CN104342503B - A kind of method simultaneously detecting 12 kinds of common Respiroviruses - Google Patents
A kind of method simultaneously detecting 12 kinds of common Respiroviruses Download PDFInfo
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Abstract
The invention discloses a kind of method simultaneously detecting 12 kinds of common Respiroviruses, the present invention is according to the gene conserved regions design primer of 12 kinds of common Respiroviruses (influenza A virus, Influenza B virus, influenza virus C, Parainfluenza type 1 virus, acute laryngo-tracheo-bronchitis virus, haemadsorption virus 1, rhinovirus, bocavirus, adenovirus, coronavirus, metapneumovirus and respiratory syncytial virus) and probe, the nucleic acid fragment extracted in testing sample expands, and finally uses capillary electrophoresis method separation sample.The present invention has that required sample size is low, sensitivity and accuracy is high, specificity is good, the advantage of low cost.Both having overcome conventional single tube multiple fluorescence PCR detection primer difficult design and multicolor fluorescence interferes the shortcoming being difficult to typing, and also overcomed the complex operation of chip detecting method, the shortcomings such as testing cost is high, the examination for Respirovirus provides new method.
Description
Technical field
The invention belongs to molecular biology method and carry out field of virus detection, be specifically related to 12 kinds of respiratory tracts of a kind of detection
The method of virus.
Background technology
Respirovirus is that a big class can be invaded respiratory tract and mainly causes the virus of the outer histoorgan pathological changes of respiratory tract.According to system
Meter, more than 90% acute respiratory infection is caused by virus, wherein most common with Respirovirus, including influenza virus, cacorhinia
Poison, respiratory syncytial virus, parainfluenza virus, adenovirus etc..Further, the newest pathogenic Respirovirus is also constantly
Be found, such as metapneumovirus, coronavirus, bocavirus etc., healthy to the mankind constitutes the biggest threat.Acute respiratory
Infect and there is the features such as incubation period is short, morbidity is anxious, appeal is strong, propagation is fast mostly, often may result in higher sickness rate and death
Rate, is particularly subject to environmental change and the impact of ecological balance destruction, and virus easily occurs various variations, often breaks out, and the state of an illness is fierce
Danger, mortality rate is the highest.The infection symptoms that Respirovirus causes is more similar, it is difficult to by clinical symptoms and common detection methods
Clarifying a diagnosis, therefore, a kind of method in the urgent need to high sensitivity, high specific carries out examination.
At present, the detection method of the Respirovirus that China is conventional mainly includes following several: 1, ultramicroscope microscopy
Directly virus in specimen is carried out Morphological Identification, shortcoming: 1. diagnosis efficiency is low, and some virus is difficult to directly from morphology
Make a distinction；2. need expensive device and have experience operator.2, virus purification is cultivated is viral diagnosis " goldstandard ", shortcoming:
1. waste time and energy, and need experienced operator；2. bio-safety risk is higher.3, immunologic test is most widely used is
Enzyme-multiplied immune technique (ELISA), the most first resists with one and occurs specific binding with antigen, then resist with the two of general enzyme labelling
With a specific combination of anti-generation, then by enzyme develop the color, observed result afterwards；Advantage: 1. sample throughput is higher, utilizes 96 hole enzymes
Target, can complete the detection of multiple sample simultaneously；The most sensitive: to utilize the characteristic that enzyme joins, can be by original antigen signals
Amplify；3. the time is shortened than traditional culture method.Shortcoming: false positive the most easily occurs；2. sensitivity is uncertain；3. virus antigen is sent out
Change Baoan Member false negative result.4 molecular biology PCR detection method: the most commonly used has PCR, real-time fluorescence
Quantitative PCRs etc., the specific target gene being both for virus detects.Wherein, fluorescent quantitative PCR detection method becomes the most
Ripe.Its advantage is: highly sensitive and can be with accurate quantification.But there is also following shortcoming: 1. flux is low, the most once can only examine
Survey a kind of virus, during multi-fluorescence detection, there is fluorescence interference effect testing result；2. testing cost is of a relatively high.
