CN111197097A - Method for detecting fusobacterium nucleatum by using PCR-ELISA - Google Patents
Method for detecting fusobacterium nucleatum by using PCR-ELISA Download PDFInfo
- Publication number
- CN111197097A CN111197097A CN202010056558.7A CN202010056558A CN111197097A CN 111197097 A CN111197097 A CN 111197097A CN 202010056558 A CN202010056558 A CN 202010056558A CN 111197097 A CN111197097 A CN 111197097A
- Authority
- CN
- China
- Prior art keywords
- pcr
- elisa
- fusobacterium nucleatum
- value
- detecting
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- 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
- C12Q1/6888—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
-
- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Urology & Nephrology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biophysics (AREA)
- Biomedical Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Hematology (AREA)
- General Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Cell Biology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for detecting fusobacterium nucleatum by using PCR-ELISA (polymerase chain reaction-enzyme-linked immunosorbent assay) aiming at non-diagnostic treatment, belonging to the technical field of molecular biological detection. The invention discloses a method for detecting fusobacterium nucleatum by utilizing PCR-ELISA aiming at non-diagnosis and treatment, which adopts a streptavidin-biotin-digoxin-anti-digoxin antibody system, has detection sensitivity 100 times that of the conventional PCR, does not generate cross reaction with other bacteria such as staphylococcus aureus, clostridium botulinum, salmonella and the like, has better specificity, and has better sensitivity, specificity and repeatability of the established fusobacterium nucleatum PCR-ELISA detection method, wherein the inter-batch and intra-batch variation coefficients of the detection method are lower than 10 percent.
Description
Technical Field
The invention relates to the technical field of molecular biological detection, in particular to a method for detecting fusobacterium nucleatum by utilizing PCR-ELISA.
Background
Fusobacterium nucleatum (f.nucleatum) is a gram-negative, non-bacillus anaerobic bacterium, named Fusobacterium because it expands in the middle of its morphology and tapers like a shuttle at both ends. Nuclear is not easy to culture, has higher requirement on environment, needs to create strict anaerobic environment during culture, and generally grows better in an anaerobic box containing 5% of hydrogen, 5% of carbon dioxide and 90% of nitrogen. In addition, the nutrient requirement is also strict, anaerobic blood plate culture is generally adopted, and after 48 hours, a bacterial colony with the diameter of about 1-2mm, a round shape, an uneven surface, a convex middle part and translucency is formed, so that the odor is emitted. In 1893, F.nuleatum is found in dental plaque by father Leiwenkeh of microscope for the first time, can cause halitosis and periodontitis, is a common bacterium in oral cavity, can cause infection of liver, lung, blood, joints and other parts of human body, and is closely related to the occurrence of esophageal cancer, oral cancer and gastric cancer. Nuclear has many characteristic factors, most typically FomA, Fap2 and FadA, wherein FadA is an adhesin of f, and the gene has a total length of 390bp, is a highly conserved gene, has no homology with other known adhesins, and can be divided into two forms, one is an undecreted form of FadA (pre-FadA) and a secreted form of mature FadA (mFadA), pre-FadA is composed of 129 amino acid residues and contains a signal peptide consisting of 18 amino acids, mFadA is composed of 111 amino acid residues and can exist in an acidic environment, and the two forms can be combined to form a complex, and are virulence factors necessary for fusobacterium nucleatum attachment and invasion into host cells.
At present, the detection of F.null usually takes PCR as a main technology, and comprises a conventional PCR technology and a multiple PCR technology, wherein the conventional PCR technology is simple and convenient to operate and widely applied, but the conventional PCR technology needs ethidium bromide for dyeing, so that certain danger exists, and the electrophoresis result is only observed by naked eyes and has low sensitivity; the multiplex PCR technique needs to design multiple pairs of primers, which is not easy to master. In addition, Loop-mediated isothermal amplification (LAMP) is a new amplification technology, and detection of fusobacterium nucleatum by LAMP is low in cost, but has high requirements for primers, small application range and serious false positive problem.
