CN115341041A - Rapid identification kit for mycobacterium fortuitum and application thereof - Google Patents

Rapid identification kit for mycobacterium fortuitum and application thereof Download PDF

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CN115341041A
CN115341041A CN202110521387.5A CN202110521387A CN115341041A CN 115341041 A CN115341041 A CN 115341041A CN 202110521387 A CN202110521387 A CN 202110521387A CN 115341041 A CN115341041 A CN 115341041A
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mycobacterium fortuitum
detected
primer
identifying
bacteria
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张福仁
刘红
暴芳芳
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Dermatology Hospital Affiliated To Shandong First Medical University
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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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    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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    • 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
    • C12Q1/6851—Quantitative amplification

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Abstract

The invention discloses a rapid identification kit for mycobacterium fortuitum and application thereof. The kit comprises a primer group for rapidly identifying the mycobacterium fortuitum, wherein the primer group consists of a primer 1, a primer 2 and a probe, and the nucleotide sequences of the primer group are respectively shown as a sequence 1, a sequence 2 and a sequence 3 in a sequence table. The invention also successfully establishes a method for detecting the mycobacterium fortuitum by adopting a real-time fluorescent quantitative PCR technology based on the primer group, and the detection method has the advantages of high speed, high sensitivity and strong specificity and provides an effective technical means for early diagnosis and treatment of patients infected by the mycobacterium fortuitum.

