CN106701966B - Rapid detection method for pathogenic microorganisms based on analysis of PCR (polymerase chain reaction) byproduct pyrophosphoric acid - Google Patents
Rapid detection method for pathogenic microorganisms based on analysis of PCR (polymerase chain reaction) byproduct pyrophosphoric acid Download PDFInfo
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Abstract
The invention discloses a rapid detection method for pathogenic microorganisms based on analysis of PCR byproduct pyrophosphoric acid, which comprises the following steps: (1) searching pathogenic microorganism characteristic nucleic acid sequence fragments; (2) designing a pair of specific PCR primers aiming at the characteristic nucleic acid sequence segment; (3) extracting microbial genomes of samples to be detected, and performing PCR amplification by using specific PCR primers; (4) analyzing pyrophosphate in PCR amplification; (5) and judging whether the PCR reaction occurs and the amount of the PCR reaction according to the analysis result, and determining whether the sample contains the pathogenic microorganism and the DNA concentration thereof. The method can accurately detect whether the sample contains the pathogenic microorganism and the content of the pathogenic microorganism; the detection and analysis method has the advantages of high sensitivity, quick detection, simple operation process and easy implementation, and can be used for quickly analyzing large samples.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a rapid detection method for pathogenic microorganisms based on analysis of PCR (polymerase chain reaction) by-product pyrophosphoric acid, which is suitable for rapid identification of specific pathogenic microorganisms such as food safety, drinking water safety monitoring, clinical sample diagnosis and the like.
Background
The detection of pathogenic microorganisms (such as salmonella, staphylococcus, streptococcus, vibrio parahaemolyticus, proteus, shigella, avian influenza virus, aspergillus flavus and viruses, foot and mouth disease virus and the like) causing human diseases and food poisoning has important significance in the fields of food, health and medical treatment. The traditional culture method has complex detection operation steps and long time consumption, and can not meet the requirement of rapid detection and diagnosis.
Molecular biology offers the possibility of rapid detection of pathogenic microorganisms. In principle, there are many mature molecular biology methods and techniques that are widely used for qualitative analysis of a certain microorganism, and these methods and techniques are roughly classified into two categories. One is real-time tracking PCR (polymerase chain reaction): such as real-time quantitative PCR technology, real-time melting curve analysis, etc.; another type is analysis of PCR products after PCR is complete: such as gold standard Sanger DNA sequencing technology for nucleic acid sequence analysis, DNA chip technology. Both of the above categories involve PCR and are both superior and inferior, the former being fast but requiring expensive instrumentation and reagents; the latter is cumbersome and time consuming. The development of rapid, low-cost detection of specific nucleic acid sequence fragments is increasingly appreciated and required in industries such as hygiene, food, and the like.
The PCR technique isA molecular biology technique for amplifying a specific DNA fragment can greatly increase a trace amount of DNA. However, in the existing technology for detecting microorganisms in molecular biology, all the objects of analysis are DNA sequences generated in PCR reaction, and the theoretical amount is 2 copies of nucleic acid in a sampleN-1(N is the number of PCR cycles), if the PCR is analyzed by a simple and rapid method such as electrophoresis, the number of nucleic acid copies in a sample needs to reach hundreds to clearly judge whether the PCR reaction occurs through an electrophoresis strip, and the low-abundance sample cannot be effectively detected.
In fact, during PCR, a PCR primer will extend one base length for each nucleotide synthesized, with the concomitant production of a by-product pyrophosphate molecule. If each primer is extended by 100 bases in a PCR process, 200 pyrophosphate molecules are generated in a PCR process on a pair of DNA templates; that is, the number of pyrophosphate molecules produced during PCR is generally several hundred times that of DNA. Therefore, if the sensitivity of detecting DNA is comparable to that of pyrophosphate, the sensitivity of detecting pyrophosphate molecules should be increased several hundred times compared to detecting DNA molecules. Therefore, the sensitivity of selectively analyzing PCR byproduct pyrophosphoric acid, but not PCR product DNA fragments is higher, and the method is more suitable for analyzing low-abundance nucleic acid sequences.