GenomeLab GeXP multiple gene expression genetic analysis systems capillary electrophoresis based on Beckman company maturation
Isolation technics and the research and development of highly sensitive laser Induced Fluorescence Technology form, and the capillary tube display and design in a branch of 8 roads makes full use of
The alignment characteristics of 96 orifice plates.Use multiple PCR method, by fuel labelling, same PCR pipe is analyzed multiple base simultaneously
The expression of cause, overcomes the defect that above-mentioned respiratory tract disease virus detection method exists, has the advantage that
1, high flux: use double 96 orifice plates, it is achieved 12 sites of single reaction detection, can do 192 reactions simultaneously, one day
Go out result；For cross infection person, this method can disposably provide accurately report, it is to avoid missing inspection.
2, accuracy is strong: this method uses capillary electrophoresis that PCR primer is carried out separation detection, can be by non-specific expansion
Volume increase thing, primer dimer separate with specific amplification products, at utmost reduce false positive；
3, sensitivity is high, and result is reproducible: it is inclined that this method overcomes that the unequal amplification of conventional PCR amplification method causes
Difference, improves and a set of genes of interest is carried out quantitative speed and sensitivity.
4, method is easy, uses economy: using primer mixed liquor, 12 sites of each sample detection only need once to be loaded；
The testing cost of single sample is low, beneficially large-scale promotion；
5, it easily is automated.
Summary of the invention
It is an object of the present invention to provide a kind of high flux, high sensitivity and high specific for 12 kinds of Respiroviruses
Rapid molecular biological detection method.
The present invention adopts the following technical scheme that one of the present invention often detects 12 kinds simultaneously to achieve these goals
See the reverse transcription primer group of Respirovirus, it is characterised in that include for 12 kinds of Respiroviruses and a kind of internal reference base
The reverse transcription primer of cause, its sequence is shown in Table 1:
Table 1
Wherein bocavirus and adenovirus are without RT primer, a kind of internal reference gene behaviour RNA internal reference, the reverse transcription of its gene
Primer is: CGGCATCTTCAAACCTCCAT.
In the PCR primer group of 12 kinds of common Respiroviruses of a kind of detection simultaneously, including for 12 kinds of respiratory tract disease
Poison and the PCR primer of 3 kinds of internal reference genes, its sequence is shown in Table 2:
Table 2
Wherein bocavirus and adenovirus have positive and negative PCR primer, the PCR primer of 3 kinds of internal reference genes: 1. people RNA internal reference
PCR primer sequence be: GCCGTGTGAACCATGTGAC；2. the primer sequence of people DNA internal reference is: 5 '
CGCTAGGAATCAGACCAACAC and 5 ' ATGGCGGTGTTTGCAGAT；3. the primer sequence reacting internal reference is: 5 '
GCCAGATATACGCGTTGACA and 5 ' AGGGCGTACTTGGCATATGAT.
A kind of method simultaneously detecting 12 kinds of common Respiroviruses of the present invention, it is characterised in that include following
Step:
(1) choose suitable genes of interest correlated series, design the special of 12 kinds of common Respiroviruses of specific detection
The nucleotide sequence such as table 3 below that property primer is concrete:
Table 3
(2) take test sample and extract total nucleic acid；
(3) reverse transcription is carried out with the total nucleic acid extracted for template；
(4) set up RT and PCR reaction system, above-mentioned primer and each reaction component are placed in PCR instrument, add tested
The DNA profiling of sample extraction or reverse transcription product, expand；
(5) sample separation, qualification are carried out with the method for capillary electrophoresis.
Described reverse transcription primer includes that the RT amplimer of 10 kinds of Respiroviruses and the RT amplification of people's RNA internal reference are drawn
Thing, described PCR primer includes forward and reverse PCR primer of 2 kinds of Respiroviruses, forward and reverse pcr amplification primer of people's DNA internal reference
Thing, forward and reverse pcr amplification primer thing of reaction internal reference and the pcr amplification primer thing of above-mentioned 10 kinds of Respiroviruses and people's RNA internal reference
Pcr amplification primer thing.
