CN113355439A - Novel combined detection method for latent infection and morbidity of mycobacterium tuberculosis - Google Patents
Novel combined detection method for latent infection and morbidity of mycobacterium tuberculosis Download PDFInfo
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- CN113355439A CN113355439A CN202110633613.9A CN202110633613A CN113355439A CN 113355439 A CN113355439 A CN 113355439A CN 202110633613 A CN202110633613 A CN 202110633613A CN 113355439 A CN113355439 A CN 113355439A
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- 241000187479 Mycobacterium tuberculosis Species 0.000 title claims abstract description 46
- 238000001514 detection method Methods 0.000 title claims abstract description 44
- 208000032420 Latent Infection Diseases 0.000 title claims abstract description 14
- 208000037771 disease arising from reactivation of latent virus Diseases 0.000 title claims abstract description 14
- 230000000694 effects Effects 0.000 claims abstract description 14
- 238000000034 method Methods 0.000 claims abstract description 11
- 238000012258 culturing Methods 0.000 claims abstract description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 8
- PLXBWHJQWKZRKG-UHFFFAOYSA-N Resazurin Chemical compound C1=CC(=O)C=C2OC3=CC(O)=CC=C3[N+]([O-])=C21 PLXBWHJQWKZRKG-UHFFFAOYSA-N 0.000 claims description 21
- 239000002824 redox indicator Substances 0.000 claims description 21
- 239000001963 growth medium Substances 0.000 claims description 19
- 241000894006 Bacteria Species 0.000 claims description 12
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 10
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 10
- COHYTHOBJLSHDF-UHFFFAOYSA-N Indigo Chemical compound N1C2=CC=CC=C2C(=O)C1=C1C(=O)C2=CC=CC=C2N1 COHYTHOBJLSHDF-UHFFFAOYSA-N 0.000 claims description 10
- 238000012408 PCR amplification Methods 0.000 claims description 10
- 239000011521 glass Substances 0.000 claims description 10
- 239000002609 medium Substances 0.000 claims description 9
- 230000035755 proliferation Effects 0.000 claims description 9
- 238000001035 drying Methods 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 6
- 102000004190 Enzymes Human genes 0.000 claims description 5
- 108090000790 Enzymes Proteins 0.000 claims description 5
- 238000002835 absorbance Methods 0.000 claims description 5
- 230000003321 amplification Effects 0.000 claims description 5
- 230000002503 metabolic effect Effects 0.000 claims description 5
- 230000035772 mutation Effects 0.000 claims description 5
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 5
- 230000003647 oxidation Effects 0.000 claims description 5
- 238000007254 oxidation reaction Methods 0.000 claims description 5
- JQXXHWHPUNPDRT-WLSIYKJHSA-N rifampicin Chemical compound O([C@](C1=O)(C)O/C=C/[C@@H]([C@H]([C@@H](OC(C)=O)[C@H](C)[C@H](O)[C@H](C)[C@@H](O)[C@@H](C)\C=C\C=C(C)/C(=O)NC=2C(O)=C3C([O-])=C4C)C)OC)C4=C1C3=C(O)C=2\C=N\N1CC[NH+](C)CC1 JQXXHWHPUNPDRT-WLSIYKJHSA-N 0.000 claims description 5
- 229960001225 rifampicin Drugs 0.000 claims description 5
- 238000007605 air drying Methods 0.000 claims description 3
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 3
- 208000015181 infectious disease Diseases 0.000 claims description 3
- 230000002458 infectious effect Effects 0.000 claims description 3
- 239000012528 membrane Substances 0.000 claims description 3
- 238000002791 soaking Methods 0.000 claims description 3
- 230000008506 pathogenesis Effects 0.000 claims 6
- 230000007547 defect Effects 0.000 abstract description 5
- 230000002062 proliferating effect Effects 0.000 description 3
- 244000052769 pathogen Species 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 201000008827 tuberculosis Diseases 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 210000003495 flagella Anatomy 0.000 description 1
- 229920006008 lipopolysaccharide Polymers 0.000 description 1
- 239000003094 microcapsule Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 208000008128 pulmonary tuberculosis Diseases 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
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- 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
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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
- 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]
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Abstract
The invention discloses a novel combined detection method for latent infection and morbidity of mycobacterium tuberculosis, which comprises the following steps: the method comprises the following steps: preparing a sample to be detected, performing activity detection on the sample to be detected, and entering the next step after the sample to be detected is qualified; step two: culturing a qualified sample to be detected by using a Roche medium, and collecting all cultures to be respectively soaked in an ethanol solution; step three: dividing the solution soaked in the step two into multiple parts, and performing operation in the step four on any one part; by the design of the invention, the activity of the detection sample can be detected in the early stage, the accuracy of the detection in the later stage is ensured, the phenomenon of missing detection is avoided, and meanwhile, on the premise of not influencing the mycobacterium tuberculosis in the sample, the single detection sample is proliferated, and is divided into a plurality of parts, so that the latent characteristic and the morbidity of the mycobacterium tuberculosis are conveniently detected in multiple ways, and the existing defects are overcome.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a novel combined detection method for latent infection and morbidity of mycobacterium tuberculosis.