A very large number of assays are available for quantitative analysis of pyrophosphate, and pyrosequencing is a typical example of the use of pyrophosphate analysis. The technology is to detect the DNA fragment amplified by PCR, namely, single-stranded DNA of a PCR product is obtained, then a sequencing primer is hybridized, the sequencing primer extends nucleotide for sequencing, and the acquisition of a sequencing signal is realized by pyrophosphoric acid molecules released by nucleotide extension reaction: whether the sequencing reaction has nucleotide synthesis and the number of syntheses can be determined by analyzing whether pyrophosphate is produced in a single sequencing reaction and the amount of pyrophosphate produced. The principle is that the reaction substrates are 5 '-phosphoryl sulfuric acid and fluorescein, pyrophosphoric acid can be combined with 5' -phosphoryl sulfuric acid to form adenosine triphosphate under the action of adenosine triphosphate sulfurylase, the generated adenosine triphosphate can be combined with the fluorescein to form oxyfluorescein under the catalysis of the luciferase, and visible light is generated at the same time. A detection peak can be obtained by the detection device, and the height of the peak is in direct proportion to the amount of pyrophosphoric acid. However, in the prior art, the DNA fragment amplified by PCR is usually detected by obtaining single-stranded DNA of the PCR product, then hybridizing a sequencing primer, extending nucleotides by the sequencing primer, and sequencing by the sequencing signal obtained by pyrophosphate molecules released by the nucleotide extension reaction, which still has the problems of complicated operation process, long time consumption, detection limit, and the like.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the problems of complex operation flow, long time consumption, detection limit and the like in the microorganism detection in the prior art, the invention provides a rapid detection method of pathogenic microorganisms based on analysis of PCR byproduct pyrophosphoric acid, which searches a segment of nucleic acid sequence segment representing the characteristics of the microorganisms from a public database according to the detected pathogenic microorganisms, designs a pair of specific PCR primers, and judges whether PCR reaction occurs and how much PCR reaction occurs through specific PCR amplification of a sample genome and analysis of total pyrophosphoric acid generated in the PCR amplification, thereby deducing whether the specific microorganisms exist and the DNA concentration thereof. The detection and analysis method has the advantages of high sensitivity, quick detection, simple operation process and easy implementation, and can be used for quickly analyzing large samples.
The technical scheme is as follows: in order to achieve the above object, the present invention provides a method for rapidly detecting pathogenic microorganisms based on analysis of pyrophosphate which is a PCR byproduct, comprising the steps of:
(1) according to the detected pathogenic microorganism, searching a characteristic nucleic acid sequence segment representing the microorganism from a database;
(2) designing a pair of specific PCR primers aiming at the characteristic nucleic acid sequence segment;
(3) extracting a sample microbial genome, and performing PCR amplification by using a specific PCR primer;
(4) analyzing pyrophosphate in PCR amplification;
(5) and judging whether the PCR reaction occurs and the amount of the PCR reaction according to the analysis result, and determining whether the sample contains the microorganism and the content of the microorganism.
Wherein, the characteristic nucleic acid sequence fragment in the step (1) is the nucleic acid sequence information which is unique to the microorganism needing to be detected, is not contained in other organisms and can be obtained through a public database.
The PCR amplification in step (3) is specific, only the characteristic nucleic acid sequence fragment in step (1) is amplified, and other sequence fragments are not amplified.
The analysis of pyrophosphate in the PCR amplification in the step (4) is realized by directly analyzing total pyrophosphate in the PCR product by a pyrophosphate chemiluminescence analysis method.
The pyrophosphate chemiluminescence analysis method is implemented by using a pyrophosphate detection kit or by using a prepared chemiluminescence reagent.
The pyrophosphate detection kit is a pyrophosphate detection kit of SIGMA-ALDRICH company.
Further, the prepared chemiluminescence reagent comprises the following main components: 0.01 to 0.5M Tris-acetate (pH 7.7),10 to 50mM EDTA,1 to 5mM Mg (Ac)20.02-0.1% Bovine Serum Albumin (BSA), 1-5 mM Dithiothreitol (DTT), 10-50 mM 5' -phosphosulfate (APS), 0.2-1.0 mg/mL polyvinylpyrrolidone (PVP), 2-10 mM fluorescein (D-luciferin), and 50-800 mM luciferase. Typically 0.01M Tris-acetate (pH 7.7),20mM EDTA,1mM Mg (Ac)20.02% Bovine Serum Albumin (BSA), 1mM Dithiothreitol (DTT), 10mM 5' -phosphosulfate (APS), 0.4mg/mL polyvinylpyrrolidone (PVP), 4mM luciferin (D-luciferin), 800mM luciferase.
