CN109706256A - A kind of primer, kit and method of inspection detecting mycoplasma pneumoniae MP - Google Patents
A kind of primer, kit and method of inspection detecting mycoplasma pneumoniae MP Download PDFInfo
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- CN109706256A CN109706256A CN201910154830.2A CN201910154830A CN109706256A CN 109706256 A CN109706256 A CN 109706256A CN 201910154830 A CN201910154830 A CN 201910154830A CN 109706256 A CN109706256 A CN 109706256A
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Abstract
The invention discloses primer, kit and the detection methods of a kind of detection mycoplasma pneumoniae (Mycoplasma Pneumonia, MP), belong to molecular biology field；By nucleotide sequence forward primer as shown in SEQ ID NO:1 and nucleotide sequence, the reverse primer as shown in SED ID NO:2 forms the primer；The forward primer is marked with biotin；The reverse primer is marked with FITC or Dig；The present invention is using recombinase-polymerase nucleic acid amplification technologies and solid phase developing technology, it only needs by isothermal duplication after clinical sample progress DNA extraction, it does not need PCR instrument and carries out thermal cycle reaction, testing result naked eyes can directly be read from solid phase colour developing test strips, primer detection mycoplasma pneumoniae MP high specificity of the invention, amplification efficiency is high, kit and detection method response procedures of the invention is simple, it can be effective, rapidly mycoplasma pneumoniae MP is detected, it is applicable in simplicity on site, quickly, accurately carries out the diagnosis of mycoplasma hominis's pneumonia.
Description
Technical field
The present invention relates to molecular biology field, in particular to a kind of primer for detecting mycoplasma pneumoniae MP, kit and
The method of inspection.
Background technique
Mycoplasma pneumoniae MP is the pathogen of mankind's Eaton agent pneumonia.Eaton agent pneumonia outstanding behaviours is tactical deployment of troops sexual stimulus
Cough, pathological change is based on interstitial pneumonia, concurrent Bronchopneumonia, referred to as primary atypical pneumonia sometimes.Mainly
Through droplet infection, incubation period 2~3 weeks, disease incidence was that children and teenager are multiple.Clinical symptoms are lighter, show as headache, pharynx
Bitterly, the general respiratory symptom such as fever, cough, but also have sporadic deaths report.Can it occur throughout the year, but mostly in the autumn and winter
Time.
Although Eaton agent pneumonia patient's self-induction symptom is heavier, the general Non Apparent Abnormality sign of chest physical examination.Nose is slight
Nasal obstruction, runny nose, pharynx moderate are congested.Ear drum membrane often has hyperemia, and about 15% has myringitis.In addition to the performance of respiratory system, mycoplasma
Pneumonia can occur together multisystem, multiple organ injury.Skin lesion can behave as maculopapule, erythema nodosum, vesicle etc..Stomach and intestine system
It unites visible vomiting, diarrhea and liver damage.The more typical hemolytic anemia of hematological.Central nervous system damage is visible more
Inflammation nerve root, meningoencephalitis and cerebellar lesion etc..Cardiovascular system lesion occasionally has myocarditis and pericarditis.Eaton agent pneumonia
Clinical manifestation and chest X-ray do not have characteristic, therefore can not pay a home visit only according to clinical manifestation and chest X-ray
It is disconnected.To clarify a diagnosis, needs to carry out the detection of pathogen, can just be made a definite diagnosis.Currently, the diagnosis master of domestic Eaton agent pneumonia
It will be by antigen in serum or antibody titer detection, but the problems such as there are sensitivity and not high specificity.And it is former for pneumonia branch
The Molecular Detection of body MP nucleic acid, predominantly PCR method detect the mycoplasma pneumoniae MP in Nasal swabs, Nasopharyngeal swabs and brush,throat
Gene, this method specificity and high sensitivity, but the time needs at least 60-90 minutes, and needs complex instrument, is not particularly suited for pinpointing
Look after detection (point-of-care test；POCT it) uses.Therefore, it is necessary to one kind can quick and precisely, simply, need not be valuable
Instrument, the detection kit and detection method that can be used for on-site test mycoplasma pneumoniae MP.
Based on recombinase-polymerase nucleic acid amplification technologies (Recombinase-polymerase amplification,
RPA intracellular nucleic acid synthesis mechanism) is simulated, within the scope of steady temperature, by recombinase, polymerase and single strand binding protein
So that DNA double chain is untwisted, and DNA fragment specific is promoted to expand, to achieve the effect that nucleic acid in vitro expands.Whole process is rapid
It (10-20 minutes) and is carried out in (37-42 DEG C) of low constant temperature.At present still in its infancy for the research of RPA technology, and it is domestic
The outer report that RPA combination solid phase developing technology is applied to mycoplasma pneumoniae MP detection not yet.The present invention is more using recombinase-
The nucleic acid amplification technologies of poly- enzyme simultaneously combine solid phase developing technology, provide one it is simple, quickly, be not necessary to expensive device can scene into
The screening mode of row mycoplasma pneumoniae MP has the characteristics that highly sensitive, specificity, is suitable for on-site quick screening.
