CN108949961A - For detecting kit and its screening of adenovirus pneumonia - Google Patents
For detecting kit and its screening of adenovirus pneumonia Download PDFInfo
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- CN108949961A CN108949961A CN201810913415.6A CN201810913415A CN108949961A CN 108949961 A CN108949961 A CN 108949961A CN 201810913415 A CN201810913415 A CN 201810913415A CN 108949961 A CN108949961 A CN 108949961A
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- 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
- C12Q1/6876—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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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
Abstract
The invention discloses for detecting adenovirus pneumonia kit and its screening, belong to biological diagnosis technical field, 4 kinds of miRNA:miR-450a-5p, miR-103a-3p, miR-103b-5p and miR-98-5p in serum in Exosome can combine as the molecular marked compound for quick and precisely detecting adenovirus pneumonia, and 4 kinds of miRNA can be used for screening or preparing the kit of adenovirus pneumonia detection；The kit includes that 4 pairs of amplimers, 4 reverse transcriptase primers, RNA extraction reagent, reverse transcription reagents and quantitative fluorescent PCR reagent, quantitative fluorescent PCR reagent include fluorescent dye.
Description
Technical field
The invention belongs to biological diagnosis technical fields, more particularly, to detect the kit and its sieve of adenovirus pneumonia
Choosing.
Background technique
Community acquired pneumonia is one of most common disease of children, less than the disease incidence about 25% of 5 years old children, every year about
2000000 children die of pneumonia.Adenovirus pneumonia is one kind of the most common and most serious of China's Community-acquired viral pneumonia,
6 months to 2 years old infants are mostly occurred in, it can be in distributing or eruption and prevalence.
Often because fever time is long, clinical manifestation is heavy and multi systemic complications easily occur, severe adenovirus pneumonia patient's
Case fatality rate is high, this brings serious spirit and financial burden to family or even society.How effectively to diagnose and treat in early stage,
It is the problem for perplexing clinician.
Adenovirus detection technique mainly includes viral-specific antigens detection, Serologic detection, polymerase chain reaction at present
It should detect, histopathology and Virus culture etc..The common first three of clinic, and latter two is not suitable for fast because of time and effort consuming
Fast, accurate clinical detection demand.However, viral-specific antigens detection, Serologic detection and polymerase chain reaction detection
Accuracy and sensitivity need to be further increased.
Exosome (excretion body) is the vesica from late endosome (also referred to as more vesica bodies) of living cells secretion,
When more vesica bodies the vesica included will be discharged into plasma membrane fusion it is extracellular.Studies have shown that from different cells
Exosome contains the functional molecular of source cell most critical.Exosome is a kind of vesicles that diameter is 30~100nm, can be by more
Kind cell secretion, the ingredients such as inner protein, lipid and microRNA.Protein contained by the Exosome of different cell origins and
RNA is different, biological function also difference.Exosome is a kind of solid-phase component that density is very low in blood, is considered taking
With biomarker information abundant.
Mature microRNA (miRNA) is a kind of small molecule non-coding RNA for being about 17~25 nucleotide, is mainly passed through
With the base pair complementarity inhibition said target mrna translation of the 3 '-non-translational regions (UTR), 5 '-UTR and coding region of said target mrna, participate in
The level modulation of expression of target gene after transcription.Bioinformatics is studies have shown that miRNA can be combined with said target mrna and be played tune
Section effect, take part in the almost all of pathology of mammal and physiological activity, as ontogeny, tissue differentiation, Apoptosis with
And energetic supersession etc..
Therefore, how to be one for detecting adenovirus pneumonia for miRNA in excretion body (Exosome) urgently to be solved to ask
Topic.
Summary of the invention
Present invention aims to overcome that the shortcomings of the prior art, and the kit for detecting adenovirus pneumonia is provided
And its screening.The present invention designs specific reverse transcriptase primer, amplimer as research object using 4 kinds of miRNA in excretion body, leads to
It crosses quantitative fluorescent PCR and quickly and accurately detects adenovirus pneumonia, and high sensitivity.
To achieve the above object, the technical scheme adopted by the invention is as follows: 4 kinds of miRNA screen or prepare for detecting gland
Application in the kit of virus bronchopneumonia, the sequence of 4 kinds of miRNA such as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3
With shown in SEQ ID NO:4.
