suppression between the different target genes amplification under same system
System.
Accompanying drawing is as follows：
Fig. 5, the amplification of sample 01（HBV is 1 with internal control concentration：1）, the two all normal amplification；
Fig. 6, the amplification of sample 02（HBV is 100 with internal control concentration：1）, the two all normal amplification；
Fig. 7, the amplification of sample 03（HBV is 1000 with internal control concentration：1）, the two all normal amplification
Fig. 8, the amplification of sample 04（HBV is 10000 with internal control concentration：1）, the two all normal amplification；
Fig. 9, the amplification of sample 05（HBV is 100000 with internal control concentration：1）, the two all normal amplification.
Embodiment two：Neck ring Tag primer system is used for the Bacteria Identification of multiple fluorescence PCR
Bacterial pneumonia is by the microbial acute pulmonary infection of pneumonia streptococcus Pneumococcal pneumonia.Account for Community-acquired lung
Scorching half.Usual hurried onset, characterized by high fever, shiver with cold, cough, bloody sputum and pectoralgia.X-ray rabat is anxious in lung section or the lobe of the lung
Property inflammatory consolidation, widely using because of antibacterials in recent years, causes this sick mode of onset symptom and x-ray changes not allusion quotation
Type.This disease is common with the early spring with winter, often with respiratory virus infection companion lines.Patient is often the between twenty and fifty or old of original health
Year and infant, male are more common.It is smoker, dement, chronic bronchitis, bronchiectasis, congestive heart failure, slow
Std patient and immunosuppressed host are easily by Streptococcus pneumoniae Invasion.When body's immunity is normal, streptococcus pneumonia is
A kind of normal flora of oral cavity and pharynx nasalis is resided in, its bacterial bearing rate often has difference with the change of age, season and immune state
It is different.The streptococcus pneumonia people that body's immunity has virulence when impaired invades human body and caused a disease.Streptococcus pneumonia in addition to pneumonia is caused,
Bacteremia or infectious shock can occur for minority, and the state of an illness of the elderly and infant are particularly acute.
The detection means of bacterial pneumonia mainly has：Chest X-ray, Bacteria Culture, blood test, immunologic test,
Bacterial nucleic acid detection etc..Quantitative fluorescent PCR has the characteristics of quick, accurate, simple, is widely used in the mirror of clinical bacteria
Not.Multiple fluorescence PCR can detect multiple bacteriums in same primary first-order equation, so as to improve detection efficiency and detection flux.But
Common multiple fluorescence PCR detects multiple template due to needing, and the primer and probe in reaction system generally has many bars, different
Primer dimer is easily produced between primer so as to influence Detection results.
In order to verify the validity of neck ring Tag primer system in multiple fluorescence PCR detection, this conceptual design one
Multiple neck ring Tag primer system is covered, while detects streptococcus pneumonia, staphylococcus aureus, klebsiella pneumoniae, influenza
Four kinds of bacteriums of haemophilus.
Sample is as follows：
The primer probe sequence that neck ring Tag primer system includes is as follows：
Streptococcus pneumonia sense primer：5'-CAAGAAGGTGGTGAActatgcagcggttgaactga-3'
Streptococcus pneumonia anti-sense primer：5'-CAAGAAGGTGGTGAAttggttggttattcgtgcaa-3'
Streptococcus pneumonia probe：FAM-5'-agctggaattaaaacgcacg-3'-BHQ1
Staphylococcus aureus sense primer：5'-CAAGAAGGTGGTGAAggttgatacacctgaaacaaagc-3'
Staphylococcus aureus anti-sense primer：5'-CAAGAAGGTGGTGAAatacgctaagccacgtccat-3'
Staphylococcus aureus probe：HEX-5'-ggtcctgaagcaagtgcatt-3'-BHQ1
Klebsiella pneumoniae sense primer：5'-CAAGAAGGTGGTGAAgcgaacaagctggatgtgta-3'
Klebsiella pneumoniae anti-sense primer：5'-CAAGAAGGTGGTGAAgtcttttgctggctggagtc-3'
Klebsiella pneumoniae probe：ROX-5'-ttgggaatctgaattctcgg-3'-BHQ2
Haemophilus influenzae sense primer：5'-CAAGAAGGTGGTGAAccgacttgcatttgctctct-3'
Haemophilus influenzae anti-sense primer：5'-CAAGAAGGTGGTGAAgcactcgggctatgtgaaac-3'
Haemophilus influenzae probe：Cy5-5'-gaggtgattgcagtagggga-3'-BHQ2
Tag primer：5'-CAAGAAGGTGGTGAA-3'
Reaction system formula is as follows：
Reaction condition is：95 DEG C of pre-degeneration 10min；95 DEG C of 15s, 60 DEG C of 40s, 15 circulations；95 DEG C of 15s, 55 DEG C of 40s, 35
Individual circulation.