Therefore, it is an urgent problem to be solved by those skilled in the art to provide a method for detecting F.nucleatum by PCR-ELISA.
Disclosure of Invention
In view of the above, the invention provides a method for detecting fusobacterium nucleatum by using PCR-ELISA, which has the advantages of high sensitivity, good specificity, high accuracy, safety, rapidness and the like, and is suitable for large-scale detection of samples.
In order to achieve the purpose, the invention adopts the following technical scheme:
a specific primer for detecting fusobacterium nucleatum by using PCR-ELISA for the purpose of non-diagnostic treatment, wherein the sequence of the primer is as follows:
F：5’-CACAAGCTGACGCTGCTAGA-3’；SEQ ID NO.1；
R：5’-TTACCAGCTCTTAAAGCTTG-3’；SEQ ID NO.2；
the 5 'end of the upstream primer F is labeled by digoxin, and the 5' end of the downstream primer R is labeled by biotin.
Further, a method for detecting fusobacterium nucleatum by using PCR-ELISA aiming at non-diagnostic treatment comprises the following specific steps:
(1) extracting sample genome DNA;
(2) performing PCR amplification on the template obtained in the step (1) by using the specific primer of claim 1 to obtain a PCR product;
(3) and (3) ELISA detection: streptavidin was expressed as 1: 800 dilution of the plates, incubation at 37 ℃ for 15min, blocking with 2% BSA at 37 ℃ for 15min, reaction at 37 ℃ for 30min for the primary antibody, and reaction with the secondary antibody at a ratio of 1: diluting at 2000 deg.C, reacting at 37 deg.C for 30min, and developing for 7 min;
(4) the cut-off value of PCR-ELISA was determined.
Further, the reaction procedure of the PCR amplification in the step (2) is as follows: 5min at 94 ℃; 30 cycles of 94 ℃ for 30s, 59 ℃ for 30s, and 72 ℃ for 30 s; 10min at 72 ℃; storing at 4 ℃.
Further, determining the PCR-ELISA threshold value in step (4): detection of OD in sample450A positive sample is judged when the value is 0.111 or more, and a negative sample is judged when the value is less than 0.111.
According to the technical scheme, compared with the prior art, the invention discloses a method for detecting fusobacterium nucleatum by utilizing PCR-ELISA (polymerase chain reaction-enzyme-linked immunosorbent assay) for the purpose of non-diagnosis treatment, a streptavidin-biotin-digoxin-anti-digoxin antibody system is adopted, the detection sensitivity is 100 times that of the conventional PCR, cross reaction with other bacteria such as staphylococcus aureus, clostridium botulinum and salmonella is avoided, the specificity is good, the variation coefficient between batches and in batches is lower than 10%, and the fusobacterium nucleatum PCR-ELISA detection method established by the invention is good in sensitivity, specificity and repeatability.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a diagram showing the result of electrophoresis of FadA gene amplification according to the invention;
wherein, M is DL-2000DNA Marker; 1: a gene of interest; 2: negative control;
FIG. 2 is a diagram showing the comparison result after sequencing of the recombinant plasmid of the present invention;
FIG. 3 is a diagram showing the result of electrophoresis in the PCR sensitivity detection of the present invention;
wherein, M is DL-2000DNA Mark; 1-11 are the recombinant plasmid dilution concentrations: 5.86X 1011copies/μL、5.86×1010copies/μL、5.86×109copies/μL、5.86×108copies/μL、5.86×107copies/μL、5.86×106copies/μL、5.86×105copies/μL、5.86×104copies/μL、5.86×103copies/μL、5.86×102copies/μL、5.86×101copies/μL。
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Fusobacterium nucleatum freeze-dried powder provided by the university of kannel in the United states; the negative control is genome DNA which is detected to be negative by the F.nucleatum FadA gene through PCR; the 4 samples to be tested were 3 stools and 1 tissue of colorectal cancer patients provided by the central hospital in the new county city.