Test sample in described step (2) includes that patient's includes that throat swab, nose swab, nasopharynx extract thing, Pharyngeal aspirate
And sputum.
Described step (3), RT reaction system and reaction condition be: the nucleic acid samples 5 μ L of 5-20ng/ μ L, DEPC water 8 μ L, 5
× RT buffer 4 μ L, RT primer 2 μ L, RT enzyme 1 μ L；Reverse transcription is carried out after mixing；Reaction condition is: 48 DEG C 1 minute, 42 DEG C
60 minutes, 95 DEG C 5 minutes, 4 DEG C until collecting RT product, in reverse transcription primer solution, the concentration of each RT primer is 500nM.
Described step (4), PCR reaction system and reaction condition: RT product 8.6 μ L, 10 × PCR buffer 2 μ L, 25mM chlorine
Change magnesium 4 μ L, PCR primer solution 2 μ L, archaeal dna polymerase 1.4 μ L, X solution 2 μ L；Join after mixing that to carry out PCR in PCR pipe anti-
Should, reaction condition is: 95 DEG C 10 minutes, 94 DEG C 30 seconds, 55 DEG C 30 seconds, 70 DEG C 1 minute, 35 circulations, 70 DEG C 1 minute, 4
DEG C until collecting PCR primer；In PCR primer solution, various PCR primer concentration are 200nM；Described X solution is for including three phosphorus
Acid Deoxydization nucleotide and universal primer, described universal primer forward amplimer sequence is that GTACGACTCACTATAGGGA is anti-
It is AGGTGACACTATAGAATA to amplimer sequence, described universal primer forward amplimer band fluorescent labeling.
Described step (5) includes preparing genetic analyzer sample, preparation separates liquid, electrocapillary phoresis separation sample, result are divided
The step of analysis.
Described reaction system sets up positive control and negative control；Described positive control is to use above-mentioned 12 kinds of respiratory tracts
The gene recombinant clone plasmid of the primer of virus, RNA internal reference, DNA internal reference and reaction internal reference；Described negative control is not make
Virus sampling liquid.
Of the present invention a kind of simultaneously detect 12 kinds of common Respiroviruses test kit, it is characterised in that: include as
Detect the reverse transcription primer group of 12 kinds of common Respiroviruses while above-mentioned, be specifically shown in Table 1 and detect 12 described above simultaneously
Plant the PCR primer group of common Respirovirus, be specifically shown in Table 2.
Specifically, it is achieved the above-mentioned purpose present invention adopts the following technical scheme that the present invention often detects 12 kinds for simultaneously
See that the method for Respirovirus comprises the following steps (1) and utilizes bio information to gain knowledge and relevant biological information software, to public affairs
Open the 12 kinds of common Respiroviruses that can retrieve in data base and choose the suitable specific sequence relevant to genes of interest,
Design the reverse transcription primer of corresponding gene fragment for 12 kinds of common Respiroviruses of specific detection, PCR primer；(2)
Take test sample and extract total nucleic acid；(3) carrying out reverse transcription with the total nucleic acid template extracted, wherein bocavirus and adenovirus need not
Design RT primer；(4) design is for the PCR primer of the corresponding gene fragment of 12 kinds of common Respiroviruses of specific detection, its
Middle bocavirus and adenovirus have positive and negative PCR primer, other 10 kinds PCR primer a kind of, then set up PCR reaction system,
Above-mentioned primer and each reaction component are placed in PCR instrument, add reverse transcription product, expand；(5) with capillary electrophoresis
Method carry out sample separation, qualification.