Background
Mycobacterium tuberculosis, commonly known as tubercle bacillus, is a pathogen causing tuberculosis, can invade all organs of the body, but causes pulmonary tuberculosis most frequently, is an ancient disease, is widely distributed in the world, is the first cause of death of bacterial infectious diseases, is a pathogen of human tuberculosis, is a bacterium with obligate oxygen, is acid-fast staining positive, has no flagella and fimbriae, has microcapsules but does not form spores, and has a bacterial wall without teichoic acid of gram-positive bacteria and lipopolysaccharide of gram-negative bacteria.
The activity of a detection sample is not detected in the conventional detection of the mycobacterium tuberculosis, once the activity of the detected sample is low, the accuracy of later detection is influenced, the phenomenon of missing detection is easy to occur, and the transmission and incidence of the mycobacterium tuberculosis cannot be correspondingly detected in the detection of the sample.
Disclosure of Invention
The invention aims to provide a novel combined detection method for latent infection and morbidity of mycobacterium tuberculosis, which aims to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme: a novel combined detection method for latent infection and morbidity of mycobacterium tuberculosis comprises the following steps:
the method comprises the following steps: preparing a sample to be detected, performing activity detection on the sample to be detected, and entering the next step after the sample to be detected is qualified;
step two: culturing a qualified sample to be detected by using a Roche medium, and collecting all cultures to be respectively soaked in an ethanol solution;
step three: dividing the solution soaked in the step two into multiple parts, and performing operation in the step four on any one part;
step four: taking a part of the soaking solution in the third step, air-drying at room temperature, collecting and drying solid matters, drying the solid matters, adding the extracting solution, mixing, and detecting whether the extracting solution contains mycobacterium tuberculosis with an amino acid sequence of NSGYIECCR;
step five: separating the biomembrane of the mycobacterium tuberculosis detected in the fourth step;
step six: and detecting whether the biological membrane is infectious or not by using a microplate reader.
As a preferred embodiment of the present invention, the activity detection method in the first step is as follows:
s1: placing a sample to be detected in a glass culture dish, adding a culture medium into the culture dish, and culturing for 10-15 min;
s2: adding AlamarBlue redox indicator, and observing the proliferation condition of the sample in an oxidation state;
s3: observing the color of the sample after adding the AlamarBlue redox indicator, wherein the AlamarBlue redox indicator taken into the bacteria is reduced by a metabolic intermediate, released to the outside of the body and dissolved in a culture medium, so that the culture medium is changed from non-fluorescent indigo blue into fluorescent pink;
s4: and (3) detecting by using a common spectrophotometer or a fluorescence spectrophotometer, wherein when the absorbance and the fluorescence intensity are in direct proportion to the number of active bacteria, the sample is qualified, and otherwise, the sample is unqualified.
Taking one solution in the second step, adopting high-fidelity DNA polymerase and non-high-fidelity DNA polymerase mixed enzyme as polymerase, carrying out PCR amplification on the DNA sample of the mycobacterium tuberculosis, and detecting whether a PCR amplification product is formed or not, wherein if the amplification product is formed, the mycobacterium tuberculosis contains rifampicin-resistant mutation.
In a preferred embodiment of the present invention, the culture medium added in S1 is a czochralski culture medium.
In a preferred embodiment of the present invention, in S2, the time for observing the proliferation of the sample is 20 to 30 min.
In a preferred embodiment of the present invention, in S4, the fluorophore is selected from FAM, ROX, HEX, CY5, TET, and CAL-Fluor.
In a preferred embodiment of the present invention, the diameter of the glass petri dish in S1 is 10-15 cm.
Compared with the prior art, the invention has the beneficial effects that:
by the design of the invention, the activity of the detection sample can be detected in the early stage, the accuracy of the detection in the later stage is ensured, the phenomenon of missing detection is avoided, and meanwhile, on the premise of not influencing the mycobacterium tuberculosis in the sample, the single detection sample is proliferated, and is divided into a plurality of parts, so that the latent characteristic and the morbidity of the mycobacterium tuberculosis are conveniently detected in multiple ways, and the existing defects are overcome.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to 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.
