CN102140550A - RT(Reverse Transcription)-PCR (Polymerase Chain Reaction) kit for detecting parainfluenza virus in one step - Google Patents
RT(Reverse Transcription)-PCR (Polymerase Chain Reaction) kit for detecting parainfluenza virus in one step Download PDFInfo
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- CN102140550A CN102140550A CN 201110072754 CN201110072754A CN102140550A CN 102140550 A CN102140550 A CN 102140550A CN 201110072754 CN201110072754 CN 201110072754 CN 201110072754 A CN201110072754 A CN 201110072754A CN 102140550 A CN102140550 A CN 102140550A
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Abstract
The invention discloses a RT (Reverse Transcription)-PCR (Polymerase Chain Reaction) kit for detecting parainfluenza virus in one step, and relates to a PCR kit in the technical field of biology. The kit comprises one-step 5xRT-PCRBuffer, a positive control substance, a negative control substance, four pairs of specific primers and EnzymeMix, wherein the sequences of the specific primers 1F and 1R designed for the parainfluenza virus A are respectively 5'-CCGGTAATTTCTCATACCTATG-3' and 5'-CCTTGGAGCGGAGTTGTTAAG-3'. The kit has the advantages of short detection time period, high detection efficiency, high specificity of the detected virus, high accuracy, simple operation, easy generalization and good repeatability of the experimental result; and the kit can carry out virus quantitative analysis, the detection sensitivity of the kit is higher than that of an immunology detection method.
Description
Technical field
The present invention relates to the PCR test kit in the biological technical field, relate in particular to a kind of parainfluenza virus one one step RT-PCR (reverse transcription-polymerase chain reaction, ThermoScript II-polymerase chain reaction) detection kit.
Background technology
Parainfluenza virus belongs to paramyxovirus section, the RNA enveloped virus of strand, and multiple viruses such as it and rhinovirus, coronavirus, adenovirus all are considered to cause the pathogenic agent of common cold.Parainfluenza virus has four kinds of serotypes: 1 type can cause serious laryngitis (pseudomembrane laryngitis), trachitis or bronchitis; 2 types cause that to 1 type the clinical manifestation of illness is similar, but not serious, only when infecting 2～4 years old children, just serious cases of infection can take place; 3 types are to be only second to the baby's bronchitis of respiratory syncytial virus and the pathogenic agent of pneumonia, often cause infant's bronchiolitis and pneumonia, also can cause the pseudomembrane laryngitis; The illness that 4 types cause is lighter, causes adult and children's upper respiratory tract infection more, does not cause pneumonia usually.
Diagnosis has the infection that two kinds of methods can the diagnosing human parainfluenza virus:
1) pass through tissue culture: virus or direct the detect virus that is present in respiratory secretions of isolation identification in cell, use methods such as immunofluorescent test, enzyme linked immunoassay mensuration.
2) the remarkable rising of IgG specific antibody or detect specific antibody IgM in the single serum specimen in two parts of serum specimens collecting of appropriate time, and reach a conclusion.
There is not effective vaccine to prevent human parainfluenza virus's infection; Yet researchers are being developed the vaccine of human parainfluenza virus's 1 type and 3 types.The baby plays important effect from human parainfluenza virus's 1 type of the parent passive acquisition in there and the antibody of 2 types in the initial some months of baby's life, emphasized breastfeeding importance.Must the tight various measure of control of noting reducing and stoping pathophoresis.The propagation of often washing one's hands and can not reduce virus with the shared utensil of the infected.Isolate those and got flu and respiratory tract infection,, what meaning the propagation that reduces the human parainfluenza virus may not had, because the propagation of virus early stage in disease all usually though also ability is participated in the active child of school of nurseries and kindergartens mechanism.In hospital, should adopt rigid measures prevents human parainfluenza virus's propagation, as regular washing one's hands, dresses work clothes that protective is arranged and gloves etc. and avoids the method that directly contacts.
Summary of the invention
Purpose of the present invention just is to overcome the shortcoming and defect that prior art exists, and a kind of parainfluenza virus one one step RT-PCR kit for testing is provided.