Based on 12 kinds of viral characteristic gene sequences, design the specific detection primer for various viruses, reverse transcription
Primer includes the RT primer with in following table 12 kinds of Respiroviruses and the RT amplimer of people's RNA internal reference, and PCR primer includes following
The pcr amplification primer thing of remaining 2 kinds of Respiroviruses in table, forward and reverse pcr amplification primer thing of people's DNA internal reference, reaction internal reference
Forward and reverse pcr amplification primer thing and the pcr amplification primer thing of above-mentioned 12 kinds of Respiroviruses and the pcr amplification primer thing of people's RNA internal reference,
Shown in gene order table described above 3；
Wherein, reaction component and the reaction condition of described reverse transcription reaction system be: the nucleic acid samples 5 μ L of 5-20ng/ μ L,
DEPC water 8 μ L, 5 × RT buffer 4 μ L, RT primer 2 μ L, RT enzyme 1 μ L；Reverse transcription is carried out after mixing；Reaction condition is: 48 DEG C 1
Minute, 42 DEG C 60 minutes, 95 DEG C 5 minutes, 4 DEG C until collecting RT product, the concentration of each RT primer in reverse transcription primer solution
For 500nM.
Reaction component and the reaction condition of described polymerase chain reaction system be: RT product 8.6 μ L, 10 × PCR buffer
Liquid 2 μ L, 25mM magnesium chloride 4 μ L, PCR primer solution 2 μ L, archaeal dna polymerase 1.4 μ L, X solution 2 μ L；PCR pipe is joined after mixing
In carry out PCR reaction, reaction condition is: 95 DEG C 10 minutes, 94 DEG C 30 seconds, 55 DEG C 30 seconds, 70 DEG C 1 minute, 35 circulations,
70 DEG C 1 minute, 4 DEG C until collecting PCR primer.In PCR primer solution, various PCR primer concentration are 200nM；Described X is molten
Liquid is for including triphosphate deoxy-nucleotide and universal primer, and described universal primer forward amplimer sequence is
GTACGACTCACTATAGGGA reverse amplimer sequence is AGGTGACACTATAGAATA, and described universal primer forward expands
Increase primer band fluorescence (Cy5) labelling.
Separation and the detection of described product are as follows: take PCR primer 1 μ L, the sample-loading buffer that GeXP genetic analyzer is supporting
38.75 μ L, DNA standard substance 0.5 μ L, join after 1 mix homogeneously of mineral oil and carry out capillary electrophoresis separation in sample panel, will
The collection of illustrative plates that the software of GeXP genetic analyzer obtains and standard diagram comparison, thus judge the kind of Respirovirus.
Compared with prior art, it is an advantage of the current invention that: the present invention can realize rapidly in unknown sample be
No carry above-mentioned 12 kinds of Respiroviruses and detect simultaneously and identify, and 192 Patient Sample A within one day, can be completed
Detection, both saved production cost and testing cost, and improve again detection efficiency and shorten the time.Also can enter as required
Row target site adjusts.
In sum, the present invention is 12 kinds of respiratory tracts of synchronous detecting based on GeXP multiple gene expression genetic analysis systems
The detection method of virus, can be simultaneous for 12 kinds of Respiroviruses and detect, and detection sensitivity is high, and specificity is good, reduces
The false positive rate of standard PCR amplification, additionally it is possible to effectively solve the easy pollution problem of Standard PCR；There is Noncompetitive internal comparison
System, highly reliable, without false negative result, utilize GeXP genetic analysis systems technical advantage sensitive, quick, high-throughout, can
It is applied to entry and exit port, Disease Control and Prevention Center, hospital and the rapid screening of other medical institutions' Respiroviruses and qualification.
Accompanying drawing explanation
Fig. 1 is the negative control electrophoresis pattern (peak is InControl) without template.
Fig. 2 is negative sample electrophoresis pattern (peak is from left to right followed successively by: huRNA/huDNA/InControl).
Fig. 3 is that (peak is from left to right followed successively by influenza A positive electrophoresis pattern: huRNA/InfA/huDNA/
InControl).
Fig. 4 is that (peak is from left to right followed successively by adenovirus positive electrophoresis pattern: huRNA/HADV/huDNA/
InControl).
Fig. 5 is that (peak is from left to right followed successively by parainfluenza 1 type positive electrophoresis pattern: huRNA/HPIV1/huDNA/
InControl).