Above-mentioned 4 kinds of miRNA are followed successively by miR-450a-5p (as shown in SEQ ID NO:1), miR-103a-3p in excretion body
(as shown in SEQ ID NO:2), miR-103b (as shown in SEQ ID NO:3) and miR-98-5p are (such as SEQ ID NO:4 institute
Show), 4 kinds of miRNA combine the biomarker quickly detected as adenovirus pneumonia, can be used for the screening or preparation of kit
In.The present invention using 4 kinds of miRNA and not single index is tested, there are many high reliablity than detecting single index.
As an improvement of the above technical solution, 4 kinds of miRNA are from excretion body (Exosome) in serum.
In addition, the present invention also provides 4 reverse transcriptase primers for pneumonia adenovirus reverse transcription, first reverse transcriptase primer
As shown in SEQ ID NO:5, second reverse transcriptase primer is as shown in SEQ ID NO:6, third reverse transcriptase primer such as SEQ ID
Shown in NO:7, the 4th reverse transcriptase primer is as shown in SEQ ID NO:8.
In addition, the present invention also provides 4 pairs of amplimers for pneumonia adenovirus amplifies, first couple of amplimer such as SEQ
Shown in ID NO:9 and SEQ ID NO:10, second pair of amplimer is as shown in SEQ ID NO:11 and SEQ ID NO:12, third
To amplimer as shown in SEQ ID NO:13 and SEQ ID NO:14, the 4th pair of amplimer such as SEQ ID NO:15 and SEQ
Shown in ID NO:16.
In addition, the present invention also provides a kind of for detecting the kit of adenovirus pneumonia comprising 4 pairs of amplifications are drawn
Object.This kit is carried out using miR-450a-5p, miR-103a-3p, miR-103b and miR-98-5p in excretion body as sample
Reverse transcription, quantitative fluorescent PCR, to quantitative determine the expression quantity of above-mentioned 4 kinds of miRNA.Firstly, the spy of above-mentioned 4 pairs of amplimers
Anisotropic strong, detection effect is more preferable when quantitative fluorescent PCR；Secondly, kit of the present invention is compareed two-by-two using 4 kinds of miRNA,
It completely eliminates because sampling method, operator operate the brings error such as not accurate, testing result is more acurrate reliable, convenient for low
Cost large-scale promotion application.
As an improvement of the above technical solution, kit further includes 4 reverse transcriptase primers.When existing RNA reverse transcription,
Universal primer Oligo (T) generally is used, kit of the present invention is in order to guarantee the measurement sensitivity of quantitative fluorescent PCR, above-mentioned 4 kinds
MiRNA separately designs 1 species specific reverse transcriptase primer.
It as a further improvement of the above technical scheme, further include that RNA extracts reagent, reverse transcription reagents and fluorescent quantitation
PCR reagent.
Further improvement as above-mentioned technical proposal, the quantitative fluorescent PCR reagent includes fluorescent dye.
The beneficial effects of the present invention are: it is first the present invention is provided to detect the kit of adenovirus pneumonia and its screening
First, the present invention is to combine the biomarker quickly detected as adenovirus pneumonia using 4 kinds of miRNA, can be used for the sieve of kit
In choosing or preparation, for single index, 4 kinds of miRNA testing results are more acurrate and reliable；Secondly, the present invention detects examination
Agent box be using miR-450a-5p, miR-103a-3p, miR-103b and miR-98-5p in excretion body as sample, carry out reverse transcription,
Quantitative fluorescent PCR is compareed two-by-two using 4 kinds of miRNA, is disappeared completely to quantitative determine the expression quantity of above-mentioned 4 kinds of miRNA
In addition to operating the brings error such as not accurate because of sampling method, operator, testing result is more acurrate reliable, big convenient for low cost
Scale promotes and applies；In order to guarantee quantitative fluorescent PCR measurement sensitivity degree, kit of the present invention devise 4 it is specific inverse
Transcription primers and 4 pairs of specific amplimers.