The fecal DNA extraction kit, the plasmid extraction kit and the bacterial genome DNA extraction kit are all purchased from OMEGA biotechnology limited company; EX Taq DNA polymerase, PMD-18-T vector and DNA Marker were purchased from Dalibao Bio Inc.; streptavidin and bovine serum albumin were purchased from Shanghai bioengineering technology, Inc.; TMB single-component color developing solution is purchased from Sigma company; horseradish peroxidase-labeled anti-digoxin antibodies were purchased from dingguo corporation.
Example 1 PCR amplification of the FadA Gene
A pair of specific primers is designed according to FadA gene of F.nuclear in GeneBank and is handed over to Shanghai bioengineering Co.
The primer sequences are as follows:
F：5’-CACAAGCTGACGCTGCTAGA-3’；SEQ ID NO.1；
R：5’-TTACCAGCTCTTAAAGCTTG-3’；SEQ ID NO.2。
activating F.nulatum freeze-dried powder, operating in an anaerobic box by using a trypticase soy liquid culture medium, collecting bacterial liquid, extracting F.nulatum genomic DNA, and carrying out PCR amplification on FadA gene by using the F.nulatum genomic DNA as a template.
Wherein, the PCR reaction system is as follows: mu.l of template, 0.5. mu.l of upstream primer, 0.5. mu.l of downstream primer, 25. mu.l of Premix Taq and 22. mu.l of double distilled water.
The PCR reaction conditions are as follows: 5min at 94 ℃; 30 cycles of 94 ℃ for 30s, 59 ℃ for 30s, and 72 ℃ for 30 s; 10min at 72 ℃; storing at 4 ℃.
The amplified products were electrophoretically detected on a 1.5% agarose gel, and the results are shown in FIG. 1, which is consistent with the expected fragment size of 232 bp.
EXAMPLE 2 construction of recombinant plasmid
Connecting the amplified target gene with a PMD-18-T vector, transforming DH5 α cells to construct a recombinant plasmid, sending the constructed recombinant plasmid to Shanghai bioengineering company Limited for sequencing, loading and unloading fusobacterium nucleatum FadA whole genome sequences from NCBI, and comparing by using DNAman sequence analysis software, wherein the result shows that the consistency is 100 percent (figure 2), the expected result is consistent, and the size of the target fragment is 232 bp.
Example 3PCR-ELISA assay
(1) Labeling the primer of example 1, wherein the 5 'end of the upstream primer F is labeled with digoxin, and the 5' end of the downstream primer R is labeled with biotin; using the recombinant plasmid constructed in example 2 as a template, PCR amplification was performed under the conditions of example 1, followed by ELISA detection.
(2) The streptavidin stock solution (1mg/ml) was diluted with carbonate buffer pH 9.5 and added per wellCoating by adding 100 μ l of ELISA plate, repeating three times for each sample, and standing overnight at 4 deg.C; washing with PBST for 3 times, each for 3 min; blocking with 2% BSA, 200. mu.l per well, standing at 37 ℃ for 2h, and washing the plate 3 times in the same way; diluting the amplification product with recombinant plasmid as template with PBST, placing 100 μ l of the amplification product in each well at 37 deg.C for 1h, washing the plate for 4 times, each time for 3 min; diluting anti-digoxin antibody labeled by horseradish peroxidase with PBST, placing 100 mul per well at 37 ℃ for 1h, and washing the plate for 5 times, 3min each time; adding TMB single-component color development liquid, wherein each hole is 50 mu l, and performing color development for 10min at room temperature in a dark place; 2M H was added2SO4Stop solution, 50. mu.l per well, was placed on an microplate reader to measure OD450The value of (c).