Example 1
The invention provides a technical scheme that: a novel combined detection method for latent infection and morbidity of mycobacterium tuberculosis comprises the following steps:
the method comprises the following steps: preparing a sample to be detected, performing activity detection on the sample to be detected, and detecting the activity of the sample to be detected in the early stage to ensure the accuracy of later detection and avoid the phenomenon of missing detection at the same time;
step two: culturing a qualified sample to be detected by using a Roche medium, and collecting all cultures to be respectively soaked in an ethanol solution;
step three: dividing the solution soaked in the step two into multiple parts, and performing operation in the step four on any one part;
step four: taking a part of the soaking solution in the third step, air-drying at room temperature, collecting and drying solid matters, drying the solid matters, adding the extracting solution, mixing, and detecting whether the extracting solution contains mycobacterium tuberculosis with an amino acid sequence of NSGYIECCR;
step five: separating the biomembrane of the mycobacterium tuberculosis detected in the fourth step;
step six: and detecting whether the biological membrane is infectious or not by using a microplate reader.
In this example, the activity detection method in step one is as follows:
s1: placing a sample to be tested in a glass culture dish, adding a culture medium into the culture dish, and culturing for 12 min;
s2: adding AlamarBlue redox indicator, and observing the proliferation condition of the sample in an oxidation state;
s3: observing the color of the sample after adding the AlamarBlue redox indicator, reducing the AlamarBlue redox indicator taken into the bacteria by a metabolic intermediate, releasing the AlamarBlue redox indicator out of the body, dissolving the AlamarBlue redox indicator in a culture medium, changing the culture medium from non-fluorescent indigo blue into fluorescent pink, proliferating the Mycobacterium tuberculosis under the premise of not influencing the Mycobacterium tuberculosis in the sample, equally dividing a single detection sample into multiple parts, facilitating the multiple detection of the latent characteristic and the morbidity of the Mycobacterium tuberculosis, and improving the existing defects;
s4: and (3) detecting by using a common spectrophotometer or a fluorescence spectrophotometer, wherein when the absorbance and the fluorescence intensity are in direct proportion to the number of active bacteria, the sample is qualified, and otherwise, the sample is unqualified.
In this embodiment, one solution of the second step is taken, high fidelity DNA polymerase and non-high fidelity DNA polymerase mixed enzyme are used as polymerase, PCR amplification is carried out on the DNA sample of the mycobacterium tuberculosis, whether PCR amplification products are formed or not is detected, wherein if amplification products are formed, the mycobacterium tuberculosis contains rifampicin-resistant mutation.
In this example, the medium added in S1 was a czochralski medium.
In this example, in S2, the time for observing the growth of the sample was 25 min.
In this example, in S4, the fluorophore is any one of FAM, ROX, HEX, CY5, TET, CAL-Fluor.
In this example, the diameter of the glass petri dish in S1 was 12 cm.
Example 2
The difference from the present embodiment 1 is that: in this example, the activity detection method in step one is as follows:
s1: placing a sample to be tested in a glass culture dish, adding a culture medium into the culture dish, and culturing for 15 min;
s2: adding AlamarBlue redox indicator, and observing the proliferation condition of the sample in an oxidation state;
s3: observing the color of the sample after adding the AlamarBlue redox indicator, reducing the AlamarBlue redox indicator taken into the bacteria by a metabolic intermediate, releasing the AlamarBlue redox indicator out of the body, dissolving the AlamarBlue redox indicator in a culture medium, changing the culture medium from non-fluorescent indigo blue into fluorescent pink, proliferating the Mycobacterium tuberculosis under the premise of not influencing the Mycobacterium tuberculosis in the sample, equally dividing a single detection sample into multiple parts, facilitating the multiple detection of the latent characteristic and the morbidity of the Mycobacterium tuberculosis, and improving the existing defects;
s4: and (3) detecting by using a common spectrophotometer or a fluorescence spectrophotometer, wherein when the absorbance and the fluorescence intensity are in direct proportion to the number of active bacteria, the sample is qualified, and otherwise, the sample is unqualified.
In this embodiment, one solution of the second step is taken, high fidelity DNA polymerase and non-high fidelity DNA polymerase mixed enzyme are used as polymerase, PCR amplification is carried out on the DNA sample of the mycobacterium tuberculosis, whether PCR amplification products are formed or not is detected, wherein if amplification products are formed, the mycobacterium tuberculosis contains rifampicin-resistant mutation.
In this example, the medium added in S1 was a czochralski medium.
In this example, in S2, the time for observing the proliferation of the sample was 30 min.
In this example, the diameter of the glass petri dish in S1 was 10 cm.
