CN108034716A - For differentiating whether patient infects and infect the kit of species - Google Patents
For differentiating whether patient infects and infect the kit of species Download PDFInfo
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- CN108034716A CN108034716A CN201810069209.1A CN201810069209A CN108034716A CN 108034716 A CN108034716 A CN 108034716A CN 201810069209 A CN201810069209 A CN 201810069209A CN 108034716 A CN108034716 A CN 108034716A
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- 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
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- 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
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- C12Q1/6851—Quantitative amplification
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- 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
- C12Q2600/00—Oligonucleotides characterized by their use
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Abstract
It is used to differentiate whether patient infects and infect the kit of species the present invention relates to a kind of, the kit includes the reagent of the reagent of the expression of the gene IFI44L and/or FAM89A of detection patient and the expression of house-keeping gene.The kit of the present invention can accurately detect the gene expression abundance of two genes of IFI44L and FAM89A, so that it is virus or bacterium to judge whether patient infects and infected, and is conducive to clinic and is targetedly treated.
Description
Technical field
The present invention relates to disease detection field, is more particularly to used to differentiate whether patient infects and infect the reagent of species
Box.
Background technology
Respiratory tract infection and alimentary canal are one of most common communicable diseases of the mankind, and childrens respiratory tract and alimentary canal sense
Dye is even more one of major public health problem for facing of children, has become the primary of less than 5 years old death of child in developing country
The cause of disease.Research report preschool child suffers from respiratory tract and infection of digestive canal 6-10 times per annual, and have 1 years old of about 3% with
Lower children due to middle severe respiratory tract and infection of digestive canal and hospitalization.
Trigger respiratory tract and the Pathogen category of infection of digestive canal complexity, existing virus, also there is bacterium, mycoplasma etc..This
After a little pathogen trigger respiratory tract infection, the clinical manifestation of patient often has fever, cough, pharyngalgia, runny nose, can be with whole body
The constitutional symptom such as powerless, dizzy, DOMS；Then there is water sample stool, or even bloody stool in infection of digestive canal.Clinically area first
Separate virus infection or bacterium infection, the selection to clinical management strategy have very great help.Such as if virus infects,
Do not have to antibiosis extract for treating then, can prevent abuse of antibiotics.
Clinically distinguishing virus and bacterium infection at present has had certain methods, such as thin by the neutral grain in peripheral blood
Born of the same parents and the change of lymphocyte content, and c reactive protein detection, substantially to distinguish bacterium or virus infection, but it is existing
These methods specificity and sensitivity it is inadequate, urgent clinical needs specificity and sensitivity better method distinguish bacterium and virus
Infection.
International academic community has attempted to distinguish from the difference of the gene expression for having core disease in the blood of host thin
Bacterium and virus infect, because after bacterium and virus infection body, there is the mechanism difference of activation host immune system approach, just must
So there are directed toward bacteria and the gene of virus infection differential expression in host cell.
The content of the invention
It is an object of the invention to overcome the shortcomings of that the prior art distinguishes bacterium and viral infectious process, IFI44L is picked
With two genes of FAM89A, for the two genes, and internal reference GAPDH genes；Devise the primer of reverse transcription PCR and glimmering
Light TaqMan probe, just can accurately detect the gene expression abundance of two genes of IFI44L and FAM89A in a tube reaction, so that
Judge that patient has infected virus or bacterium.
In one embodiment, the present invention is provided to differentiate whether patient infects and infect the kit of species, institute
Stating kit includes detecting the expression of the reagent and house-keeping gene of the expression of the gene IFI44L and/or FAM89A of patient
Horizontal reagent, the house-keeping gene is preferably Gene A CTB and GAPDH.
In one embodiment, the kit is detection gene IFI44L, FAM89A and house-keeping gene GAPDH tri-
Gene one-step method reverse transcription PCR kit.