The object of the present invention is achieved like this:
1, parainfluenza virus one one step RT-PCR kit for testing (abbreviation test kit)
This test kit is on the basis to the parainfluenza virus nucleic acid sequence analysis, chooses the conservative gene sequences Design and goes out four pairs of Auele Specific Primers, utilizes round pcr that influenza virus is detected.This technology has not only shortened the operating time, has reduced pollution, has also reduced the sample diagnosing cost, has the potential using value.
In the presence of the primer of design as follows, in the PCR instrument, viral nucleic acid is carried out pcr amplification, realize detection to parainfluenza virus.
Specifically, this test kit comprises a step 5xRT-PCR Buffer, positive control, negative control, the four pairs of Auele Specific Primers and Enzyme Mix;
1. Parainfluenza type 1 virus designs Auele Specific Primer
1F：5’-CCGGTAATTTCTCATACCTATG-3’，
1R：5’-CCTTGGAGCGGAGTTGTTAAG-3’；
Acute laryngo-tracheo-bronchitis virus design Auele Specific Primer
2F：5’-AACAATCTGCTGCAGCATTT-3’，
2R：5’-ATGTCAGACAATGGGCAAAT-3’；
Haemadsorption virus 1 design Auele Specific Primer
3F：5’-CTCGAGGTTGTCAGGATATAG-3’，
3R：5’-CTTTGGGAGTTGAACACAGTT-3’；
Parainfluenza virus type 4 design Auele Specific Primer
4F：5’-CTGAACGGTTGCATTCAGGT-3’，
4R：5’-TTGCATCAAGAATGAGTCCT-3’。
2. positive control
Be connected with the pGBKT-7 cloning vector of the gene conserved sequence of design parainfluenza virus 1,2,3,4 type primers respectively;
3. negative control
RNase?Free?H
2O；
4. 5 * RT-PCR Buffer preparation
Configuration mixes the back in refrigerator-20 ℃ preservation;
5. Enzyme Mix preparation
Configuration mixes the back in refrigerator-20 ℃ preservation.
2, the using method of test kit
1. the extraction of viral nucleic acid:
A, at first in damping fluid 1,2, add dehydrated alcohol, add 25ml and 30ml dehydrated alcohol respectively; In rinsing liquid, add 30 μ g/ml carrier RNA;
B, get 30 μ l proteolytic enzyme and put into the 1.5ml centrifuge tube;
C, sample (as throat swab, sputum etc.) is got 200 μ l add in this pipe, fully mixing;
D, add 200 μ l rinsing liquids (containing 30 μ g/ml carrier RNA) respectively at every pipe again, mixing vibration 30s, 70 ℃ of incubation 10min;
E, adding 250 μ l dehydrated alcohols, abundant mixing vibration 30s, room temperature cracking 5min;
F, above-mentioned lysate is added in the centrifugal post, 8000rpm, centrifugal 1min abandons the centrifugate in the collection tube.The filter post is still put back on the collection tube, and the 3. remaining mixed solution of step is all sucked in the filter post, abandons centrifugate after centrifugal;
Add 500 μ l damping fluids 1 in G, the filter post, 12000rpm, centrifugal 1min abandons the centrifugate in the collection tube;
H, get a clean 2ml collection tube in addition, the filter post after centrifugal is moved on on the new collection tube, add 500 μ l damping fluids 2 in the filter post, 12000rpm, centrifugal 1min, repeating step are 8. once;
I, will filter post and move on in the clean collection tube, 12000rpm, centrifugal 3min, after will filter post and be placed on 37 ℃ of 15min with dry filter membrane;
J, will filter post and be placed on the 1.5ml Eppendorf pipe, in the filter post, add 50ulRNase-free H
2O, room temperature leaves standstill 2min.12000rpm, centrifugal 2min collects the nucleic acid that centrifugate is extraction.
2. one step RT-PCR amplification (every part of 25ul system)
Get the 5ul viral nucleic acid as template, add 20ul one one step RT-PCR reaction solution simultaneously and to the PCR pipe, carry out pcr amplification.