Fig. 6 is that (peak is from left to right followed successively by influenza C positive electrophoresis pattern: huRNA/InfC/huDNA/
InControl).
Fig. 7 is that (peak is from left to right followed successively by bocavirus positive electrophoresis pattern: huRNA/Boca/huDNA/
InControl).
Fig. 8 is that (peak is from left to right followed successively by rhinovirus positive electrophoresis pattern: huRNA/HRV/huDNA/
InControl).
Fig. 9 is that (peak is from left to right followed successively by parainfluenza 3 type positive electrophoresis pattern: huRNA/HPIV3/huDNA/
InControl).
Figure 10 is that (peak is from left to right followed successively by parainfluenza 2 type positive electrophoresis pattern: huRNA/HPIV2/huDNA/
InControl).
Figure 11 is that (peak is from left to right followed successively by metapneumovirus positive electrophoresis pattern: huRNA/HMPV/huDNA/
InControl).
Figure 12 is that (peak is from left to right followed successively by influenza B positive electrophoresis pattern: huRNA/InfB/huDNA/
InControl).
Figure 13 be coronavirus positive electrophoresis pattern (peak is from left to right followed successively by: huRNA/huDNA/HCOV/
InControl).
Figure 14 be respiratory syncytial virus positive electrophoresis pattern (peak is from left to right followed successively by: huRNA/huDNA/
HRSV/InControl).
Figure 15 is that (peak is from left to right followed successively by all positive: huRNA/InfA/HADV/ with comparison electrophoresis pattern
HPIV1/InfC/Boca/HRV/HPIV3/HPIV2/HMPV/InfB/huDNA/HCOV/HRS V/InControl).
Figure 16 is that influenza A virus susceptiveness tests electrophoresis pattern, in figure: A. 40 copy/ μ L, B. 80 copy/ μ
L, C. 160 copy/ μ L, A, B, C are the electrophoresis pattern of different susceptivenesss.
Figure 17 is specific test electrophoresis pattern (peak is from left to right followed successively by: huRNA/huDNA/ InControl).
Detailed description of the invention
For making the present invention easier to understand, below in conjunction with specific embodiment, the present invention is expanded on further.Should be understood that this
A little embodiments are merely to illustrate the present invention rather than limit the scope of the present invention, NM specific experiment in the following example
Method, is generally carried out according to normal experiment method.
It is embodied as one:
Sample nucleic acid extracts
Gather clinical samples and extract total nucleic acid: gathering the Nasopharyngeal swabs of patient, the isolated culture of sputum, from separating training
Support and thing extracts total nucleic acid.The present invention designs the nucleoside that the specific primer of 12 kinds of common Respiroviruses of specific detection is concrete
Acid sequence.
The present invention is directed to 12 kinds of Respiroviruses and the reverse transcription primer of a kind of internal reference gene, its sequence is shown in Table 1:
Table 1
Wherein bocavirus and adenovirus are without RT primer, a kind of internal reference behaviour RNA internal reference, the reverse transcription primer of its gene
For: CGGCATCTTCAAACCTCCAT.
The present invention is directed to 12 kinds of Respiroviruses and the PCR primer of 3 kinds of internal reference genes, its sequence is shown in Table 2.
Table 2
Wherein bocavirus and adenovirus have positive and negative PCR primer, the PCR primer of 3 kinds of internal reference genes: 1. people RNA internal reference
PCR primer sequence be: GCCGTGTGAACCATGTGAC；2. the primer sequence of people DNA internal reference is: 5 '
CGCTAGGAATCAGACCAACAC and 5 ' ATGGCGGTGTTTGCAGAT；3. the primer sequence reacting internal reference is: 5 '
GCCAGATATACGCGTTGACA and 5 ' AGGGCGTACTTGGCATATGAT.
It is embodied as two: carry out reverse transcription with the total nucleic acid extracted for template
RT reaction system and reaction condition be: the nucleic acid samples 5 μ L of 5-20ng/ μ L, the present embodiment uses 10 ng/ μ L's
Nucleic acid samples 5 μ L.