In one embodiment, the kit includes following primer：Primer for the amplification of people GAPDH gene PCRs:
Forward primer SEQ ID NO:1, gctcatttcctggtatgacaacg, reverse primer SEQ ID NO:2,
ctccccagcagtgagggt；Primer for the amplification of people IFI44L gene PCRs：Forward primer SEQ ID NO:3,
Tgcactgaggcagatgct, SEQ ID NO:4, gtacaggctgggccaaat；With expand for people IFI44L gene PCRs
Primer：Forward primer SEQ ID NO:5, aagcctcccaacctggac；Reverse primer SEQ ID NO:
6tcatcgaagaagccgttc。
In one embodiment, the kit includes following probe：Probe SEQ for people's GAPDH genetic tests
ID NO:9：ccctgttgctgtagccaaattcgt；Probe SEQ ID NO for people's IFI44L genetic tests:10：
aactggtgcaattgagagagcgt；With the probe SEQ ID NO for people's FAM89A genetic tests:11：
agaccaaccatctctttgcgga。
In one embodiment, the kit includes following probe：SEQ ID NO:9 fluorescence probes mark is as follows：
FAM-ccctgttgctgtagccaaattcgt-Dabcyl；SEQ ID NO:10 fluorescence probes mark is as follows：TAMRA-
aactggtgcaattgagagagcgt-BHQ2；With SEQ ID NO:11 fluorescence probes mark is as follows：JOE-
agaccaaccatctctttgcgga-BHQ1。
Brief description of the drawings
, below will be to needed in the embodiment in order to illustrate more clearly of the technical solution in the embodiment of the present application
Attached drawing is briefly described, it should be apparent that, drawings in the following description are only some embodiments described in the application, right
For those of ordinary skill in the art, without creative efforts, it can also be obtained according to these attached drawings
Its attached drawing.
Fig. 1 a are the primer PCR amplification figures before FAM89A optimizations, and Fig. 1 b are the primer PCR expansions after FAM89A optimizations
Increase result figure；
Fig. 2 is fluorescence quantitative RT-RCR response diagram, wherein inner line-GAPDH genes, direct lines-IFI44L genes,
Outer lines-FAM89A genes；
Fig. 3 is gene expression abundance scatter diagram of the IFI44L genes in normal population and viral infection population；With
Fig. 4 is gene expression abundance scatter diagram of Fig. 4 FAM89A genes in normal population and bacterium infection crowd.
Embodiment
In order to make art technology field personnel more fully understand the technical solution in the application, below in conjunction with following reality
Applying example, the invention will be further described, it is clear that and described embodiments are only a part of embodiments of the present application, rather than entirely
The embodiment in portion.Based on the embodiment in the application, those of ordinary skill in the art are without making creative work
The all other embodiment obtained, should all belong to the scope of the application protection.
First, multiple PCR primer designs
Gene expression abundance except needing detection IFI44L and FAM89A genes, it is also necessary to detect the table of house-keeping gene GAPDH
Reference up to abundance as data normalization, it is therefore desirable to while one-step method reverse transcription PCR amplifies three genes in a pipe,
So as to first need to design the one-step method reverse transcription PCR primer of three genes, referring to table 1.
Table 1. detects the one-step method reverse transcription PCR primer of three genes
Above-mentioned primer is in optimized selection by the present inventor, they not only ensure that above three gene in one-step method
Expanded in reverse transcription PCR, and above-mentioned primer greatly strengthen the specificity of amplification so that amplified production specificity is more
It is good,
It is the front and rear sequence of FAM89A gene primers optimization below：
Sequence before the optimization of table 2FAM89A gene primers
Fig. 1 a and Fig. 1 b are the front and rear comparisons of FAM89A gene primers optimization, and the primer in Fig. 1 a before FAM89A optimizations produces
Many non-specific amplifications, background are high；Primer specificity after Fig. 1 b optimizations is strong, and background is low.
2nd, probe designs
For the TaqMan probe sequence such as table 3 of three genes.
The TaqMan probe sequence of 3. 3 genes of table
3rd, PCR amplification
The clinical sample for the specimen types periphery whole blood that the present invention is detected, blood sample were placed in in vitro 30 minutes
(hundred Tyke Bioisystech Co., Ltd of Beijing, 200L whole bloods are placed on 750L's in TRIpure LS Reagent whole blood lysates
In lysate), -20 DEG C can preserve for a long time.When needing to use RNA, it can be carried with TRIZOL (Life Technologies companies)
Take sample rna.One-step method reverse transcription PCR reaction system uses the one-step method reverse transcription PCR system of Dalian Bao Sheng biotech firms, such as
Table 4, reaction condition such as table 5.