The preparation of A, an one step RT-PCR reaction solution (mix):
C, detecting instrument:
The present invention uses ABI Veriti Thermal Cycler PCR instrument to detect.
D, result judge:
Resulting DNA product uses 2% sepharose leakage of electricity swimming, uses according to the glue instrument and comes analyzing and testing.
Then negative as no band demonstration in the swimming lane, there is band to be shown as the positive;
Having only a band to be shown as 317bp as swimming lane is Parainfluenza type 1 virus;
Having only a band to be shown as 507bp as swimming lane is acute laryngo-tracheo-bronchitis virus;
Having only a band to be shown as 189bp as swimming lane is haemadsorption virus 1;
Having only a band to be shown as 451bp as swimming lane is parainfluenza virus type 4.
The present invention has following advantage and positively effect:
1. cycle detection time is short, detection efficiency is high;
2. detect the virus-specific height, the accuracy rate height;
3. can carry out viral qualitative analysis;
4. detection sensitivity is more highly sensitive than immunological detection method;
5. simple to operate, be easy to promote;
6. experimental result good reproducibility.
Embodiment:
One, the secondary susceptible malicious one step RT-PCR kit for testing of embodiment 1---
Detect 12 parts of the susceptible malicious infected patient throat swab samples of doubtful pair, respectively get specimen fluids 200 μ l and carry out the viral nucleic acid extraction.
1, the composition of test kit:
This test kit comprises a step 5xRT-PCR Buffer, positive control, negative control, the four pairs of Auele Specific Primers and Enzyme Mix;
2, Parainfluenza type 1 virus design Auele Specific Primer
1F：5’-CCGGTAATTTCTCATACCTATG-3’，
1R：5’-CCTTGGAGCGGAGTTGTTAAG-3’；
Acute laryngo-tracheo-bronchitis virus design Auele Specific Primer
2F：5’-AACAATCTGCTGCAGCATTT-3’，
2R：5’-ATGTCAGACAATGGGCAAAT-3’；
Haemadsorption virus 1 design Auele Specific Primer
3F：5’-CTCGAGGTTGTCAGGATATAG-3’，
3R：5’-CTTTGGGAGTTGAACACAGTT-3’；
Parainfluenza virus type 4 design Auele Specific Primer
4F：5’-CTGAACGGTTGCATTCAGGT-3’，
4R：5’-TTGCATCAAGAATGAGTCCT-3’。
3,5 * RT-PCR Buffer preparation:
Configuration mixes the back in refrigerator-20 ℃ preservation.
4, Enzyme Mix preparation:
Configuration mixes the back in refrigerator-20 ℃ preservation.
5, the extraction of viral nucleic acid:
1. at first in damping fluid 1,2, add dehydrated alcohol, add 25ml and 30ml dehydrated alcohol respectively; In rinsing liquid, add 30 μ g/ml carrier RNA.
2. get 30 μ l proteolytic enzyme and put into the 1.5ml centrifuge tube.
3. sample (as the sputum of nose/throat swab, liquefaction, hydrothorax, irrigating solution etc.) is got 200 μ l and add in this pipe, fully mixing.
4. add 200 μ l rinsing liquids (containing 30 μ g/ml carrier RNA) respectively at every pipe again, mixing vibration 30s, 70 ℃ of incubation 10min.
5. add 250 μ l dehydrated alcohols, abundant mixing vibration 30s, room temperature cracking 5min.
6. above-mentioned lysate is added in the centrifugal post, 8000rpm, centrifugal 1min abandons the centrifugate in the collection tube.The filter post is still put back on the collection tube, and the 3. remaining mixed solution of step is all sucked in the filter post, abandons centrifugate after centrifugal.
7. filter adding 500 μ l damping fluids 1 in the post, 12000rpm, centrifugal 1min abandons the centrifugate in the collection tube.
8. get a clean 2ml collection tube in addition, the filter post after centrifugal is moved on on the new collection tube, in the filter post, add 500 μ l damping fluids 2,12000rpm, centrifugal 1min.Repeating step 8. once.