Interpretation of result：After employing neck ring Tag primer system, quadruple Fluorescence PCR, no matter template concentrations height,
It can guarantee that the amplification of each passage is normally carried out, detection sensitivity can reach 5.0 × 102copies/mL。
Accompanying drawing is as follows：
Figure 10, the amplification of sample 06（Concentration is 5.0 × 102copies/mL）, quadruple amplified fluorescence it is more all normal and compared with
To be homogeneous；
Figure 11, the amplification of sample 07（Concentration is 5.0 × 104copies/mL）, quadruple amplified fluorescence it is more all normal and compared with
To be homogeneous；
Figure 12, the amplification of sample 08（Concentration is 5.0 × 105copies/mL）, quadruple amplified fluorescence it is more all normal and compared with
To be homogeneous；
Figure 13, the amplification of sample 09（Concentration is 5.0 × 106copies/mL）, quadruple amplified fluorescence it is more all normal and compared with
To be homogeneous；
Presently preferred embodiments of the present invention is the foregoing is only, all equivalent changes done according to scope of the present invention patent are with repairing
Decorations, it should all belong to the covering scope of the present invention.
SEQUENCE LISTING
<110>Jiaxing Ya Kangbobeinan bio tech ltd
<120>It is a kind of for the neck ring primer of multiplexed PCR amplification and its application
<130> 19
<160> 19
<170> PatentIn version 3.3
<210> 1
<211> 15
<212> DNA
<213>Artificial sequence
<400> 1
caagaaggtg gtgaa 15
<210> 2
<211> 35
<212> DNA
<213>Artificial sequence
<400> 2
caagaaggtg gtgaatcctg tcctccaatt tgtcc 35
<210> 3
<211> 35
<212> DNA
<213>Artificial sequence
<400> 3
caagaaggtg gtgaaggtcc cgtactggtt gttgt 35
<210> 4
<211> 20
<212> DNA
<213>Artificial sequence
<400> 4
ttcctcttca tcctgctgct 20
<210> 5
<211> 33
<212> DNA
<213>Artificial sequence
<400> 5
caagaaggtg gtgaaaccgg actgaaggag ctg 33
<210> 6
<211> 34
<212> DNA
<213>Artificial sequence
<400> 6
caagaaggtg gtgaaactgc tgactatgtc ccgc 34
<210> 7
<211> 20
<212> DNA
<213>Artificial sequence
<400> 7
cctgccctgt gcaacgtgga 20
<210> 8
<211> 35
<212> DNA
<213>Artificial sequence
<400> 8
caagaaggtg gtgaactatg cagcggttga actga 35
<210> 9
<211> 35
<212> DNA
<213>Artificial sequence
<400> 9
caagaaggtg gtgaattggt tggttattcg tgcaa 35
<210> 10
<211> 20
<212> DNA
<213>Artificial sequence
<400> 10
agctggaatt aaaacgcacg 20
<210> 11
<211> 38
<212> DNA
<213>Artificial sequence
<400> 11
caagaaggtg gtgaaggttg atacacctga aacaaagc 38
<210> 12
<211> 35
<212> DNA
<213>Artificial sequence
<400> 12
caagaaggtg gtgaaatacg ctaagccacg tccat 35
<210> 13
<211> 20
<212> DNA
<213>Artificial sequence
<400> 13
ggtcctgaag caagtgcatt 20
<210> 14
<211> 35
<212> DNA
<213>Artificial sequence
<400> 14
caagaaggtg gtgaagcgaa caagctggat gtgta 35
<210> 15
<211> 35
<212> DNA
<213>Artificial sequence
<400> 15
caagaaggtg gtgaagtctt ttgctggctg gagtc 35
<210> 16
<211> 20
<212> DNA
<213>Artificial sequence
<400> 16
ttgggaatct gaattctcgg 20
<210> 17
<211> 35
<212> DNA
<213>Artificial sequence
<400> 17
caagaaggtg gtgaaccgac ttgcatttgc tctct 35
<210> 18
<211> 35
<212> DNA
<213>Artificial sequence
<400> 18
caagaaggtg gtgaagcact cgggctatgt gaaac 35
<210> 19
<211> 20
<212> DNA
<213>Artificial sequence
<400> 19
gaggtgattg cagtagggga 20
Claims (4)
- A kind of 1. neck ring primer for multiplexed PCR amplification, it is characterised in that：The primer is that a segment length is 35 ~ 50bp's Oligonucleotides；3 ' ends of the oligonucleotides are the sequence complementary with target gene, and 5 ' ends are one section of sequence artificially edited.