Further, a photodiode or a charge-coupled device (CCD) is used for the detection antigen of the optical signal in the pyrophosphate chemiluminescence analysis method; the detection can be carried out singly or in parallel.
Wherein the determination of whether the microorganism is contained in the sample in the step (5) is determined by a set value of CV of pyrophosphate: the pyro-analysis signal intensity is greater than the CV value and is judged to be present, and is corresponding to the sample containing the microorganism, and is less than the CV value and is judged to be absent, and is corresponding to the sample not containing the microorganism;
the step (5) of determining the quantitative analysis of the content of the microorganisms in the sample means that a series of known concentrations of the microorganisms pass through the steps (1) - (5), a relation curve of concentration and signal intensity is established, then the signal intensity obtained by analyzing the sample is analyzed, and the specific concentration of the microorganisms is obtained from the relation curve.
The present invention is based on the use of pyrophosphate produced in several hundred times the amount of DNA during PCR to determine whether the final PCR reaction occurs and the extent of the reaction, and at the same time, to deduce whether a certain microorganism and the amount thereof are contained in a sample participating in the PCR reaction. Since pyrophosphate detection is a well-established technology, corresponding kits are commercially available (SIGMA-ALDRICH, Inc., etc.). Therefore, only a simple optical signal detection device is required to be constructed to realize the quantitative analysis of the pyrophosphate.
Has the advantages that: compared with the prior art, the method directly detects a large amount of pyrophosphoric acid generated in the PCR process to judge whether the PCR reaction occurs; the prior art discloses a pyrosequencing technology, which detects a DNA fragment amplified by PCR, namely, obtains a single-stranded DNA of a PCR product, then hybridizes with a sequencing primer, and the sequencing primer extends nucleotides to perform sequencing, wherein the obtaining of a sequencing signal is realized by pyromolecules released by a nucleotide extension reaction. Therefore, although all the detection methods detect pyrophosphate molecules, the differences are that: 1. the pyrosequencing technology disclosed in the prior art is to detect pyromolecules of a single sequencing reaction in real time; in contrast, the present invention detects total pyrophosphate molecules produced by PCR reactions. 2. The detection objects are different, and the detection objects of the pyrosequencing technology disclosed in the prior art are DNA sequences of PCR products; the target of detection in the present invention is pyrophosphate of the PCR product.
The invention has the following advantages:
1. the invention selects the characteristic nucleic acid sequence segment of the microorganism to design a specific primer, and can accurately detect whether the microorganism and the DNA concentration thereof are contained in the sample by detecting the pyrophosphoric acid in the PCR by-product.
2. The invention adopts pyrophosphoric acid for detecting hundreds of times of DNA molecules to track PCR reaction, and has higher sensitivity than the traditional method for detecting DNA molecules.
3. The invention adopts direct detection of the pyrophosphate content in the PCR product, the PCR is completed, the analysis of the sample can be completed within 3 minutes, and the detection speed is faster than that of the traditional DNA molecule detection method.
4. The method can be used for quickly analyzing a large sample, and can be used for primary screening and detection.
5. The invention can realize quantitative analysis of pyrophosphoric acid by adopting a simple optical detection device, and has simple operation flow and easy implementation.
Drawings
FIG. 1 is a standard curve of the concentration C (ng/. mu.L) of the shigella DNA template and the intensity (Y) of the fluorescence signal in example 1 of the present invention;
FIG. 2 is the result of agarose gel electrophoresis detection of PCR products of different shigella contents in example 1 of the present invention.
Detailed Description
The invention is further illustrated by the following figures and examples.
Example 1: detection of shigella in chicken
1. Preparation of a genome DNA sample: washing bacteria on the surface of chicken by PBS (pH 7.2), extracting the washed bacterial DNA by a bacterial DNA extraction kit, and preserving at low temperature for later use.
