CN109680088A - A kind of primer, kit and detection method detecting Bordetella pertussis BP - Google Patents
A kind of primer, kit and detection method detecting Bordetella pertussis BP Download PDFInfo
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- CN109680088A CN109680088A CN201910154887.2A CN201910154887A CN109680088A CN 109680088 A CN109680088 A CN 109680088A CN 201910154887 A CN201910154887 A CN 201910154887A CN 109680088 A CN109680088 A CN 109680088A
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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
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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Abstract
The invention discloses primer, kit and the detection methods of a kind of detection Bordetella pertussis (Bordetella Pertussis, BP), 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 is only necessary to by not needing PCR instrument and carrying out thermal cycle reaction, testing result naked eyes can directly be read from solid phase colour developing test strips to isothermal duplication after clinical sample progress DNA extraction.Primer detection Bordetella pertussis BP high specificity of the invention, amplification efficiency are high.Kit and detection method response procedures of the invention is simple, can be effective, rapidly detects to Bordetella pertussis BP, is applicable in simplicity on site, quickly, accurately carries out the detection of pertussis.
Description
Technical field
The present invention relates to molecular biology field, in particular to a kind of primer for detecting Bordetella pertussis BP, kit and
Detection method.
Background technique
Bordetella pertussis is the pathogen of mankind's pertussis, mainly passes through droplet transmission.Pertussis incubation period is 1~2 week.
Morbidity early stage (catarrhal period) only has slight cough.Bacterium mass propagation and is arranged on trachea and bronchus mucous membrane with droplet at this time
Out, infectiousness is maximum.Occur paroxysmal spasmodic cough (spasm period) after 1~2 week, at this moment bacterium discharges toxin, leads to mucous membrane
Epithelial cell ciliary movement imbalance, a large amount of thick secretions cannot be discharged, and stimulate the receptor in mucous membrane to generate strong paroxysmal spasmodic cough, are in
Reveal special high-pitched tone crow sample roar.The mucus embolus of formation, which can also block bronchium, causes atelectasis and breathing tired
Difficult, cyanosis.It furthermore can be with vomiting, convulsions.Gradually it is transferred to convalescence after 4~6 weeks, paroxysmal cough mitigates, tend to recovery from illness, but have 1~
The infection of 10% patient Yi Jifa hemolytic streptococcus, Bacillus influenzae etc..This disease course of disease is longer, therefore named pertussis, wherein one-year-old
Following infant case fatality rate is high.Therefore, the early diagnosis of pertussis is particularly important.
Currently, the diagnosis of domestic pertussis relies primarily on based on being separately cultured, it is reachable that catarrhal period separates positive rate
91.5%, and convalescence only has about 26%.Sample uses nasopharynx swab or cough plate method, is incubated on culture plate, according to bacterium colony
Form, smear staining microscopy make tentative diagnosis.But there are complicated for operation, sensitivity is low with specificity, and interval between diagnosis is long etc. lacks
Point.The domestic Molecular Detection means for Bordetella pertussis BP nucleic acid are used less at present, and predominantly PCR method detection nasal cavity is wiped
Bordetella pertussis BP gene in son, Nasopharyngeal swabs and brush,throat, this method specificity and high sensitivity, but the time needs at least
60-90 minutes, and complex instrument is needed, it is not particularly suited for point-of-care detection (point-of-care test；POCT it) uses.Cause
This, needs a kind of can quick and precisely, simply, without expensive instrument, the detection that can be used for on-site test Bordetella pertussis BP try
Agent box and detection method.
Summary of the invention
Primer, the examination of a kind of detection Bordetella pertussis BP 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 Bordetella pertussis BP detection existing in the prior art is cumbersome and are difficult to meet demand.
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 Bordetella pertussis BP 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 Bordetella pertussis BP has the characteristics that highly sensitive, specificity, is suitable for on-site quick screening.
The invention is realized in this way it is a kind of detect Bordetella pertussis BP primer, the primer by nucleotide sequence such as
Forward primer shown in SEQ ID NO:1 and the nucleotide sequence reverse primer as shown in SED ID NO:2 composition；The forward direction
Primer mark has 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 Bordetella pertussis BP.
The present invention also provides a kind of kits for detecting Bordetella pertussis BP comprising above-mentioned primer.
The kit and detection method response procedures of above-mentioned technical proposal are simple, can be effective, rapidly to Bordetella pertussis
BP is detected, and is applicable in simplicity on site, quickly, is accurately carried out the diagnosis of the disease.