Example 3
The difference from the above embodiment is that: in this example, the activity detection method in step one is as follows:
s1: placing a sample to be tested in a glass culture dish, adding a culture medium into the culture dish, and culturing for 10 min;
s2: adding AlamarBlue redox indicator, and observing the proliferation condition of the sample in an oxidation state;
s3: observing the color of the sample after adding the AlamarBlue redox indicator, reducing the AlamarBlue redox indicator taken into the bacteria by a metabolic intermediate, releasing the AlamarBlue redox indicator out of the body, dissolving the AlamarBlue redox indicator in a culture medium, changing the culture medium from non-fluorescent indigo blue into fluorescent pink, proliferating the Mycobacterium tuberculosis under the premise of not influencing the Mycobacterium tuberculosis in the sample, equally dividing a single detection sample into multiple parts, facilitating the multiple detection of the latent characteristic and the morbidity of the Mycobacterium tuberculosis, and improving the existing defects;
s4: and (3) detecting by using a common spectrophotometer or a fluorescence spectrophotometer, wherein when the absorbance and the fluorescence intensity are in direct proportion to the number of active bacteria, the sample is qualified, and otherwise, the sample is unqualified.
In this embodiment, one solution of the second step is taken, high fidelity DNA polymerase and non-high fidelity DNA polymerase mixed enzyme are used as polymerase, PCR amplification is carried out on the DNA sample of the mycobacterium tuberculosis, whether PCR amplification products are formed or not is detected, wherein if amplification products are formed, the mycobacterium tuberculosis contains rifampicin-resistant mutation.
In this example, the medium added in S1 was a czochralski medium.
In this example, in S2, the time for observing the proliferation of the sample was 20 min.
In this example, the diameter of the glass petri dish in S1 was 10 cm.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (7)
1. A novel combined detection method for latent infection and morbidity of Mycobacterium tuberculosis is characterized by comprising the following steps: the method comprises the following steps:
the method comprises the following steps: preparing a sample to be detected, performing activity detection on the sample to be detected, and entering the next step after the sample to be detected is qualified;
step two: culturing a qualified sample to be detected by using a Roche medium, and collecting all cultures to be respectively soaked in an ethanol solution;
step three: dividing the solution soaked in the step two into multiple parts, and performing operation in the step four on any one part;
step four: taking a part of the soaking solution in the third step, air-drying at room temperature, collecting and drying solid matters, drying the solid matters, adding the extracting solution, mixing, and detecting whether the extracting solution contains mycobacterium tuberculosis with an amino acid sequence of NSGYIECCR;
step five: separating the biomembrane of the mycobacterium tuberculosis detected in the fourth step;
step six: and detecting whether the biological membrane is infectious or not by using a microplate reader.
2. The method of claim 1, wherein the combination of the detection of latent infection and pathogenesis of mycobacterium tuberculosis comprises: the activity detection method in the first step is as follows:
s1: placing a sample to be detected in a glass culture dish, adding a culture medium into the culture dish, and culturing for 10-15 min;
s2: adding AlamarBlue redox indicator, and observing the proliferation condition of the sample in an oxidation state;
s3: observing the color of the sample after adding the AlamarBlue redox indicator, wherein the AlamarBlue redox indicator taken into the bacteria is reduced by a metabolic intermediate, released to the outside of the body and dissolved in a culture medium, so that the culture medium is changed from non-fluorescent indigo blue into fluorescent pink;
s4: and (3) detecting by using a common spectrophotometer or a fluorescence spectrophotometer, wherein when the absorbance and the fluorescence intensity are in direct proportion to the number of active bacteria, the sample is qualified, and otherwise, the sample is unqualified.
3. The method of claim 1, wherein the combination of the detection of latent infection and pathogenesis of mycobacterium tuberculosis comprises: taking one solution in the second step, adopting high-fidelity DNA polymerase and non-high-fidelity DNA polymerase mixed enzyme as polymerase, carrying out PCR amplification on the DNA sample of the mycobacterium tuberculosis, and detecting whether PCR amplification products are formed or not, wherein if the amplification products are formed, the mycobacterium tuberculosis contains rifampicin-resistant mutation.
4. The method of claim 1, wherein the combination of the detection of latent infection and pathogenesis of mycobacterium tuberculosis comprises: the culture medium added in the S1 is a Czochralski culture medium.
5. The method of claim 1, wherein the combination of the detection of latent infection and pathogenesis of mycobacterium tuberculosis comprises: in the S2, the time for observing the proliferation of the sample is 20-30 min.
6. The method of claim 1, wherein the combination of the detection of latent infection and pathogenesis of mycobacterium tuberculosis comprises: in S4, the fluorescent group is any one of FAM, ROX, HEX, CY5, TET and CAL-Fluor.
7. The method of claim 1, wherein the combination of the detection of latent infection and pathogenesis of mycobacterium tuberculosis comprises: the diameter of the glass culture dish in the S1 is 10-15 cm.
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