2. PCR primer design and PCR reaction
Aiming at a characteristic nucleic acid sequence fragment of the shigella, the ipaH gene of the shigella is a gene for coding and secreting a virulence protein, is closely related to the pathogenicity of the shigella, is peculiar to the shigella, and a forward primer and a reverse primer are designed according to GenBank accession No. M76445.1:
(sequences of amplified genes were searched in GeneBank database, submitted to primer design software Primer5.0 for primer design, the specificity of which can be compared with the homologous sequence by NCBI BLAST (http:// www.ncbi.nlm.nih.gov/BLAST /), the primer specificity was required to be strong, no or less primer dimer, miscitation, etc.
The designed primers were synthesized and subjected to the following PCR reaction:
(1) determination of the standard curve: a25. mu.L PCR amplification system comprised: the genome DNAs were 126, 1.26, and 1.26X 10, respectively-2、1.26×10-4、1.26×10-60 ng/. mu.L, 0.2mM dNTP, 0.8. mu.M forward and 0.8. mu.M reverse primers, 2U Taq DNA polymerase, 1 Xamplification buffer, 1.5mM MgCl2. Amplification conditions: pre-denaturation at 94 ℃ for 5min, 35 thermal cycles (denaturation at 94 ℃ for 30 sec-54 ℃ annealing for 30 sec-72 ℃ extension for 30 sec), and final extension at 72 ℃ for 5 min.
(2) Detection of the actual sample: a25. mu.L PCR amplification system comprised: 200ng genomic DNA, 0.2mM dNTP, 0.8. mu.M forward and 0.8. mu.M reverse primer, 2U Taq DNA polymerase, 1 Xamplification buffer, 1.5mM MgCl2. Amplification conditions: pre-denaturation at 94 ℃ for 5min, 35 thermal cycles (denaturation at 94 ℃ for 30 sec-54 ℃ annealing for 30 sec-72 ℃ extension for 30 sec), and final extension at 72 ℃ for 5 min.
3. PCR product detection
(1) Preparation of chemiluminescent system
The following self-made formula reaction system (S-mix) was used: 0.01M Tris-acetate (pH 7.7),20mM EDTA,1mM Mg (Ac)20.02% Bovine Serum Albumin (BSA), 1mM Dithiothreitol (DTT), 10mM 5' -phosphosulfate (APS), 0.4mg/mL polyvinylpyrrolidone (PVP), 4mM luciferin (D-luciferin), 800mM luciferase.
(2) PCR product detection
A. And starting the weak light-emitting measuring instrument, and setting the temperature and the high-voltage value.
B. A1.5 mL centrifuge tube was prepared and 400. mu.L of chemiluminescent system (S-mix) solution was added.
C. And (3) checking whether a shutter of the weak luminescence measuring instrument is closed, opening a cover of the sample chamber when the shutter is closed, putting the centrifuge tube into the sample chamber, covering an upper cover, opening the shutter, and measuring the background signal of the chemiluminescence system solution.
D. And (4) closing the shutter, opening the upper cover, and taking out the centrifuge tube. Adding 1 mu L of PCR amplification product into the solution of the chemiluminescence system, and shaking and mixing uniformly. Then the sample is put into a sample chamber, a shutter is opened, and the fluorescence signal value of the PCR amplification product is measured. And recording the measured data.
E. And after the measurement is finished, closing the shutter and the measuring instrument.
F. And (3) according to the CV value measured under the standard concentration of the DNA of the shigella, comparing the fluorescence signal value of the measured sample with the CV value in an evolutionary manner, judging that the sample contains the microorganism if the signal intensity is greater than the CV value, and judging that the sample does not contain the microorganism if the signal intensity is less than the CV value.
(3) Result judgment
A. And (3) measuring results: the results of the standard curve and the actual sample are shown in Table 1. A positive sample is obtained when the signal intensity of the measured sample is higher than 45000 according to the fluorescence signal values at different concentrations, which indicates that the sample contains the microorganism. Signal intensities between 45000 and 56000 are lightly contaminated; signals with signal strengths between 56000 and 88000 were moderately contaminated, and signals with signal strengths greater than 88000 were heavily contaminated.
TABLE 1 fluorescence signal intensity of PCR products at different DNA template concentrations.