To be of the present invention for detecting the preferred embodiment of the kit of Bordetella pertussis BP, 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 Bordetella pertussis BP, 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 Bordetella pertussis BP, 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 Bordetella pertussis BP, 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 Bordetella pertussis BP, 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 Bordetella pertussis BP, 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 Bordetella pertussis BP, 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 Bordetella pertussis BP 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 Bordetella pertussis BP reflecting point, test sample is the positive；If test strips reaction zone is not
It shows Bordetella pertussis BP 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.
Compared with prior art, the beneficial effects of the present invention are:
The present invention combines recombinase-polymerase nucleic acid amplification technologies with solid phase developing technology, provide one it is simple,
Quickly, being not necessary to expensive device can the live screening mode for carrying out Bordetella pertussis BP.Without thermal cycle reaction and special instrument
Device, testing result can directly be observed by vision, had the characteristics that highly sensitive, specificity, are suitable for instant quick diagnosis,
Screening can be 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 Bordetella pertussis BP 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 Bordetella pertussis BP, 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 used for (table 1) of the invention.
2, primer synthesizes
According to primer sequence shown in table 1, sequent synthesis is carried out.It is characterized in that, its Bordetella pertussis BP 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 Bordetella pertussis BP 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: BSA5wt% and PBS
Buffer 10mM.
Embodiment 2: detection Bordetella pertussis BP
The method that the present embodiment uses the detection Bordetella pertussis BP 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 (48) with Bordetella pertussis BP afterwards, and is confirmed as no Bordetella pertussis BP's after inspection
Negative clinical sample (52).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, sequencing are extracted with PlasmidDNAPreparation Mini Kit (QIAGEN, Valencia, CA)
Correct recombinant plasmid 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/ μ L | 1μL |
| Bsu archaeal dna polymerase, 30ng/ μ L | 1μL |
| T4gp32,900ng/ μ L | 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 Bordetella pertussis BP is anti-
Ying Dian, then test sample is the positive；If test strips reaction zone does not show Bordetella pertussis BP reflecting point, sample is detected as yin
Property.
8, result interpretation
The primer sets designed by the present invention are used for quickly detecting Bordetella pertussis BP, through laboratory quantitative fluorescent PCR side
The sample of the positive Bordetella pertussis BP of 48 of method identification has 48 display Bordetella pertussis BP 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 Bordetella pertussis BP, which is characterized in that the primer is made of forward primer and reverse primer, institute
It states forward primer and is marked with biotin, the reverse primer is marked with FITC or Dig,
The forward primer is nucleotide sequence SEQ ID NO:1:
5'Biotin-ACATCGACATCAAGAAGCTGGGACGTATC 3'；
The reverse primer is nucleotide sequence SEQ ID NO:2:
5’FITC/DIG-TAGTAGGCCACTGCGTCCTTGAGGAACTG 3’。
2. a kind of kit for detecting Bordetella pertussis BP, which is characterized in that the kit includes described in claim 1 draws
Object.
3. a kind of kit for detecting Bordetella pertussis BP according to claim 2, which is characterized 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 Bordetella pertussis BP 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 Bordetella pertussis BP 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 5100mM, the second
Sour magnesium density is 514mM, and the dithiothreitol (DTT) concentration is 52mM, and the dNTPs concentration is 5200 μM, and the ATP concentration is
53mM, the phosphocreatine concentration are 550mM, and the creatine kinase concentration is 5100ng/ μ L, the Carbowax20M concentration
For 55wt%.
6. a kind of kit for detecting Bordetella pertussis BP 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.
7. a kind of kit for detecting Bordetella pertussis BP according to claim 6, 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.
8. a kind of kit for detecting Bordetella pertussis BP according to claim 3, which is characterized in that the positive endoplasm
Control includes the primer pair being made of the nucleotide sequence as shown in SEQ ID NO:3 and SED ID NO:4.
9. a kind of kit for detecting Bordetella pertussis BP according to claim 3, which is characterized in that in the positive
Quality Control is the recombinant plasmid for inserting people's GAPDH gene.
10. a kind of method of the detection Bordetella pertussis BP using the described in any item kits of claim 2~9, feature
It is, 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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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113388689A (en) * | 2021-06-23 | 2021-09-14 | 国药(武汉)医学实验室有限公司 | Primer combination, probe, kit and use method thereof |
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2019
- 2019-03-01 CN CN201910154887.2A patent/CN109680088A/en not_active Withdrawn
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113388689A (en) * | 2021-06-23 | 2021-09-14 | 国药(武汉)医学实验室有限公司 | Primer combination, probe, kit and use method thereof |
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