Detailed description of the invention
Fig. 1 shows Exosome in healthy child (Control) and adenovirus pneumonia infant (ADV) serum；Wherein, 1A is aobvious
Show the morphological feature of healthy child and Exosome in ADV infant serum under transmission electron microscope, 1B shows Western blot detection
The content of CD9 and HSP90 α, S indicate serum (serum), and Ex indicates Exosome；
Fig. 2 shows that miRNA Microarray analyzes result in Exosome in healthy child and ADV infant equivalent serum；
2A shows high-flux sequence result volcano figure, wherein red point (point i.e. in figure in two rectangles in top) is poor in ADV infant
The miRNAs of different expression；2B shows the miRNAs for significantly raising or lowering in ADV infant；Wherein, ADV is adenovirus pneumonia trouble
Youngster, Control are healthy child；
Fig. 3 be 5 ADV infants in exosome in 5 health child serum to microRNA Microarray result
Quantitative fluorescent PCR verifying is carried out, two-by-two the t inspection result of interaction reference；
Fig. 4 is the inspection of detection effect；15 healthy children and 15 adenovirus lungs are detected using fluorescence quantifying PCR method
In scorching infant serum in Exosome 4 kinds of miRNA content, and with Ct (miR-103b-5p)-Ct (miR-98-5p) difference and Ct
(miR-450a-5p)-Ct (miR-103a-3p) difference is two axial two-dimentional mappings of progress；Bluepoint is the data of healthy child,
Red fork is adenovirus pneumonia infant data.
Specific embodiment
Purposes, technical schemes and advantages in order to better illustrate the present invention, below in conjunction with specific embodiments and the drawings pair
The present invention is described further.
It should be understood that various to describe using term " first ", " second ", " third " and " the 4th " etc. in the present invention
Information, but these information should not necessarily be limited by these terms, and these terms are only used to for same type of information being distinguished from each other out.Example
Such as, without departing from the present invention, " first " information can also be referred to as " second " information, similar, " second "
Information can also be referred to as " first " information.
The screening of quick diagnosis adenovirus pneumonia nucleic acid markers
One, primary dcreening operation
(1) 500 μ L of healthy child and ADV infant blood is acquired respectively, and 4 DEG C of refrigerators stand 1~2h, 2000rpm centrifugation
After 5min, serum is taken out；
(2) detection method (System Biosciences Inc, Mountain View, CA) is extracted according to Exosome,
Extract the Exosome in serum；
(3) form (as shown in Figure 1A) of Exosome is observed under transmission electron microscope；In addition, being detected with Western Blot
The expression (as shown in Figure 1B) of the significant PROTEIN C D9 and HSP90 α of two of Exosome；
(4) with Trizol reagent (Life Tech Inc, USA) extract Exosome in RNA, and it is carried out concentration and
The measurement of purity；
(5) primary dcreening operation of nucleic acid markers: by above-mentioned analysis method, the equivalent of healthy child and adenovirus pneumonia infant
RNA in pooled serum in Exosome carries out microRNA Microarray analysis, according to up-regulation or lowers differential expression >
200 times of standard has carried out the primary dcreening operation of microRNA, is analyzed and is raised or lower mileometer adjustment by microRNA Microarray
Up to the apparent miRNAs of difference.
As shown in Figure 1A, the diameter of Exosome is all between 30~100nm；As shown in Figure 1B, Exosome expresses its mark
Will PROTEIN C D9 and HSP90 α；It can be seen that being successfully extracted Exosome in serum.As shown in Fig. 2, up-regulation or downward expression
The apparent miRNAs of difference include miR-450a-5p,
MiR-103a-3p, miR-103b and miR-98-5p are marked with black box in Fig. 2 B；The nucleic acid of 4 kinds of miRNA
Sequence is as shown in table 1.
The nucleic acid sequence of table 1 miR-450a-5p, miR-103a-3p, miR-103b and miR-98-5p
Two, secondary screening
The secondary screening of nucleic acid markers: random selection healthy children and adenovirus pneumonia infant serum extract in serum
Exosome carries out fluorescence quantitative PCR detection to the above-mentioned miRNAs just sifted out.
Reverse transcription obtains cDNA according to a conventional method, then carries out quantitative fluorescent PCR reaction, and concrete operations are as follows:
(1) reverse transcription obtains cDNA:
1) RNA extracted in above-mentioned Exosome is used as template, be added in the PCR pipe for removing RNase 1.0 μ g of RNA template with
RNA free H2O to total volume be 8 μ L；
2) above-mentioned solution is mixed, 85 DEG C of incubation 5min, to open RNA secondary structure, be immediately placed on ice, to prevent
Only RNA renaturation restores secondary structure again；
3) reverse transcription: in process of reverse-transcription, the specific reverse transcriptase RT primer sequence of the miRNAs just sifted out is as shown in table 2
(primer sequence of miR-450a-5p, miR-103a-3p, miR-103b-5p and miR-98-5p are only gived, others are not given
Out).
The specific reverse transcriptase primer sequence of table 2 miR-450a-5p, miR-103a-3p, miR-103b and miR-98-5p
Following solution is configured in another PCR pipe for removing RNase:
4) 3) solution in is added in solution 2), 42 DEG C of incubation 60min after mixing；
5) 85 DEG C of incubation 10min inactivate reverse transcriptase, can be obtained cDNA.