(3) PCR-ELISA reaction conditions
Streptavidin was expressed as 1: 800 dilution plates (concentration 1.25 ng/. mu.l), incubation at 37 ℃ for 15min, blocking with 2% BSA at 37 ℃ for 15min, primary antibody reaction at 37 ℃ for 30min, secondary antibody reaction at 1: diluting at 2000 deg.C, reacting at 37 deg.C for 30min, and developing for 7 min.
Under the same conditions, comparative tests were carried out for each reaction condition:
A. mixing a streptavidin mother solution according to the proportion of 1: 50. 1: 100. 1: 200. 1:400, 1:1600, 1: 3200 dilution, the results are given in table 1.
TABLE 1 streptavidin coating concentration
Table 1 the results show that with the coating concentration 1 of the present invention: 800, concentration 1.25 ng/. mu.l, OD450The value of (positive mean) is closest to 1 and the negative value is smaller.
B. The streptavidin coating conditions are respectively as follows: 4 ℃ overnight, 37 ℃ for 2h, 37 ℃ for 1h, 37 ℃ for 45min, 37 ℃ for 30min, 37 ℃ for 10min, the results are shown in Table 2.
TABLE 2 streptavidin coating conditions
Table 2 results show that the invention can be usedIncubation for 15min at 37 ℃ under streptavidin coating conditions, OD450The value of (positive mean) is closest to 1 and the negative value is smaller.
C. The sealing time is respectively as follows: 10min, 30min, 45min, 1h and 2h, and the results are shown in Table 3.
TABLE 3 blocking time
The results in Table 3 show that the sealing time is 15min, OD450The value of (positive mean) is closest to 1 and the negative value is smaller.
D. The primary antibody reaction time is respectively as follows: 5min, 10min, 15min, 45min and 1h, and the results are shown in Table 4.
TABLE 4 Primary antibody reaction time
The results in Table 4 show that the primary antibody of the present invention has a reaction time of 30min and OD450The value of (positive mean) is closest to 1 and the negative value is smaller.
E. The reaction concentration of the secondary antibody is as follows: 500. 1: 1000. 1: 4000. 1: 8000. 1: 16000. 1: 32000 dilution, the results are shown in Table 5.
TABLE 5 Secondary antibody reaction concentration
Table 5 the results show that with the secondary antibody of the present invention, the reaction concentration is 1: 2000, OD450The value of (positive mean) is closest to 1 and the negative value is smaller.
F. The reaction time of the secondary antibody is 10min, 15min, 45min and 1h respectively, and the results are shown in Table 6.
TABLE 6 Secondary antibody reaction time
The results in Table 6 show that the secondary antibody of the present invention has a reaction time of 30min and OD450The value of (positive mean) is closest to 1 and the negative value is smaller.
G. The color development time was 1min, 3min, 5min, 10min, 15min, respectively, and the results are shown in Table 7.
TABLE 7 color development time
The results in Table 7 show that the color development time is 7min, OD450The value of (positive mean) is closest to 1 and the negative value is smaller.
(4) Establishment of Positive and negative cut-off values
Taking 30 samples with negative common PCR detection, applying the established PCR-ELISA method to detect, and determining the OD450The value of (c). According to the formula: the critical value is the mean value (X) +3 Standard Deviations (SD) of the detected samples, and samples greater than or equal to the critical value are positive for fusobacterium nucleatum, and negative otherwise.