Post transcription cloning (RT) reacts
(1) in PCR pipe, kit sample is added in the following proportions
| RT reaction reagent | Amount/hole |
| DEPC water | 8μL |
| 5 × RT buffer | 4μL |
| RT primer (each RT primer concentration is 500nM) | 2μL |
| RT enzyme | 1μL |
| Sample (5-20ng/ μ L) | 5μL |
| Total | 20μL |
Note: in reverse transcription primer solution, the concentration of each RT primer is 500nM；Positive reference substance, sun is added in RT reacts
Property reference substance be by each target viral clone gained, comprise purpose fragment of plasmid, consumption be 1 μ L/ reaction.By following temperature after mixing
Degree is hatched:
| Step | Temperature | Time |
| 1 | 48℃ | 1 minute |
| 2 | 42℃ | 60 minutes |
| 3 | 95℃ | 5 minutes |
| 4 | 4℃ | Continue: until collecting RT product |
It is embodied as three
Polymerase chain reaction (PCR) amplified reaction, PCR reaction system and reaction condition: use the RT being embodied as two
Product 8.6 μ L.
PCR reaction is carried out for template with reverse transcription product.Addition kit sample in PCR pipe the most in the following proportions:
| PCR reaction reagent | Amount/hole |
| 10 × PCR buffer | 2μL |
| 25mM magnesium chloride solution | 4μL |
| PCR primer | 2μL |
| Archaeal dna polymerase | 1.4μL |
| X solution | 2μL |
| RT product | 8.6μL |
| Total | 20μL |
Note: in PCR primer solution, various PCR primer concentration are 200nM；X solution is for including deoxyadenosine triphosphate acid
(dNTPs) and universal primer, described universal primer forward amplimer sequence is GTACGACTCACTATAGGGA；Reversely expand
Increasing primer sequence is AGGTGACACTATAGAATA.Described universal primer forward amplimer band fluorescent labeling.
2. thermal cycle reaction is carried out by temperature below after mixing:
| Step | Temperature | Time |
| 1 | 95℃ | 10 minutes |
| 2 | 94℃ | 30 seconds |
| 3 | 55℃ | 30 seconds |
| 4 | 70℃ | 1 minute |
| 5 | N/A | Repeat 2-4 step 34 time (totally 35 times) |
| 6 | 70℃ | 1 minute |
| 7 | 4℃ | Continue: until collecting PCR primer |
It is embodied as four
GeXP genetic analyzer electrocapillary phoresis carries out virus purification, qualification
1. GeXP sample is prepared:
| GeXP sample | Amount/hole |
| Sample-loading buffer | 38.75μL |
| DNA size criteria 400 | 0.5μL |
| PCR primer | 1μL |
| Mineral oil | 1 |
2. electrocapillary phoresis separation sample.GeXP sample is added in sample panel and an appropriate number of hole carries out capillary electrophoresis
Separate；Capillary electrophoresis separation is the class Novel liquid-phase with capillary tube as split tunnel, with high-voltage dc as driving force
Isolation technics, specific procedure is 90 DEG C of degeneration 120 seconds, and sample introduction voltage is 2kV, 30 seconds, and separation voltage is 6kV, 35 minutes.
Fig. 1-Figure 14 is shown in by each concrete sample electrophoresis collection of illustrative plates.
It is embodied as five
Interpretation of result
Have the parameter of acquiescence on software by oneself according to GeXP genetic analyzer and result is carried out clip size analysis, its abscissa
Representing clip size, vertical coordinate represents that signal is strong and weak.The collection of illustrative plates that the software of GeXP genetic analyzer is obtained and standard diagram pair
Ratio, it is judged that the kind of Respirovirus.As shown in figure 15, its result can accurately detect 12 kinds of Respiroviruses to standard diagram,
Each target fragment size interval is moderate, and signal is unlikely to supersaturation, and between each target, signal is the most fair, and does not has broad peak, double
The phenomenons such as peak.