B. Drawing a standard curve: the measurement graph 1 is plotted according to the fluorescence signal intensity (Y) measured according to the known DNA template concentration (C) in table 1, and the curve equation obtained by fitting is Y11750X +86300, X is the logarithm of the DNA concentration (lgC), and Y is the measured fluorescence signal intensity minus the fluorescence signal intensity of the blank (24000).
C. And (3) actual sample detection result: in this example 2, the signal intensity of the sample 1 was 45000, and the detected shigella DNA concentration was 3.98 × 10 according to the standard curve (fig. 1)-6ng/μ L, mild contamination; the signal intensity of sample 2 was 22000, which is equivalent to that of the blank 24000, and shigella was not detected and no contamination was observed.
Comparative example 1: shigella contrast detection method
(1) PCR product electrophoresis detection of known shigella genome DNA
1. Preparation of a genome DNA sample: bacteria on the surface of chicken were washed out with PBS (PH 7.2), and then the washed-out bacterial DNA was extracted with a bacterial DNA extraction kit and the concentration of genomic DNA was determined.
2. PCR primer design and PCR reaction
Aiming at a characteristic nucleic acid sequence fragment of the shigella, the ipaH gene of the shigella is a gene for coding and secreting a virulence protein, is closely related to the pathogenicity of the shigella, is peculiar to the shigella, and a forward primer and a reverse primer are designed according to GenBank accession No. M76445.1:
(sequences of amplified genes were searched in GeneBank database, submitted to primer design software Primer5.0 for primer design, the specificity of which can be compared with the homologous sequence by NCBI BLAST (http:// www.ncbi.nlm.nih.gov/BLAST /), the primer specificity was required to be strong, no or less primer dimer, miscitation, etc.
The designed primers were synthesized and subjected to the following PCR reaction (blank control without DNA template):
a25. mu.L PCR amplification system comprised: the concentrations of the genomic DNA templates were 126, 1.26, and 1.26X 10, respectively-2、1.26×10-4、1.26×10-60 ng/. mu.L, 0.2mM dNTP, 0.8. mu.M forward and 0.8. mu.M reverse primers, 2U Taq DNA polymerase, 1 Xamplification buffer, 1.5mM MgCl2. Amplification conditions: pre-denaturation at 94 ℃ for 5min, 35 thermal cycles (denaturation at 94 ℃ for 30 sec-54 ℃ annealing for 30 sec-72 ℃ extension for 30 sec), and final extension at 72 ℃ for 5 min.
3. Agarose gel electrophoresis detection of PCR products
5 μ l of the PCR product was applied to a 1% agarose gel, and the PCR product was subjected to agarose gel electrophoresis (electrophoresis conditions: 120V, 20 minutes), and whether the band was clear or not was observed, and the results were shown in FIG. 2. In FIG. 2, aAnd (3) detecting the result of agarose gel electrophoresis of the PCR product of the shigella of the same content. In the figure, M, 1, 2, 3, 4, 5 and 6 are DNA fragments (Marker) with known lengths respectively, and the concentrations of PCR templates are 126, 1.26 and 1.26 multiplied by 10 respectively-2、1.26×10-4、1.26×10-6、0ng/μL。
(2) Shigella culture detection method
1. Preparing a BPW culture medium: weighing 20.1g of BPW culture medium, adding 1L of distilled water, stirring, heating, boiling to dissolve completely, subpackaging in 90 mL/bottle into each conical flask, sterilizing at 121 deg.C for 15min, and cooling to room temperature.
2. After the culture medium is cooled to room temperature, the culture medium and the whole room are subjected to ultraviolet sterilization for 15 minutes, and the lamp is turned off for 60 minutes for use.
3. Pre-enrichment: weighing 10g of chicken samples into conical flasks containing BPW culture medium, covering the conical flasks with a cover in time, shaking at 150rpm for 10min, and culturing at 36 +/-1 ℃ for about 18 h.
4. Preparing TTB enrichment fluid: and (3) taking 93.6g of TTB enrichment medium base, adding 1L of distilled water, stirring, heating, boiling until the TTB enrichment medium base is completely dissolved, and subpackaging in conical flasks, wherein each flask is 100 mL. Placing the conical flask into a sterilizing pot, and sterilizing at 121 deg.C for 20 min; cooling to 30 deg.C, adding TTB reagent iodine solution and Brilliant green (75% alcohol cotton is used to sterilize the surface of penicillin bottle before opening) into each 100mL of basic culture medium, and mixing.