(2) quantitative fluorescent PCR
In quantitative fluorescent PCR, the amplimer sequence of the quantitative fluorescent PCR of the miRNAs just sifted out is as shown in table 3 (only
The primer sequence of miR-450a-5p, miR-103a-3p, miR-103b-5p and miR-98-5p are given, others do not provide).
The amplification of the quantitative fluorescent PCR of table 3 miR-450a-5p, miR-103a-3p, miR-103b and miR-98-5p is drawn
Object sequence
1) quantitative fluorescent PCR is carried out respectively to the miRNAs just sifted out by following reaction system:
2) quantitative fluorescent PCR reaction condition: 50 DEG C, 2min；95 DEG C, 2min；95 DEG C, 15s；60 DEG C, 32s；Read plate 40
Circulation；Melt curve analysis analysis: 60~95 DEG C of temperature.
Then the Ct value of these miRNAs of primary dcreening operation is compared two-by-two, interaction reference, to eliminate because of sampling method, behaviour
Make the brings error such as error.As shown in figure 3, to the microRNA of Exosome in ADV infant and healthy child's serum
Microarray result carries out quantitative fluorescent PCR verifying, two-by-two the t inspection result of interaction reference；In comparison procedure two-by-two, meaning
The Ct difference of outer discovery miR-450a-5p/miR-103a-3p and the Ct difference of miR-103b-5p/miR-98-5p are at healthy group
There is significant difference (P < 0.01) in adenovirus pneumonia group.
In conclusion selecting Ct (miR-450a-5p)-Ct (miR-103a-3p) difference and Ct (miR-103b-5p)-Ct
(miR-98-5p) index that difference is used as adenovirus pneumonia to detect simultaneously, can increasing adenovirus pneumonia detection, (especially early stage is examined
Survey) robustness and judgement accuracy.
The quickly verifying of detection adenovirus pneumonia nucleic acid markers
In order to further confirm that filter out 4 kinds of miRNAs (miR-450a-5p, miR-103a-3p, miR-103b-5p and
MiR-98-5p) whether closely related with the quick and precisely detection of adenovirus pneumonia, it is extracted 15 healthy children and 15 respectively
The RNA of Exosome in adenovirus pneumonia infant fresh serum, the RNA of Exosome carries out quantitative fluorescent PCR in this several groups of serum
Detection detects the Ct value of miR-450a-5p, miR-103a-3p, miR-103b-5p and miR-98-5p in each group, and carries out two
Two compare, interaction reference, to eliminate because of the brings error such as sampling method, operating error.Calculate the Ct (miR- of each sample
450a-5p)-Ct (miR-103a-3p) difference and Ct (miR-103b-5p)-Ct (miR-98-5p), and with the two differences point
Not Wei X, Y coordinate draws.
As shown in figure 4, experimental result shows that normal group (Bluepoint), adenovirus pneumonia group (red fork) can distinguish completely.
And according to the statistics of sample, it can tentatively obtain and know to draw a conclusion: enable x=Ct (miR-450a-5p)-Ct (miR-103a-3p),
Y=Ct (miR-103b-5p)-Ct (miR-98-5p)；
If when x≤- 2 and y≤0, suffering from adenovirus pneumonia；
If being normal when x > -2 and y > 0.
In conclusion the present invention has been surprisingly found that miR-450a-5p, miR- in serum in Exosome by largely screening
There are conspicuousness differential expressions between healthy person and adenovirus pneumonia patient by 103a-3p, miR-103b-5p and miR-98-5p；
In addition, having been surprisingly found that: Ct (miR-450a-5p)-Ct (miR-103a-3p) difference and Ct (miR-103b-5p)-Ct (miR-98-
5p) there are significant differences between healthy person and adenovirus pneumonia patient.MiR-450a-5p in serum in Exosome,
MiR-103a-3p, miR-103b-5p and miR-98-5p can combine as the molecule mark for quick and precisely detecting adenovirus pneumonia
Remember object, can be used for screening or preparing the kit of adenovirus pneumonia；The kit draws including 4 pairs of amplimers, 4 reverse transcriptions
It includes fluorescent dye that object, RNA, which extract reagent, reverse transcription reagents and quantitative fluorescent PCR reagent, quantitative fluorescent PCR reagent,；First pair
Amplimer is as shown in SEQ ID NO:9 and SEQ ID NO:10, second pair of amplimer such as SEQ ID NO:11 and SEQ ID
Shown in NO:12, third to amplimer as shown in SEQ ID NO:13 and SEQ ID NO:14, the 4th couple of amplimer such as SEQ
Shown in ID NO:15 and SEQ ID NO:16；First reverse transcriptase primer is as shown in SEQ ID NO:5, second reverse transcriptase primer
As shown in SEQ ID NO:6, third reverse transcriptase primer is as shown in SEQ ID NO:7, the 4th reverse transcriptase primer such as SEQ ID
Shown in NO:8.