Streptavidin was expressed as 1: 800 dilution plates (concentration 1.25 ng/. mu.l), incubation at 37 ℃ for 15min, 2% BSA blocking at 37 ℃ for 15min, primary antibody reaction at 37 ℃ for 30min, secondary antibody at 1: diluting at 2000 deg.C, reacting at 37 deg.C for 30min, and developing for 7 min. Under the reaction conditions, 30 samples that were negative to the f.nuclearum PCR assay were subjected to PCR-ELISA assay, and the OD of 30 negative samples450The values are respectively: 0.076, 0.065, 0.087, 0.090, 0.067, 0.090, 0.064, 0.086, 0.083, 0.075, 0.058, 0.079, 0.069, 0.089, 0.094, 0.096, 0.068, 0.054, 0.088, 0.068, 0.077, 0.087, 0.089, 0.070, 0.062, 0.082, 0.072, 0.054, 0.060, 0.059, 30 negative samples OD450Has an average value of 0.075 and a standard deviation of 0.012, according to the formula: when the cut-off value is X +3SD, the cut-off value is 0.111, i.e., more than 0.111, the test piece is positive, and the test piece is negative.
EXAMPLE 4 sensitivity test
Diluting the successfully constructed recombinant plasmid mother liquor by multiple times, respectively taking different concentrations as templates to carry out PCR and PCR-ELISA detection, and comparing the sensitivity.
The constructed positive recombinant plasmid (plasmid concentration: 149 ng/. mu.l, copy number: 5.86X 10)11copies/. mu.L) were diluted 10-fold in order, i.e., 5.86X 1011copies/μL-5.86×101The samples/. mu.L were subjected to PCR and PCR-ELISA using different concentrations as templates. The result of PCR is shown in FIG. 3, which shows that 5.86X 10 PCR can be detected6The results of the PCR-ELISA are shown in Table 8, and the results show that the PCR-ELISA method can detect 5.86X 10 at the lowest4copies/. mu.L; the results show that the sensitivity of the PCR-ELISA detection method is 100 times higher than that of the PCR detection method.
TABLE 8 PCR-ELISA sensitivity test results
The PCR-ELISA can detect 5.86 x 10 when the PCR-ELISA is judged to be positive according to the condition that the PCR-ELISA is larger than the critical value of 0.1114copies/μL。
EXAMPLE 5 specificity test
In order to verify the specificity of the established PCR-ELISA detection method, F.tuberculosis, Escherichia coli, Staphylococcus aureus, Clostridium botulinum, Salmonella type, triple vaccine (Clostridium perfringens type A, Clostridium perfringens type D, Clostridium welchii) were detected by PCR-ELISA method, respectively, and the results are shown in Table 9.
TABLE 9 PCR-ELISA specificity test results
Note: the + number indicates positive, the-number indicates negative, and the product was judged to be positive when the value was greater than the threshold value of 0.111.
According to the PCR-ELISA detection result, only the F.null result shows positive, other bacteria show negative, and the result does not generate cross reaction, which indicates that the PCR-ELISA detection method has good specificity.
Example 6 repeatability evaluation
The method comprises the steps of carrying out PCR-ELISA tests by taking DNAs of 4 positive samples and 1 negative sample as templates, carrying out batch repeat tests by using an ELISA plate coated at the same time and carrying out batch repeat tests by using an ELISA plate coated at different times, using the same samples for the batch and batch tests, setting three repeats for each sample, and analyzing detection results to further determine the repeatability effect of the method, wherein the batch repeat test results are shown in a table 10, and the batch repeat test results are shown in a table 11.
TABLE 10 repeatability tests between batches
Note: the mean and standard deviation were kept to three decimal places.
TABLE 11 repeatability test in batches
Note: the mean and standard deviation were kept to three decimal places.
The result of a repeatability test shows that the PCR-ELISA detection method has the inter-batch variation coefficient of 1.20-6.99 percent and the intra-batch variation coefficient of 6.07-8.72 percent which are both lower than 10 percent, thereby showing that the repeatability is better.
Example 7 sample testing
The established PCR-ELISA method and the PCR method are used for detecting clinical samples, and the reliability of the results is compared.