5. Enrichment of bacteria
6. Inoculation and culture: the cultured sample mixture is gently shaken, 1mL of the sample mixture is transferred into 9mL of TTB enrichment medium, and the mixture is cultured for 18 to 24 hours at the temperature of 42 +/-1 ℃.
7. The culture phenomenon was observed.
8. And (4) preparing a shigella chromogenic medium plate.
9. 4.75g of dry powder in the bottle is taken and dissolved by 100mL of distilled water, and the dry powder can be proportionally expanded or reduced.
10. Mixing, heating to 100 deg.C, stirring, heating to dissolve completely, cooling to 50 deg.C, and pouring.
11. The samples were inoculated by either streaking or plating and incubated at 37 ℃ for 24 hours.
12. The colony color was observed.
Through comparison and analysis with electrophoresis detection of samples with known Shigella DNA concentration, the genome DNA template with the lowest concentration of 1.26 × 10 can be detected by the rapid detection method for pathogenic microorganisms based on the analysis of PCR byproduct pyrophosphoric acid- 6ng/. mu.L, whereas the traditional PCR product electrophoresis detection method cannot detect the concentration of the genomic DNA template as 1.26X 10-2ng/. mu.L of the sample was tested and thus more sensitive.
Through the comparative analysis of the actual sample culture detection method, the result of the rapid detection method for pathogenic microorganisms based on the analysis of the pyrophosphate as the PCR by-product is consistent with that of the culture detection method.
The culture detection method is a strict and accurate microorganism detection method, but the method has complex operation flow and long time consumption, and is not suitable for the rapid detection of microorganisms; the electrophoretic detection method of the PCR product of the microorganism is a qualitative analysis method for rapid detection at present, and compared with the electrophoretic detection method of the PCR product of the microorganism, the method of example 1 not only takes shorter time, but also can carry out quantitative analysis on the detected microorganism. Therefore, the rapid detection method for pathogenic microorganisms based on the analysis of the pyrophosphoric acid as the PCR byproduct has the advantages of rapid detection, simple operation process, easy implementation, capability of performing rapid analysis on a large sample, and suitability for rapid detection of microorganisms in the industries of food, sanitation and the like.
Example 2
Example 2 the same procedure as in example 1 was followed, except that the pyrophosphate chemiluminescence analysis method used a pyrophosphate detection kit purchased from SIGMA-ALDRICH, and the detection results were similar to those of example 1.
SEQUENCE LISTING
<110> university of southeast
<120> quick detection method of pathogenic microorganism based on analysis of PCR byproduct pyrophosphoric acid
<130> 2017.01.20
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 19
<212> DNA
<213> Artificial Synthesis
<400> 1
actattggct ctggatgtt 19
<210> 2
<211> 17
<212> DNA
<213> Artificial Synthesis
<400> 2
gggcgatgga gtgtatt 17
Claims (1)
1. A rapid detection method for pathogenic microorganisms based on analysis of pyrophosphate as a PCR byproduct is characterized by comprising the following steps:
step 1: preparation of a genome DNA sample: washing bacteria on the surface of chicken by PBS (phosphate buffer solution) pH =7.2, extracting the washed bacterial DNA by a bacterial DNA extraction kit, and storing at a low temperature for later use;
step 2: PCR primer design and PCR reaction
Aiming at the characteristic nucleic acid sequence fragment of the Shigella, the ipaH gene of the Shigella is a gene for coding and secreting virulence protein, is closely related to the pathogenicity of the shigella, is peculiar to the Shigella, and a forward primer and a reverse primer are designed according to GenBank accession No. M76445.1:
SEQ ID NO 1 is a forward primer F5 'ACTATTGGCTCTGGATGTT 3',
SEQ ID NO 2 is a reverse primer R5 'GGGCGATGGAGTGTATT 3';