Compared with the various clinical testing procedures used in current, molecular marked compound miR-450a-5p provided by the invention,
MiR-103a-3p, miR-103b-5p and miR-98-5p can quick and precisely detect adenovirus pneumonia infant, it is a kind of objective
Evaluation method has many advantages, such as convenient, easy to operate, high specificity of drawing materials, therefore the present invention has great clinical application
May, it can be used to the detection of adenovirus.
Finally, it should be noted that above embodiments protect the present invention to illustrate technical solution of the present invention
The limitation of range, although the invention is described in detail with reference to the preferred embodiments, those skilled in the art should be managed
Solution, can modify to technical solution of the present invention or replace on an equal basis, without departing from technical solution of the present invention essence and
Range.
SEQUENCE LISTING
<110>Guangzhou Xin Yuan Biotechnology Co., Ltd
<120>for detecting kit and its screening of adenovirus pneumonia
<130> 2018.7.10
<160> 16
<170> PatentIn version 3.3
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Claims (8)
1.4 kinds of miRNA are screening or are preparing the application in the kit for detecting adenovirus pneumonia, which is characterized in that 4 kinds
The sequence of miRNA is as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.
2. application as described in claim 1, which is characterized in that 4 kinds of miRNA are from excretion body in serum.
3. being used for 4 reverse transcriptase primers of pneumonia adenovirus reverse transcription, which is characterized in that first reverse transcriptase primer such as SEQ ID
Shown in NO:5, second reverse transcriptase primer as shown in SEQ ID NO:6, third reverse transcriptase primer as shown in SEQ ID NO:7,
4th reverse transcriptase primer is as shown in SEQ ID NO:8.
4. being used for 4 pairs of amplimers of pneumonia adenovirus amplifies, which is characterized in that first pair of amplimer such as SEQ ID NO:9
With shown in SEQ ID NO:10, second pair of amplimer is as shown in SEQ ID NO:11 and SEQ ID NO:12, and third is to amplification
Primer is as shown in SEQ ID NO:13 and SEQ ID NO:14, the 4th pair of amplimer such as SEQ ID NO:15 and SEQ ID NO:
Shown in 16.
5. a kind of for detecting the kit of adenovirus pneumonia, which is characterized in that including 4 pairs of amplifications as shown in claim 4
Primer.
6. kit as claimed in claim 5, which is characterized in that further include that 4 reverse transcriptions as claimed in claim 3 draw
Object.
7. such as kit described in claim 5 or 6, which is characterized in that further include that RNA extracts reagent, reverse transcription reagents and glimmering
Fluorescent Quantitative PCR reagent.
8. kit as claimed in claim 7, which is characterized in that the quantitative fluorescent PCR reagent includes fluorescent dye.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110016522A (en) * | 2019-03-18 | 2019-07-16 | 广州市妇女儿童医疗中心 | The relevant excretion body miRNA of children's adenovirus pneumonia and application |
| CN114381509A (en) * | 2021-12-27 | 2022-04-22 | 深圳大学 | Plasma miRNA marker related to non-tuberculous pneumonia and application thereof |
| CN114525331A (en) * | 2021-11-23 | 2022-05-24 | 中山大学 | Detection product for rapidly identifying severe pneumonia |
| CN114958722A (en) * | 2021-06-22 | 2022-08-30 | 姜海涛 | Colorectal targeted drug-loaded exosome, application thereof and drug for treating colorectal diseases |
| CN114525331B (en) * | 2021-11-23 | 2024-04-26 | 中山大学 | Detection product for rapidly identifying severe pneumonia |
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| CN114525331B (en) * | 2021-11-23 | 2024-04-26 | 中山大学 | Detection product for rapidly identifying severe pneumonia |
| CN114381509A (en) * | 2021-12-27 | 2022-04-22 | 深圳大学 | Plasma miRNA marker related to non-tuberculous pneumonia and application thereof |
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Application publication date: 20181207 |