As clinical samples are difficult to collect, three fecal samples and three tissue samples of colorectal cancer patients are temporarily used, 4 samples are detected by the established PCR-ELISA detection method, F.tuberculosis is detected in the tissue samples by the PCR-ELISA and the PCR method, the fecal samples are not detected, the positive detection rate is 25%, and in addition, the bacterial colony in the fecal samples is identified as bacteroides ovatus by sequencing.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Sequence listing
<110> college of New county
<120> a method for detecting fusobacterium nucleatum by using PCR-ELISA
<160>2
<170>SIPOSequenceListing 1.0
<210>1
<211>20
<212>DNA
<213>Artificial Sequence
<400>1
cacaagctga cgctgctaga 20
<210>2
<211>20
<212>DNA
<213>Artificial Sequence
<400>2
ttaccagctc ttaaagcttg 20
Claims (4)
1. A specific primer for detecting fusobacterium nucleatum by using PCR-ELISA for the purpose of non-diagnostic treatment, which is characterized in that the sequence of the primer is as follows:
F：5’-CACAAGCTGACGCTGCTAGA-3’；SEQ ID NO.1；
R：5’-TTACCAGCTCTTAAAGCTTG-3’；SEQ ID NO.2；
the 5 'end of the upstream primer F is labeled by digoxin, and the 5' end of the downstream primer R is labeled by biotin.
2. A method for detecting fusobacterium nucleatum by utilizing PCR-ELISA aiming at non-diagnostic treatment is characterized by comprising the following specific steps:
(1) extracting sample genome DNA;
(2) performing PCR amplification on the template obtained in the step (1) by using the specific primer of claim 1 to obtain a PCR product;
(3) and (3) ELISA detection: streptavidin was expressed as 1: 800 dilution of the plates, incubation at 37 ℃ for 15min, blocking with 2% BSA at 37 ℃ for 15min, reaction at 37 ℃ for 30min for the primary antibody, and reaction with the secondary antibody at a ratio of 1: diluting at 2000 deg.C, reacting at 37 deg.C for 30min, and developing for 7 min;
(4) the cut-off value of PCR-ELISA was determined.
3. The method for detecting F.nucleatum by PCR-ELISA for the purpose of non-diagnostic treatment as claimed in claim 2, wherein the reaction procedure of PCR amplification in step (2) is as follows: 5min at 94 ℃; 30 cycles of 94 ℃ for 30s, 59 ℃ for 30s, and 72 ℃ for 30 s; 10min at 72 ℃; storing at 4 ℃.
4. The method for detecting F.nucleatum by PCR-ELISA according to claim 2, wherein the critical value of PCR-ELISA is determined in step (4): detection of OD in sample450A positive sample is judged when the value is 0.111 or more, and a negative sample is judged when the value is less than 0.111.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010056558.7A CN111197097A (en) | 2020-01-18 | 2020-01-18 | Method for detecting fusobacterium nucleatum by using PCR-ELISA |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010056558.7A CN111197097A (en) | 2020-01-18 | 2020-01-18 | Method for detecting fusobacterium nucleatum by using PCR-ELISA |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111197097A true CN111197097A (en) | 2020-05-26 |
Family
ID=70744875
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010056558.7A Pending CN111197097A (en) | 2020-01-18 | 2020-01-18 | Method for detecting fusobacterium nucleatum by using PCR-ELISA |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111197097A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040197849A1 (en) * | 2002-12-10 | 2004-10-07 | Kao Corporation | Primers for detecting fusobacterium nucleatum by PCR methods and methods for detection |
| CN104745703A (en) * | 2015-03-31 | 2015-07-01 | 大连工业大学 | Vbrio parahaemolyticus PCR-ELISA detection kit and detection method |
| CN107312869A (en) * | 2017-08-31 | 2017-11-03 | 西南大学 | The kit and its detection method of PCR ELISA method detection nosema bombycis |
-
2020