The A reaction systems of table 4.RT-PCR cloning RNA viruses
| Title | Volume (25 μ L reaction systems) | Final concentration |
| 2×SuperRT OneStep Buffer | 12.5 | 1× |
| Forward Primer mix (every kind of 10 μM) | 1 | Every kind of 0.4M |
| Reverse Primer mix (every kind of 10 μM) | 1 | Every kind of 0.4M |
| SuperRT OneStep EnzymeMix | 0.5 | |
| It is nucleic acid-templated | 2 | |
| Moisturizing | 8 |
Table 5.RT-PCR reaction conditions
On quantitative fluorescent PCR instrument ABI7500, the Representative fluorescence quantitative reaction curve map ginseng of three genes of one-time detection
See Fig. 2.Fig. 2 fluorescence quantitative RT-RCR response diagrams.Inner line-GAPDH genes, direct lines-IFI44L genes, out conductor
Bar-FAM89A genes
5. the application of kit of the present invention
Examined with the fluorescent quantitative RT-PCR method for IFI44L, FAM89A and GAPDH gene designed by the present invention
Survey human peripheral karyocyte gene expression abundance.First be directed to normal population peripheral blood, detection wherein IFI44L, FAM89A and
The gene expression abundance of GAPDH genes；Then there is the patient of respiratory tract infection and diarrhea infection symptoms for clinic, we are first with 13
Respiratory pathogen liquid-phase chip, and the pathogen classification that 12 diarrhoeal diseases substance chip detection patients specifically infect, Ran Houzai
Test and analyze the gene expression abundance of peripheral blood in patients IFI44L, FAM89A and GAPDH gene.13 respiratory pathogen liquid-phase chips
Including 8 kinds of virus 5 kind bacteriums, it is specifically：Influenza A virus (INF-A), influenza virus B type (INF-B), parainfluenza virus
(PIV), human metapneumovirus (hMPV), rhinovirus (HRV), bocavirus (hBoV), adenovirus (R-Adv), respiratory syncystial disease
Malicious (RSV), mycoplasma pneumoniae (MP), moraxelle catarrhalis (MC), haemophilus influenzae (Hi), streptococcus pneumonia (SP), pertussis
Bacillus (BP), 11 diarrhoeal diseases substance chips include 5 kinds of virus, 6 kinds of bacteriums, are specifically adenovirus (D-Adv), astrovirus
(AsV), norovirus (GI), rotavirus (RoV), bed ripples viral (SapV), Listeria monocytogenes (Lis), salmonella
(Sal), Shigella (SC), campylobacter jejuni (CJ), comma bacillus (VC), clostridium difficile (CD).
The detection data of 20 samples in 6. normal population of table
The detection data of 50 samples in 7. infection population of table
The data preparation of table 6, table 7 into figure, respectively such as Fig. 3 and Fig. 4.
It can be found that analysis IFI44L gene expression doses, in the crowd of virus infection, hence it is evident that higher than normal population；
And FAM89A gene expression doses, in the crowd of bacterium infection, hence it is evident that higher than normal population.Therefore IFI44L expression rise,
It may determine that and infected for virus；And FAM89A gene expressions raise, it can be determined that be bacterium infection.
It should be understood that the present invention disclosed is not limited only to specific method, scheme and the material of description, because these
Equal alterable.It will also be understood that purpose of the terminology used here just for the sake of the specific embodiment scheme of description, rather than
It is intended to limit the scope of the invention, the scope of the present invention is limited solely by appended claim.
Those skilled in the art, which will also be appreciated that or be able to confirm that, uses no more than normal experiment, institute herein
The many equivalents for the specific embodiment of the invention stated.These equivalents are also contained in appended claim.
Sequence table
<110>Guangzhou Ao Baiquepu bio tech ltd
<120>For differentiating whether patient infects and infect the kit of species
<130> BR3665
<141> 2018-01-24
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
gctcatttcc tggtatgaca acg 23
<210> 2
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
ctccccagca gtgagggt 18
<210> 3
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
tgcactgagg cagatgct 18
<210> 4
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
gtacaggctg ggccaaat 18
<210> 5
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
aagcctccca acctggac 18
<210> 6
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 6
tcatcgaaga agccgttc 18
<210> 7
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 7
caacctggac gccgctct 18
<210> 8
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 8
ccccttgtac tcctgaatcg act 23
<210> 9
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 9
ccctgttgct gtagccaaat tcgt 24
<210> 10
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 10
aactggtgca attgagagag cgt 23
<210> 11
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 11
agaccaacca tctctttgcg ga 22
Claims (5)
1. for differentiating whether patient infects and infect the kit of species, it is characterised in that the kit includes detection patient
Gene IFI44L and/or FAM89A expression reagent and house-keeping gene expression reagent, it is described special
Gene is preferably Gene A CTB and GAPDH.