It is embodied as six
The sensitivity of detection method and specificity analyses
Sensitive analysis: by positive control according to certain copy number doubling dilution after, through PCR amplification and capillary electrophoresis examine
Surveying until can't detect signal, this copy number is lowest detection line, the namely sensitivity of test kit.Maximum sensitivity can be examined
Measure 40 copies.See Figure 16.
Specificity analyses: substance PCR amplification is detected as the unimodal of target fragment size through capillary electrophoresis.Detect after optimization
Carrying out the detection of single template system internal specific in system to evaluate, have no non-specific signals, system internal specific is good；Optimize
Rear detection system adds staphylococcus aureus, Pseudomonas aeruginosa, escherichia coli, the mixed nucleus of Klebsiella Pneumoniae
Acid, has no non-specific signals, and the outer specificity of system is good.See Figure 17.
It is embodied as seven
The 50 parts of throat swab clinical samples having heating paresthesia crowd are detected by the method using the present invention, testing result
It is shown in Table 4.The judgement of testing result should meet following 2 conditions simultaneously, and otherwise result of the test is invalid:
1. 3 kinds of internal reference peak-to-peak signal intensity equal ＞ 2000A.U；
2. negative control peak-to-peak signal intensity all≤2000A.U.
It is judged as positive findings as target peak-to-peak signal intensity ＞ 2000A.U.
It is judged as negative findings when target peak-to-peak signal intensity is more than≤2000A.U.
Table 4 throat swab clinical sample (50 parts) carries out testing result
Note: "+" represent: peak-to-peak signal intensity ＞ 2000A.U；" " represents: peak-to-peak signal intensity≤2000A.U.
Described above not limitation of the present invention, present invention is also not necessarily limited to the example above.The common skill of the art
Art personnel, in the essential scope of the present invention, the change made, retrofit, add and replace, and also should belong to the protection model of the present invention
Enclose.
Last institute is it should be noted that, the present invention is only protected by above example in order to technical scheme to be described
Protecting the restriction of scope, although being explained in detail the present invention with reference to preferred embodiment, those of ordinary skill in the art should
Understand, technical scheme can be modified or equivalent, without deviating from the essence of technical solution of the present invention
And scope.
Claims (1)
1. the test kit simultaneously detecting 12 kinds of common Respiroviruses, it is characterised in that: include the RT primer sets in table 1,
PCR primer group in table 2, reverse transcription buffer, PCR buffer, 25mM magnesium chloride, reverse transcription, archaeal dna polymerase, fluorescence mark
Note thing, positive control and X solution, described X solution includes triphosphate deoxy-nucleotide and universal primer, described universal primer
Forward amplimer sequence be GTACGACTCACTATAGGGA reverse amplimer sequence be AGGTGACACTATAGAATA, institute
The universal primer forward amplimer band fluorescent labeling stated；
Table 1
Table 2
。
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| CN105018488B (en) * | 2015-08-03 | 2017-10-27 | 博奥生物集团有限公司 | Kit and its application for detecting Respirovirus |
| CN105734168B (en) * | 2015-10-28 | 2019-05-17 | 南京美宁康诚生物科技有限公司 | A kind of 12 kinds of Respirovirus nucleic acid multiple PCR detection kits |
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| CN113817870B (en) * | 2021-09-10 | 2023-12-22 | 宁波海尔施基因科技股份有限公司 | Primer composition for simultaneously detecting seven respiratory tract related viruses and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101985665B (en) * | 2010-11-12 | 2013-11-06 | 复旦大学 | Method for detecting various respiratory viruses and primers and probes thereof |
| CN102839223B (en) * | 2011-06-22 | 2015-04-22 | 中国疾病预防控制中心病毒病预防控制所 | Application of GeXP multiplex gene expression genetic analysis system in genotyping of 16 common respiratory viruses |
| CN102994648B (en) * | 2012-06-21 | 2015-04-08 | 宁波海尔施基因科技有限公司 | Multi-gene detection method of respiratory viruses based on capillary electrophoresis |
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