Summary of the invention
Primer, the examination of a kind of detection mycoplasma pneumoniae MP are provided it is an object of the invention to overcome the deficiencies in the prior art
Agent box and the method for inspection solve the problems, such as that mycoplasma pneumoniae MP detection existing in the prior art is cumbersome and are difficult to meet demand.
It is a kind of detect mycoplasma pneumoniae MP primer, the primer as nucleotide sequence as shown in SEQ ID NO:1 just
To primer and nucleotide sequence, the reverse primer as shown in SED ID NO:2 is formed；The forward primer is marked with biotin
(Biotin)；The reverse primer is marked with FITC or Dig.
Primer of the invention is the pathogenic genes sequence and TwistDx that inventor refers to NCBI gene database
Instruction manual and Primer-BLAST design of primers principle, a plurality of primer of design and synthesis.In design process
In, consider not only the formation that avoid primer dimer, hairpin structure, it is also contemplated that different target gene is anti-at one
With answering in system coamplification problem.Primer screening experiment shows that primer specificity of the invention is strong, amplification efficiency is high, can effectively,
Rapidly detect mycoplasma pneumoniae MP.
The present invention also provides a kind of kits for detecting mycoplasma pneumoniae MP comprising above-mentioned primer.
The kit and detection method response procedures of above-mentioned technical proposal are simple, can be effective, rapidly to mycoplasma pneumoniae
MP is detected, and is applicable in simplicity on site, quickly, is accurately carried out the detection of the virus.
To be of the present invention for detecting the preferred embodiment of the kit of mycoplasma pneumoniae MP, the kit is also
Including Quality Control, ddH in recombinase, polymerase, DNA binding protein, buffer solution, the positive2O and solid phase chromogenic detection reagents.
To be of the present invention for detecting the preferred embodiment of the kit of mycoplasma pneumoniae MP, the positive endoplasm
Control contains the primer pair being made of the nucleotide sequence as shown in SEQ ID NO:3 and SED ID NO:4.
To be of the present invention for detecting the preferred embodiment of the kit of mycoplasma pneumoniae MP, the recombinase is
T4uvsX/uvsY；The polymerase is Bsu archaeal dna polymerase；The DNA binding protein is T4gp32；The buffer solution contains
Have following components: Tris-HCl buffer, potassium acetate, magnesium acetate, dithiothreitol (DTT), dNTPs, ATP, phosphocreatine, creatine swash
Enzyme and Carbowax20M.
To be of the present invention for detecting the preferred embodiment of the kit of mycoplasma pneumoniae MP, the buffer contains
Have a component of following concentration: Tris-HCl buffer 50mM, potassium acetate 100mM, magnesium acetate 14mM, dithiothreitol (DTT) 2mM,
DNTPs200 μM, ATP3mM, phosphocreatine 50mM, creatine kinase 100ng/ μ L, Carbowax20M5wt%；The Tris-HCl
The pH value of buffer is 7.9.
To be of the present invention for detecting the preferred embodiment of the kit of mycoplasma pneumoniae MP, the solid phase colour developing
Detection reagent includes solid phase colour developing test strips and color developing detection buffer.
To be of the present invention for detecting the preferred embodiment of the kit of mycoplasma pneumoniae MP, the colour developing inspection
It surveys buffer to be made of the component of following concentration: BSA 5wt% and PBS buffer solution 10mM.
As of the present invention for detecting the preferred embodiment of the kit of mycoplasma pneumoniae MP, the positive
Interior Quality Control is the recombinant plasmid for inserting people's GAPDH gene.
It is described the positive in Quality Control the preparation method comprises the following steps: respectively with nucleotide sequence such as SEQ ID No.3 and SEQ ID No.4
Shown in primer be upstream and downstream primer its PCR product genetic fragment is recombinated to plastid (pMD- with people's GAPDH gene cDNA
In 20T), to filter out single bacterium colony with antibiotic (ampicillin), then with Plasmid DNA Preparation Mini
Kit (QIAGEN, Valencia, CA) extracts Plasmid DNA, and correct recombinant plasmid is sequenced as Quality Control in the positive.