- A kind of 2. neck ring primer for multiplexed PCR amplification according to claim 1, it is characterised in that：Include two widows Nucleotide primer, upstream neck ring Tag primer and downstream neck ring Tag primer；Neck ring Tag primer is made up of two parts, 3 ' ends For the specific primer designed for not target-gene sequence；5 ' ends are sequence label, and the sequence label of all primers is consistent.
- A kind of 3. neck ring primer for multiplexed PCR amplification according to claim 1, it is characterised in that：5 ' described ends Sequence label is CAAGAAGGTGGTGAA.
- 4. a kind of neck ring primer for multiplexed PCR amplification as claimed in claim 1 is in multiple real time fluorescence PCR reactions Using.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711402111.5A CN107858404A (en) | 2017-12-22 | 2017-12-22 | It is a kind of for the neck ring primer of multiplexed PCR amplification and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711402111.5A CN107858404A (en) | 2017-12-22 | 2017-12-22 | It is a kind of for the neck ring primer of multiplexed PCR amplification and its application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107858404A true CN107858404A (en) | 2018-03-30 |
Family
ID=61706817
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711402111.5A Pending CN107858404A (en) | 2017-12-22 | 2017-12-22 | It is a kind of for the neck ring primer of multiplexed PCR amplification and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107858404A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110607357A (en) * | 2019-11-07 | 2019-12-24 | 益善生物技术股份有限公司 | Human PCDH10 gene methylation detection kit |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003000839A2 (en) * | 2001-06-21 | 2003-01-03 | Institut Belka Of The Russian Federation | Clinical assay for nucleic acids amplified in solid matrices to produce colonies of the progeny of individual target molecules |
| CN101671674A (en) * | 2009-03-27 | 2010-03-17 | 厦门艾德生物医药科技有限公司 | Annular primer for amplification of nucleic acid and application thereof |
| CN101842494A (en) * | 2007-07-03 | 2010-09-22 | 吉纳珀莱有限公司 | Chimeric primers for improved nucleic acid amplification reactions |
| WO2015063154A1 (en) * | 2013-10-31 | 2015-05-07 | Multiplicom N.V. | Blocking primers in multiplex pcr based assays |
| CN105524983A (en) * | 2014-09-30 | 2016-04-27 | 大连晶泰生物技术有限公司 | Marker based on high-throughput sequencing and method and kit for capturing one or multiple specific genes of multiple samples |
| CN106795569A (en) * | 2015-07-07 | 2017-05-31 | 上海真固生物科技有限公司 | The method for reducing primer dimer amplification |
-
2017
- 2017-12-22 CN CN201711402111.5A patent/CN107858404A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003000839A2 (en) * | 2001-06-21 | 2003-01-03 | Institut Belka Of The Russian Federation | Clinical assay for nucleic acids amplified in solid matrices to produce colonies of the progeny of individual target molecules |
| CN101842494A (en) * | 2007-07-03 | 2010-09-22 | 吉纳珀莱有限公司 | Chimeric primers for improved nucleic acid amplification reactions |
| CN101671674A (en) * | 2009-03-27 | 2010-03-17 | 厦门艾德生物医药科技有限公司 | Annular primer for amplification of nucleic acid and application thereof |
| WO2015063154A1 (en) * | 2013-10-31 | 2015-05-07 | Multiplicom N.V. | Blocking primers in multiplex pcr based assays |
| CN105524983A (en) * | 2014-09-30 | 2016-04-27 | 大连晶泰生物技术有限公司 | Marker based on high-throughput sequencing and method and kit for capturing one or multiple specific genes of multiple samples |
| CN106795569A (en) * | 2015-07-07 | 2017-05-31 | 上海真固生物科技有限公司 | The method for reducing primer dimer amplification |
Non-Patent Citations (7)
| Title |
|---|
| JANNINE BROWNIE等: "The elimination of primer-dimer accumulation in PCR", 《NUCLEIC ACIDS RESEARCH》 * |