9. will filter post and move on in the clean collection tube, 12000rpm, centrifugal 3min, after will filter post and be placed on 37 ℃ of 15min with dry filter membrane.
10. will filter post and be placed on the 1.5ml Eppendorf pipe, in the filter post, add 50ulRNase-free H
2O, room temperature leaves standstill 2min.12000rpm, centrifugal 2min collects the nucleic acid that centrifugate is extraction.
6, one step RT-PCR amplification (every part of 25ul system)
Get the 5ul viral nucleic acid as template, add 20ul one one step RT-PCR reaction solution simultaneously and to the PCR pipe, carry out pcr amplification.
The preparation of A, an one step RT-PCR reaction solution (mix):
C, detecting instrument:
The present invention uses ABI Veriti Thermal Cycler PCR instrument to detect.
D, result judge:
Resulting DNA product uses 2% sepharose leakage of electricity swimming, uses according to the glue instrument and comes analyzing and testing.
Blob of viscose has 1 swimming lane to have only a band to be shown as 317bp, then is judged as Parainfluenza type 1 virus;
Blob of viscose has 1 swimming lane to have only a band to be shown as 507bp, then is judged as acute laryngo-tracheo-bronchitis virus;
Blob of viscose has 2 swimming lanes to have only a band to be shown as 189bp, then is judged as haemadsorption virus 1;
Blob of viscose has 1 swimming lane to have only a band to be shown as 451bp, then is judged as parainfluenza virus type 4;
Finally, detect positive 5 parts of parainfluenza virus in 12 parts of doubtful parainfluenza virus infected patient throat swab samples, wherein positive 1 example of Parainfluenza type 1 virus, positive 1 example of acute laryngo-tracheo-bronchitis virus, positive 2 examples of haemadsorption virus 1, positive 1 example of parainfluenza virus type 4, recall rate is 100%.
Two, embodiment 2
With aforesaid method other 20 parts of doubtful parainfluenza virus infected patient sputum samples are detected, detect positive 8 parts of parainfluenza virus, positive 2 examples of Parainfluenza type 1 virus wherein, positive 2 examples of acute laryngo-tracheo-bronchitis virus, positive 3 examples of haemadsorption virus 1, positive 1 example of parainfluenza virus type 4, recall rate is 100%.
Sequence table
＜110〉Wuhan University
＜120〉parainfluenza virus one one step RT-PCR kit for testing
<140>
<141>
<160>?8
<210>?1
<211>?22
<212>?DNA
＜213〉Parainfluenza type 1 virus upstream primer
<400>
5'-CCGGTAATTTCTCATACCTATG-3'。
<210>?2
<211>?21
<212>?DNA
＜213〉Parainfluenza type 1 virus downstream primer
<400>
5'-CCTTGGAGCGGAGTTGTTAAG-3'。
<210>?3
<211>?20
<212>?DNA
＜213〉acute laryngo-tracheo-bronchitis virus upstream primer
<400>
5'-AACAATCTGCTGCAGCATTT-3'。
<210>?4
<211>?20
<212>?DNA
＜213〉acute laryngo-tracheo-bronchitis virus downstream primer
<400>
5'-ATGTCAGACAATGGGCAAAT-3'。
<210>?5
<211>?21
<212>?DNA
＜213〉haemadsorption virus 1 upstream primer
<400>
5'-CTCGAGGTTGTCAGGATATAG-3'。
<210>?6
<211>?21
<212>?DNA
＜213〉haemadsorption virus 1 downstream primer
<400>
5'-CTTTGGGAGTTGAACACAGTT-3'。
<210>?7
<211>?21
<212>?DNA
＜213〉parainfluenza virus type 4 upstream primer
<400>
5'-CTGAACGGTTGCATTCAGGT-3'。
<210>?8
<211>?21
<212>?DNA
＜213〉parainfluenza virus type 4 downstream primer
<400>
5'-TTGCATCAAGAATGAGTCCT-3'。
Claims (1)
1. parainfluenza virus one one step RT-PCR kit for testing is characterized in that:
This test kit comprises a step 5xRT-PCR Buffer, positive control, negative control, the four pairs of Auele Specific Primers and Enzyme Mix;
1. Parainfluenza type 1 virus designs Auele Specific Primer
1F：5’-CCGGTAATTTCTCATACCTATG-3’，
1R：5’-CCTTGGAGCGGAGTTGTTAAG-3’；
Acute laryngo-tracheo-bronchitis virus design Auele Specific Primer
2F：5’-AACAATCTGCTGCAGCATTT-3’，
2R：5’-ATGTCAGACAATGGGCAAAT-3’；
Haemadsorption virus 1 design Auele Specific Primer
3F：5’-CTCGAGGTTGTCAGGATATAG-3’，
3R：5’-CTTTGGGAGTTGAACACAGTT-3’；
Parainfluenza virus type 4 design Auele Specific Primer
4F：5’-CTGAACGGTTGCATTCAGGT-3’，
4R：5’-TTGCATCAAGAATGAGTCCT-3’。
2. positive control
Be connected with the pGBKT-7 cloning vector of the gene conserved sequence of design parainfluenza virus 1,2,3,4 type primers respectively;
3. negative control
RNase?Free?H
2O；
4. 5 * RT-PCR Buffer preparation
Configuration mixes the back in refrigerator-20 ℃ preservation;
5. Enzyme Mix preparation
Configuration mixes the back in refrigerator-20 ℃ preservation.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102876810A (en) * | 2012-10-19 | 2013-01-16 | 中国水产科学研究院长江水产研究所 | RT-PCR (reverse transcription-polymerase chain reaction) detection kit and detection method for spring viraemia of carp virus |
| CN103993104A (en) * | 2014-05-27 | 2014-08-20 | 福建省立医院 | Primer group and probe group for detecting acute respiratory infectious diseases as well as application method and kit thereof |
| CN106048096A (en) * | 2016-08-16 | 2016-10-26 | 无锡市疾病预防控制中心 | Four pairs of upstream and downstream primers used in RT-PCR method for simultaneously detecting four respiratory viruses |
| WO2020132774A1 (en) | 2018-12-28 | 2020-07-02 | Pontificia Universidad Católica De Chile | Monoclonal antibody or antigen binding fragment thereof that binds to the l protein of the human parainfluenza virus (piv); method and kit for detecting piv |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101985665A (en) * | 2010-11-12 | 2011-03-16 | 复旦大学 | Method for detecting various respiratory viruses and primers and probes thereof |
-
2011
- 2011-03-25 CN CN 201110072754 patent/CN102140550A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101985665A (en) * | 2010-11-12 | 2011-03-16 | 复旦大学 | Method for detecting various respiratory viruses and primers and probes thereof |
Non-Patent Citations (2)
| Title |
|---|
| 《临床输血与检验》 20101030 胡晓武等 一步法实时荧光RT-PCR在快速检测甲型流感病毒中的应用 296-299 1 第12卷, 第4期 * |
| 《华南预防医学》 20080229 吴德等 人副流感病毒HPIV多重RT-PCR快速检测方法的建立 18-21 1 第34卷, 第1期 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102876810A (en) * | 2012-10-19 | 2013-01-16 | 中国水产科学研究院长江水产研究所 | RT-PCR (reverse transcription-polymerase chain reaction) detection kit and detection method for spring viraemia of carp virus |
| CN103993104A (en) * | 2014-05-27 | 2014-08-20 | 福建省立医院 | Primer group and probe group for detecting acute respiratory infectious diseases as well as application method and kit thereof |
| CN106048096A (en) * | 2016-08-16 | 2016-10-26 | 无锡市疾病预防控制中心 | Four pairs of upstream and downstream primers used in RT-PCR method for simultaneously detecting four respiratory viruses |
| WO2020132774A1 (en) | 2018-12-28 | 2020-07-02 | Pontificia Universidad Católica De Chile | Monoclonal antibody or antigen binding fragment thereof that binds to the l protein of the human parainfluenza virus (piv); method and kit for detecting piv |
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Application publication date: 20110803 |