The present invention also provides the methods of detection mycoplasma pneumoniae MP using above-mentioned kit a kind of, including following step
It is rapid:
(1) DNA of sample to be tested is extracted as detection template；
(2) detection template being expanded, obtains DNA amplification sequence: preparing reaction system, concussion mixes reaction system,
Sample reacts 20 minutes in 37~42 DEG C, is expanded, obtains DNA amplification sequence；
(3) amplified production is detected using solid phase colour developing test strips: takes a certain number of solid phase colour developing test strips, needle
Different samples to be tested are marked, each sample takes 50 μ L color developing detection buffers, is added in reaction tube；20 μ L amplification is taken to produce
Object mixes in centrifuge tube, amplified production and color developing detection buffer totally 70 μ L；Test strips sample application zone is added in above-mentioned reaction solution,
It is incubated for 5 minutes；
(4) point is presented according to test strips reaction zone and judges testing result: after incubation, naked eyes are observed immediately；If examination
Paper slip reaction zone shows test paper quality control point (Marker) and Quality Control point (Internal Control), then this reaction is effectively anti-
It answers；If test strips reaction zone shows mycoplasma pneumoniae MP reflecting point, test sample is the positive；If test strips reaction zone is not
It shows mycoplasma pneumoniae MP reflecting point, then detects sample as feminine gender.
Above-mentioned detection method is combined using recombinase-polymerase nucleic acid amplification technologies with solid phase developing technology, simple,
Quickly, has the characteristics that highly sensitive, specificity.
Sample to be tested derives from human respiratory secretion, such as Nasal swabs, Nasopharyngeal swabs and brush,throat.
The beneficial effects of the present invention are:
1, the present invention combines recombinase-polymerase nucleic acid amplification technologies with solid phase developing technology, provides a letter
It is single, quickly, be not necessary to expensive device can scene carry out the screening mode of mycoplasma pneumoniae MP, without thermal cycle reaction and special
Instrument, testing result can directly be observed by vision；
2, primer detection mycoplasma pneumoniae MP high specificity of the invention, amplification efficiency are high.
3, kit of the invention and detection method response procedures are simple, can be effective, rapidly to mycoplasma pneumoniae MP into
Row detection is applicable in simplicity on site, quickly, accurately carries out the diagnosis of mycoplasma hominis's pneumonia, is suitable for instant quick diagnosis, can
Screening is carried out in a non-laboratory environment, it is possible to provide the quick detection of live First Line.
Detailed description of the invention
Fig. 1 is the test strips that recombinase polymeric enzymatic amplification detection mycoplasma pneumoniae MP is carried out using kit of the present invention
Diagram；
Fig. 2 is to carry out recombinase polymeric enzymatic amplification using kit of the present invention to detect mycoplasma pneumoniae MP, and be directed to
The result figure that difference detection sample is marked.
Appended drawing reference: A. siphon area, B. reaction zone, the sample application zone C..
Specific embodiment
Purposes, technical schemes and advantages in order to better illustrate the present invention, below in conjunction with the drawings and specific embodiments pair
The present invention further illustrates.It will be appreciated by those skilled in the art that specific embodiment described herein is only to explain this hair
It is bright, it is not intended to limit the present invention.
Test method as used in the following examples is conventional method unless otherwise specified；Institute in following embodiments
Material, reagent for using etc., are commercially available unless otherwise specified.
Embodiment 1: primer and kit
1, design of primers
According to the respiratory tract infection pathogenic genes sequence of NCBI gene database, with reference to TwistDx instruction
Manual and Primer-BLAST design of primers principle, design primer.Primer length is about 30-35nt, due to currently without needle
To the primer-design software of RPA, is designed during previous work of the present invention and synthesized a large amount of primers, be screened out from it high sensitivity
And specific good primer is for the present invention.It is shown in Table 1.
2, primer synthesizes
According to primer sequence shown in table 1, sequent synthesis is carried out.It is characterized in that, its mycoplasma pneumoniae MP gene is positive
Primer sequence is modified and Biotin is added as shown in SEQ ID NO:1；Reverse primer sequences are repaired as shown in SEQ ID NO:2
FITC or Dig is added in decorations.Its interior Quality Control forward primer sequence modifies and Biotin is added as shown in SEQ ID NO:3；Reversely draw
Object sequence modifies and TAMRA is added as shown in SEQ ID NO:4.