| 兰全学等: "多重PCR联合同源加尾技术检测四种食源性致病菌", 《中国食品工业》 * |
| 张群: "肉制品致病菌多重PCR快速检测技术研究", 《食品与生物技术学报》 * |
| 滕勇勇等: "基于HAND系统的腹泻病原体多重PCR检测方法的建立和应用研究", 《中国优秀硕士学位论文全文数据库》 * |
| 滕勇勇等: "基于同源加尾系统的4种腹泻病毒的多重RT-PCR检测方法", 《J SOUTH MED UNI》 * |
| 马光华等: "多重荧光定量ＲT-PCＲ检测高致病性 H5N6禽流感病毒", 《临床检验杂志》 * |
| 黄松洁等: "多重荧光PCR应用于下呼吸道感染病原菌检测的研究", 《中国卫生管理标准》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110607357A (en) * | 2019-11-07 | 2019-12-24 | 益善生物技术股份有限公司 | Human PCDH10 gene methylation detection kit |
| CN110607357B (en) * | 2019-11-07 | 2023-09-19 | 益善生物技术股份有限公司 | Human PCDH10 gene methylation detection kit |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112280897A (en) | Respiratory tract infection pathogen nucleic acid joint detection kit | |
| CN105400907A (en) | Kit for nucleic acid combined detection of influenza virus A, influenza virus B and respiratory syncytial virus | |
| CN107630098B (en) | Fluorescent PCR detection architecture, kit and detection method for joint-detection various respiratory road bacterium | |
| CN104846125B (en) | A kind of fluorescence RT PCR primers, probe and kit and detection method for being used to detect MERS | |
| CN102337351B (en) | Typing detection kit for influenza virus | |
| CN103060451A (en) | Detection kit for mycoplasma pneumonia (MP) | |
| CN110408725A (en) | Kit for respiratory pathogen Multiple detection | |
| CN110273026A (en) | Respiratory tract infection Multiple detection kit and detection method | |
| CN110331232A (en) | A kind of respiratory pathogen multiple nucleic acid detection kit | |
| CN110172529A (en) | A kind of Respirovirus nucleic acid combined detection kit and detection method | |
| CN101948931A (en) | Single-tube multiplex fluorescence PCR (Polymerase Chain Reaction) detection method and kit for 1,2,3-type parainfluenza viruses | |
| CN109913564A (en) | It is a kind of for detecting primer combination of probe object, kit and the method for chlamydia pneumoniae | |
| CN105132584B (en) | For the kit and its production method of parting detection varicellazoster virus and application | |
| Horimoto et al. | Direct reverse transcriptase PCR to determine virulence potential of influenza A viruses in birds | |
| CN107858404A (en) | It is a kind of for the neck ring primer of multiplexed PCR amplification and its application | |
| CN108411014A (en) | Differentiate the primer and probe and detection method of A types and the dual real-time fluorescence quantitative PCR of Type B ox pasteurella multocida | |
| CN105950785A (en) | Ternary fluorescence RT-PCR detection kit of avian influenza virus, new castle disease virus and infectious bronchitis virus, primers and probes | |
| CN110305975A (en) | A kind of RPA kit and application thereof of quick detection chicken Mycoplasma synoviae | |
| Saputri et al. | Phylogenetic studies of Newcastle disease virus isolated from poultry flocks in South Sulawesi Province, Indonesia, in 2019 | |
| CN109371148A (en) | Identify the fluorescent PCR kit and quantitative detecting method of three kinds of porcine respiratory bacteriums | |
| CN105525038B (en) | Newcastle disease virus is strong, weak malicious one-step method real-time fluorescent RT-PCR detection kit | |
| CN105936945B (en) | A kind of multiple reverse transcription quantitative PCR kit of four kinds of Respirovirus of joint inspection | |
| CN110468223A (en) | Primer, probe, kit and the method for mycoplasma pneumoniae and Bao Te Pseudomonas detection of nucleic acids based on dUTP/UNG method | |
| CN113817870B (en) | Primer composition for simultaneously detecting seven respiratory tract related viruses and application thereof | |
| CN108950072A (en) | A kind of Porcine epidemic diarrhea virus fluorescence LAMP primer group, kit and detection method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180330 |