- 2020-01-18 CN CN202010056558.7A patent/CN111197097A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040197849A1 (en) * | 2002-12-10 | 2004-10-07 | Kao Corporation | Primers for detecting fusobacterium nucleatum by PCR methods and methods for detection |
| CN104745703A (en) * | 2015-03-31 | 2015-07-01 | 大连工业大学 | Vbrio parahaemolyticus PCR-ELISA detection kit and detection method |
| CN107312869A (en) * | 2017-08-31 | 2017-11-03 | 西南大学 | The kit and its detection method of PCR ELISA method detection nosema bombycis |
Non-Patent Citations (2)
| Title |
|---|
| 朱绍平等: "正畸治疗中具核梭杆菌及其毒力因子 FadA 粘附素的检测", 《实用口腔医学杂志》 * |
| 王瑞飞: "具核梭杆菌PCR-ELISA检测方法的建立", 《中国知网硕士电子期刊》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105603123B (en) | Real-time fluorescent RPA kit and test strip RPA kit for rapidly detecting porcine parvovirus and application thereof | |
| CN110904250B (en) | Multiplex fluorescent quantitative PCR primer, kit and detection method for detecting multiple bacteria | |
| CN108504780A (en) | 8 kinds of sheep common virus and bacterium Taqman PCR kit for fluorescence quantitative and method are detected simultaneously | |
| CN113025734A (en) | Primer and probe for identifying Brucella vaccine strain A19 and wild strain and application | |
| CN107164557A (en) | It is a kind of while detecting primer and the application of pasteurella multocida and its capsular serotype A type dual real-time fluorescence quantifying PCR method | |
| CN106636470A (en) | Special primer for double PCR of contagious pustular dermatitis virus and mycoplasma ovipneumoniae | |
| KR102675157B1 (en) | Method and Kit for Diagnosing Bovine tuberculosis using PNA | |
| CN113046476A (en) | Primer composition and kit for rapidly detecting N501Y mutation of novel coronavirus | |
| CN111518931A (en) | Multiple PCR diagnostic kit for 3 kinds of pathogenic bacteria in milk | |
| CN111197097A (en) | Method for detecting fusobacterium nucleatum by using PCR-ELISA | |
| NL2031160B1 (en) | Primer Set, Probe and Application for Distinguishing Brucella S2 Vaccine Strain from Wild Strain | |
| CN112961926B (en) | Primer, kit and method for simultaneously detecting listeria monocytogenes CC87 and listeria monocytogenes type 88 strains | |
| CN116334081A (en) | Method for detecting pathogenic microorganisms based on CRISPR technology | |
| CN114959074B (en) | Specific sequence of clostridium difficile with high toxigenic and application thereof in identification and typing | |
| CN108676902A (en) | A kind of composite PCR kit of detection HPS, SS2, Pm, APP | |
| KR102502129B1 (en) | Method and Kit for Identifying the serotype of Actinobacillus pleuropneumoniae causing pneumonia in Pigs using PNA probe | |
| CN111088379B (en) | Specific primer group for detecting salmonella abortus equi and application thereof | |
| CN117778596A (en) | Primer for identifying bifidobacterium infantis YLGB-1496 strain and application thereof | |
| Cuong et al. | Research Article The Production of Standardized Samples with Known Concentrations for Severe Acute Respiratory Syndrome Coronavirus 2 RT-qPCR Testing Validation for Developing Countries in the Period of the Pandemic Era | |
| CN110951901A (en) | Primer, probe and method for triple fluorescence PCR detection of streptococcus suis, haemophilus parasuis and bordetella bronchiseptica | |
| CN118064614A (en) | Primer probe group for helicobacter pylori ERA detection and detection method | |
| CN111996270A (en) | Reagent for detecting bifidobacterium in feces and application thereof | |
| CN117106940A (en) | Primer group for identifying brucella as well as detection method and application thereof | |
| CN116536434A (en) | Primer probe for specific detection and identification detection of lactobacillus rhamnosus and application thereof | |
| CN110982917A (en) | Double PCR detection kit for Escherichia coli O8 and O9 serotypes and detection method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200526 |
|
| RJ01 | Rejection of invention patent application after publication |