Table 1
Kit described in the present embodiment for mycoplasma pneumoniae MP quick diagnosis includes that above-mentioned upstream primer and downstream are drawn
Object, the kit further include recombinase, polymerase, DNA binding protein, buffer solution, the positive in Quality Control, ddH2O and solid phase
Chromogenic detection reagents.The recombinase is T4uvsX/uvsY, and the polymerase is Bsu archaeal dna polymerase, the DNA combination egg
White is T4gp32；The buffer contains the component of following concentration: Tris-HCl buffer 50mM, potassium acetate 100mM, magnesium acetate
14mM, 2mM, dNTPs200 μM of dithiothreitol (DTT), ATP3mM, phosphocreatine 50mM, creatine kinase 100ng/ μ L,
Carbowax20M5wt%；The pH value of the Tris-HCl buffer is 7.9.The solid phase chromogenic detection reagents include that solid phase is aobvious
Color test strips and color developing detection buffer, the color developing detection buffer are made of the component of following concentration: BSA 5wt% and
PBS buffer solution 10mM.
Embodiment 2 detects mycoplasma pneumoniae MP
The method that the present embodiment uses the detection mycoplasma pneumoniae MP detection of kit described in embodiment 1 to use is poly- for recombinase
Synthase constant temperature gene amplification method simultaneously combines solid phase developing technology, the specific steps are as follows:
1, clinical sample prepares
Collect Nasal swabs, Nasopharyngeal swabs and brush,throat, collection condition are as follows: check (quantitative fluorescent PCR) through laboratory
It is confirmed as the positive clinical sample (40) with mycoplasma pneumoniae MP afterwards, and is confirmed as no mycoplasma pneumoniae MP's after inspection
Negative clinical sample (89).Clinical sample is placed in -80 DEG C of preservations.
2, the nucleic acid extraction of sample
The present embodiment is extracted or is automated sample nucleic acid extraction apparatus using DNA col-umn chromatography, carries out the nucleic acid of clinical sample
It extracts.
3, prepared by positive reference product nucleic acid
Respectively using primer shown in SEQ ID NO:3 and SEQ ID NO:4 as upstream and downstream primer, with people's GAPDH gene
CDNA recombinates its PCR product genetic fragment into plastid (pMD-20T), to filter out list with antibiotic (ampicillin)
One bacterium colony, then Plasmid DNA is extracted with Plasmid DNA Preparation Mini Kit (QIAGEN, Valencia, CA), it surveys
The correct recombinant plasmid of sequence is Quality Control in the positive.
4, reaction system configures
By 2 proportional arrangement reaction system of table:
Table 2
| Sample nucleic acid | 2μg |
| Quality Control in the positive | 0.5μg |
| Positive and reverse primer | Each 1 μ L |
| Buffer solution | 25μL |
| T4uvsX/uvsY,120ng/μL | 1μL |
| Bsu archaeal dna polymerase, 30ng/ μ L | 1μL |
| T4gp32,900ng/μL | 1μL |
| ddH2O | Complement to 50 μ L |
5, concussion mixes made from step 4 after reaction system, sample in 39 DEG C isothermal reaction 20 minutes, it is poly- to carry out recombinase
Synthase isothermal duplication.
6, a certain number of solid phase colour developing test strips are taken, are marked for different detection samples.Each detection sample
50 μ L color developing detection buffers (5wt%BSA in PBS) are taken, are added in reaction tube.The resulting amplification of 20 μ L steps (5) is taken to produce
Object mixes in centrifuge tube, amplified production and color developing detection buffer totally 70 μ L.Test strips sample application zone is added in above-mentioned reaction solution,
It is incubated for 5 minutes.
7, after being incubated for, naked eyes are observed immediately.If test strips reaction zone shows test paper quality control point (Marker) and matter
It controls point (Internal Control), then this reaction is effecting reaction；If test strips reaction zone shows that mycoplasma pneumoniae MP is anti-
Ying Dian, then test sample is the positive；If test strips reaction zone does not show mycoplasma pneumoniae MP reflecting point, sample is detected as yin
Property.
8, result interpretation
The primer sets designed by the present invention are used for quickly detecting mycoplasma pneumoniae MP, through laboratory quantitative fluorescent PCR side
The sample of 40 positive S mycoplasma MP of method identification has 39 display mycoplasma pneumoniae MP anti-in solid phase colour developing test strips
Ying Dian, all negative samples do not show this reflecting point (Fig. 2, table 3), it can thus be appreciated that wherein sample 1-6 is positive sample, sample
7 be negative sample.