the designed primers were synthesized and subjected to the following PCR reaction:
(1) determination of the standard curve: a25. mu.L PCR amplification system comprised: the genome DNAs were 126, 1.26, and 1.26X 10, respectively-2、1.26× 10-4、1.26×10-60 ng/. mu.L, 0.2mM dNTP, 0.8. mu.M forward and 0.8. mu.M reverse primers, 2U Taq DNA polymerase, 1 Xamplification buffer, 1.5mM MgCl2(ii) a Amplification conditions: pre-denaturation at 94 ℃ for 5 minutes, denaturation at 94 ℃ for 30 seconds to 54 ℃ for annealing at 30 seconds to 72 ℃ for 30 seconds by 35 thermal cycles, and finally extension at 72 ℃ for 5 minutes;
(2) detection of the actual sample: a25. mu.L PCR amplification system comprised: 200ng genomic DNA, 0.2mM dNTP, 0.8. mu.M forward and 0.8. mu.M reverse primer, 2U Taq DNA polymerase, 1 Xamplification buffer, 1.5mM MgCl2(ii) a Amplification conditions: pre-denaturation at 94 ℃ for 5 minutes, denaturation at 94 ℃ for 30 seconds to 54 ℃ for annealing at 30 seconds to 72 ℃ for 30 seconds by 35 thermal cycles, and finally extension at 72 ℃ for 5 minutes;
and step 3: PCR product detection
(1) Preparation of chemiluminescent system
The following self-made formula reaction system is adopted: 0.01M Tris-acetate pH 7.7, 20mM EDTA,1mM Mg (Ac)20.02% bovine serum albumin BSA, 1mM dithiothreitol, 10mM 5' -phosphosulfuric acid, 0.4mg/mL polyvinylpyrrolidone, 4mM luciferin, 800mM luciferase;
(2) PCR product detection
A. Starting a weak light-emitting measuring instrument, and setting a temperature value and a high-voltage value;
B. preparing a 1.5mL centrifuge tube, and adding 400 mu L of chemiluminescent system S-mix solution;
C. checking whether a shutter of the weak luminescence measuring instrument is closed, opening a cover of the sample chamber when the shutter is closed, putting the centrifuge tube into the sample chamber, covering an upper cover, opening the shutter, and measuring a background signal of a chemiluminescence system solution;
D. closing the shutter, opening the upper cover, taking out the centrifuge tube, adding 1 mu L of PCR amplification product into the solution added with the chemiluminescence system, oscillating and mixing uniformly, then putting the mixture into a sample chamber, opening the shutter, measuring to obtain a fluorescence signal value of the PCR amplification product, and recording the measured data;
E. after the measurement is finished, closing the shutter and the measuring instrument;
F. according to the CV value measured under the standard concentration of the DNA of the shigella, comparing the fluorescence signal value of the measured sample with the CV value in an evolutionary manner, judging that the sample contains the microorganism if the signal intensity is greater than the CV value, and judging that the sample does not contain the microorganism if the signal intensity is less than the CV value; and simultaneously establishing a relation curve of concentration and signal intensity, analyzing the signal intensity obtained by the sample, and obtaining the specific concentration of the microorganism DNA from the relation curve.
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| CN1398988A (en) * | 2002-08-09 | 2003-02-26 | 周国华 | Gene expression amount comparing analysis method |
| CN1852990A (en) * | 2003-01-14 | 2006-10-25 | 劳伦斯·C·第希 | Method for determining the amount of template nucleic acid present in a sample |
| CN101974616A (en) * | 2009-12-10 | 2011-02-16 | 西华大学 | Kit and method for quickly detecting enterotoxin generating escherichia coli |
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| CN1398988A (en) * | 2002-08-09 | 2003-02-26 | 周国华 | Gene expression amount comparing analysis method |
| CN1852990A (en) * | 2003-01-14 | 2006-10-25 | 劳伦斯·C·第希 | Method for determining the amount of template nucleic acid present in a sample |
| CN101974616A (en) * | 2009-12-10 | 2011-02-16 | 西华大学 | Kit and method for quickly detecting enterotoxin generating escherichia coli |
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| 《应用生物发光技术定量检测PCR 扩增产物》;姚群峰 宁勇 徐顺清 周宜开;《临床输血与检验》;20030831;第5卷(第3期);第5,14-16段 * |
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