2. according to the kit described in claim 1, it is characterised in that the kit is detection gene IFI44L, FAM89A
With tri- gene one-step method reverse transcription PCR kits of house-keeping gene GAPDH.
3. according to the kit described in claim 2, it is characterised in that the kit includes following primer：For people
The primer of GAPDH gene PCRs amplification:Forward primer SEQ ID NO:1, gctcatttcctggtatgacaacg, reverse primer
SEQ ID NO:2, ctccccagcagtgagggt；Primer for the amplification of people IFI44L gene PCRs：Forward primer SEQ ID
NO:3, tgcactgaggcagatgct, SEQ ID NO:4, gtacaggctgggccaaat；With for people's IFI44L gene PCRs
The primer of amplification：Forward primer SEQ ID NO:5, aagcctcccaacctggac；Reverse primer SEQ ID NO:
6tcatcgaagaagccgttc。
4. according to the kit described in claim 3, it is characterised in that the kit includes following probe：For people
The probe SEQ ID NO of GAPDH genetic tests:9：ccctgttgctgtagccaaattcgt；For people's IFI44L genetic tests
Probe SEQ ID NO:10：aactggtgcaattgagagagcgt；With the probe SEQ ID for people's FAM89A genetic tests
NO:11：agaccaaccatctctttgcgga.
5. according to the kit described in claim 4, it is characterised in that the kit includes following probe：SEQ ID NO:
9 fluorescence probes mark is as follows：FAM-ccctgttgctgtagccaaattcgt-Dabcyl；SEQ ID NO:10 fluorescence probe marks
Note is as follows：TAMRA-aactggtgcaattgagagagcgt-BHQ2；With SEQ ID NO:11 fluorescence probes mark is as follows：JOE-
agaccaaccatctctttgcgga-BHQ1。
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810069209.1A CN108034716B (en) | 2018-01-24 | 2018-01-24 | Kit for identifying infection and infection type of patient |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810069209.1A CN108034716B (en) | 2018-01-24 | 2018-01-24 | Kit for identifying infection and infection type of patient |
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| Publication Number | Publication Date |
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| CN108034716A true CN108034716A (en) | 2018-05-15 |
| CN108034716B CN108034716B (en) | 2021-06-25 |
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| CN201810069209.1A Active CN108034716B (en) | 2018-01-24 | 2018-01-24 | Kit for identifying infection and infection type of patient |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103119180A (en) * | 2011-01-12 | 2013-05-22 | 森永乳业株式会社 | Method for screening for feed which enables production of milk having immunomodulating activity |
| CN103436621A (en) * | 2013-04-27 | 2013-12-11 | 北京明谛生物医药科技有限公司 | Method and kit for quickly detecting expression quantity of CK19 mRNA (messenger ribonucleic acid) |
| US20140329704A1 (en) * | 2013-03-28 | 2014-11-06 | President And Fellows Of Harvard College | Markers for mature beta-cells and methods of using the same |
| CN107058521A (en) * | 2017-03-17 | 2017-08-18 | 中国科学院北京基因组研究所 | A kind of detecting system for detecting human immunity state |
-
2018
- 2018-01-24 CN CN201810069209.1A patent/CN108034716B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103119180A (en) * | 2011-01-12 | 2013-05-22 | 森永乳业株式会社 | Method for screening for feed which enables production of milk having immunomodulating activity |
| US20140329704A1 (en) * | 2013-03-28 | 2014-11-06 | President And Fellows Of Harvard College | Markers for mature beta-cells and methods of using the same |
| CN103436621A (en) * | 2013-04-27 | 2013-12-11 | 北京明谛生物医药科技有限公司 | Method and kit for quickly detecting expression quantity of CK19 mRNA (messenger ribonucleic acid) |
| CN107058521A (en) * | 2017-03-17 | 2017-08-18 | 中国科学院北京基因组研究所 | A kind of detecting system for detecting human immunity state |
Non-Patent Citations (2)
| Title |
|---|
| JETHRO A. HERBERG ET AL.: "Diagnostic Test Accuracy of a 2-Transcript Host RNA Signature for Discriminating Bacterial vs Viral Infection in Febrile Children", 《JAMA-JOURNAL OF THE AMERICAN MEDICAL ASSOCIATION》 * |
| 张磊等: "感染性疾病诊断新技术的应用", 《中华临床实验室管理电子杂志》 * |
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