Table 3
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than protects to the present invention
The limitation of range is protected, although the invention is described in detail with reference to the preferred embodiments, those skilled in the art should
Understand, it can be with modification or equivalent replacement of the technical solution of the present invention are made, without departing from the essence of technical solution of the present invention
And range.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (10)
1. a kind of primer for detecting mycoplasma pneumoniae MP, it is characterised in that: the primer is made of forward primer and reverse primer,
The forward primer is marked with biotin, and the reverse primer is marked with FITC or Dig,
The forward primer is nucleotide sequence SEQ ID NO:1:
5'Biotin-TGAACGTATCGTAACACGAGCTTTTCCTCC3'；
The reverse primer is nucleotide sequence SEQ ID NO:2:
5’FITC/DIG-GGGTTTAACTTCGAAAAATCGAGGTCGTATAA3’。
2. a kind of kit for detecting mycoplasma pneumoniae MP, it is characterised in that: the kit includes described in claim 1 draws
Object.
3. a kind of kit for detecting mycoplasma pneumoniae MP according to claim 2, it is characterised in that: the kit also wraps
Include recombinase, polymerase, DNA binding protein, buffer solution, the positive in Quality Control, ddH2O and solid phase chromogenic detection reagents.
4. a kind of kit for detecting mycoplasma pneumoniae MP according to claim 3, which is characterized in that the recombinase is
T4 uvsX/uvsY；
The polymerase is Bsu archaeal dna polymerase；
The DNA binding protein is T4 gp32；
The buffer solution includes Tris-HCl buffer, potassium acetate, magnesium acetate, dithiothreitol (DTT), dNTPs, ATP, phosphoric acid flesh
Acid, creatine kinase and Carbowax20M.
5. a kind of kit for detecting mycoplasma pneumoniae MP according to claim 4, which is characterized in that the Tris-HCl
Buffer concentration is 50mM, and the pH value of the Tris-HCl buffer is 7.9, and the acetic acid potassium concn is 100mM, the acetic acid
Magnesium density is 14mM, and the dithiothreitol (DTT) concentration is 2mM, and the dNTPs concentration is 200 μM, and the ATP concentration is 3mM, institute
Stating phosphocreatine concentration is 50mM, and the creatine kinase concentration is 100ng/ μ L, and the Carbowax20M concentration is 5wt%.
6. a kind of kit for detecting mycoplasma pneumoniae MP according to claim 3, it is characterised in that: the positive endoplasm
Control includes the primer pair that the nucleotide sequence shown in SEQ ID NO:3 and SED ID NO:4 forms.
7. a kind of kit for detecting mycoplasma pneumoniae MP according to claim 3, which is characterized in that in the positive
Quality Control is the recombinant plasmid for inserting people's GAPDH gene.
8. a kind of kit for detecting mycoplasma pneumoniae MP according to claim 3, which is characterized in that the solid phase colour developing
Detection reagent includes solid phase colour developing test strips and color developing detection buffer.
9. a kind of kit for detecting mycoplasma pneumoniae MP according to claim 8, which is characterized in that the color developing detection
The PBS buffer solution that the BSA and concentration that buffer is 5wt% by concentration are 10mM forms.
10. a kind of method using the described in any item kit detection mycoplasma pneumoniae MP of claim 2~9, feature exist
In, comprising the following steps:
(1) DNA of sample to be tested is extracted as detection template；
(2) detection template is expanded, obtains DNA amplification sequence；
(3) amplified production is detected using solid phase colour developing test strips；
(4) point is presented according to test strips reaction zone and judges testing result.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201910154830.2A CN109706256A (en) | 2019-03-01 | 2019-03-01 | A kind of primer, kit and method of inspection detecting mycoplasma pneumoniae MP |
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| CN201910154830.2A CN109706256A (en) | 2019-03-01 | 2019-03-01 | A kind of primer, kit and method of inspection detecting mycoplasma pneumoniae MP |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116515961A (en) * | 2023-06-25 | 2023-08-01 | 广东忠信生物科技有限公司 | RPA nucleic acid detection method based on chemiluminescence immunoassay |
| NL2033870B1 (en) * | 2022-09-01 | 2024-03-12 | Baoding Childrens Hospital | Kit for detection of mycoplasma pneumoniae, detection method and use thereof |
-
2019
- 2019-03-01 CN CN201910154830.2A patent/CN109706256A/en not_active Withdrawn
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| NL2033870B1 (en) * | 2022-09-01 | 2024-03-12 | Baoding Childrens Hospital | Kit for detection of mycoplasma pneumoniae, detection method and use thereof |
| CN116515961A (en) * | 2023-06-25 | 2023-08-01 | 广东忠信生物科技有限公司 | RPA nucleic acid detection method based on chemiluminescence immunoassay |
| CN116515961B (en) * | 2023-06-25 | 2023-11-03 | 广东忠信生物科技有限公司 | RPA nucleic acid detection method based on chemiluminescence immunoassay |
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Application publication date: 20190503 |