Description

Rapid identification kit for mycobacterium fortuitum and application thereof
Technical Field
The invention relates to the technical field of biology, in particular to a rapid identification kit for mycobacterium fortuitum and application thereof.
Background
Mycobacterium fortuitum is a Rapid Growing Mycobacterium (RGM) among atypical mycobacteria, and 75 pathogenic bacteria are currently known to be included in RGM, with Mycobacterium fortuitum being one of the most common RGM. The bacteria are widely present in the nature (such as water source and soil), and human infection is often caused by infection from medical equipment to wounds, such as injection, operation and the like.
The traditional diagnosis method of the mycobacterium fortuitum mainly depends on the culture of pathogenic bacteria, but takes long time, needs 3-5 days, and is not beneficial to early diagnosis and treatment of diseases. The PCR-based molecular identification method provides a new technical means for diagnosing mycobacteria due to the advantages of high sensitivity and rapid diagnosis.
Disclosure of Invention
The invention aims to solve the technical problem of establishing a method for detecting mycobacterium fortuitum by adopting a real-time fluorescent quantitative PCR technology.
In a first aspect, the present invention protects a primer set for identifying or aiding in identifying mycobacterium fortuitum.
The primer group for identifying or assisting in identifying the mycobacterium fortuitum, which is protected by the invention, consists of a primer 1, a primer 2 and a probe;
the primer 1 is a 1) or a 2) as follows:
a1 A single-stranded DNA molecule shown in a sequence 1 in a sequence table;
a2 A single-stranded DNA molecule obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 1 and having the same function as the sequence 1;
the primer 2 is a 3) or a 4) as follows:
a3 A single-stranded DNA molecule shown in a sequence 2 in a sequence table;
a4 A single-stranded DNA molecule obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 2 and having the same function as the sequence 2;
the probe is a 5) or a 6) as follows:
a5 A single-stranded DNA molecule shown in a sequence 3 in a sequence table;
a6 Single-stranded DNA molecule obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 3 and having the same function as the sequence 3.
In the primer set, the molar ratio of the primer 1, the primer 2 and the probe is 5.
In a second aspect, the present invention protects the use of the above primer set in any one of the following b 1) to b 8):
b1 Preparing a product for identifying or assisting in identifying whether the bacteria to be detected are mycobacterium fortuitum;
b2 Identifying or assisting in identifying whether the test bacterium is mycobacterium fortuitum;
b3 Preparing a product for diagnosing or assisting in diagnosing whether a subject is infected with mycobacterium fortuitum;
b4 ) diagnosing or aiding in diagnosing whether the subject is infected with Mycobacterium fortuitum;
b5 Preparing a product for detecting or assisting in detecting whether a sample to be detected contains mycobacterium fortuitum;
b6 Detecting or aiding in detecting whether the sample to be detected contains mycobacterium fortuitum;
b7 Preparing a product for identifying or assisting in identifying the mycobacterium fortuitum and other mycobacteria;
b8 ) or to aid in the identification of mycobacterium fortuitum from other mycobacteria.
In a third aspect, the present invention provides a kit comprising the above primer set;
the kit has the functions of any one of c 1) to c 4) as follows:
c1 Identifying or assisting in identifying whether the test bacterium is mycobacterium fortuitum;
c2 To diagnose or aid in diagnosing whether a subject is infected with mycobacterium fortuitum;
c3 Detecting or aiding in detecting whether the sample to be detected contains mycobacterium fortuitum;
c4 ) or to aid in the identification of mycobacterium fortuitum from other mycobacteria.
Further, the kit may further comprise other reagents for identifying mycobacterium fortuitum. In the present invention, the other reagent for identifying Mycobacterium fortuitum may be TaqMan Gene Expression Master Mix.
Still further, the kit may include a negative control (e.g., ddH) 2 O) and positive control (e.g.M.fortuitum Standard Strain: (
Figure BDA0003064107720000021
19709 TM ) Genomic DNA of (4).
In a fourth aspect, the present invention provides a preparation method of the above kit, wherein the preparation method is d 1) or d 2):
d1 Respectively and individually packaging each primer in the primer group;
d2 Each primer in the primer set is mixed together in proportion.
Further, in the d 2), the primer 1, the primer 2 and the probe in the primer set are mixed together according to a molar ratio of 5.
In a fifth aspect, the present invention provides a method for identifying or assisting in identifying whether a test bacterium is mycobacterium fortuitum.
The method for identifying or assisting in identifying whether the bacteria to be detected are mycobacterium fortuitum or not comprises the following steps: extracting nucleic acid of bacteria to be detected, and performing real-time fluorescence quantitative PCR by using the primer group by using the nucleic acid of the bacteria to be detected as a template; and (3) judging whether the bacteria to be detected are mycobacterium fortuitum or not through the amplification curve and the CT value after the reaction is finished: if the bacteria to be detected have an S-type amplification curve and the CT value is less than 35, the bacteria to be detected are or are selected as the accidental mycobacteria; otherwise, the bacteria to be tested is not or is not candidate to be the mycobacterium fortuitum.
In a sixth aspect, the invention features a method of diagnosing or aiding in diagnosing whether a subject is infected with mycobacterium fortuitum.
The method for diagnosing or assisting in diagnosing whether a person to be tested is infected with mycobacterium fortuitum comprises the following steps: extracting nucleic acid of a person to be detected, and performing real-time fluorescence quantitative PCR by using the primer group by using the nucleic acid of the person to be detected as a template; and (3) judging whether the person to be detected is infected with mycobacterium fortuitum or not through the amplification curve and the CT value after the reaction is finished: if the person to be tested has an S-type amplification curve and the CT value is less than 35, the person to be tested is infected or candidate infected with mycobacterium fortuitum; otherwise, the tested person is not infected or candidate is not infected with mycobacterium fortuitum.
In a seventh aspect, the present invention provides a method for detecting or aiding in the detection of whether a test sample contains mycobacterium fortuitum.
The method for detecting or assisting in detecting whether the sample to be detected contains the mycobacterium fortuitum comprises the following steps: extracting nucleic acid of a sample to be detected, and performing real-time fluorescence quantitative PCR by using the primer group by using the nucleic acid of the sample to be detected as a template; and (3) judging whether the sample to be detected contains mycobacterium fortuitum or not through the amplification curve and the CT value after the reaction is finished: if the sample to be detected has an S-shaped amplification curve and the CT value is less than 35, the sample to be detected contains or is candidate to contain the mycobacterium fortuitum; otherwise, the sample to be tested does not contain or candidate does not contain the mycobacterium fortuitum.
In an eighth aspect, the invention features a method for identifying or aiding in identifying mycobacterium fortuitum and other mycobacteria.
The method for identifying or assisting in identifying the mycobacterium fortuitum and other mycobacteria comprises the following steps: extracting nucleic acid of bacteria to be detected, and performing real-time fluorescence quantitative PCR by using the primer group by using the nucleic acid of the bacteria to be detected as a template; and after the reaction is finished, judging whether the bacteria to be detected are the mycobacterium fortuitum or other mycobacteria according to the amplification curve and the CT value: if the bacteria to be detected have an S-type amplification curve and the CT value is less than 35, the bacteria to be detected are or are selected as the accidental mycobacteria; otherwise, the bacteria to be tested are or are selected as other mycobacteria.
Any of the above methods further comprises the steps of: and (3) judging the result on the premise that the negative control has no amplification curve or the CT value is more than 35, the positive control has an S-shaped amplification curve and the CT value is less than 35.
In any of the above methods, the final concentration of each of the primer 1 and the primer 2 in the real-time fluorescence quantitative PCR system may be 7.5 μ M, and the final concentration of the probe in the real-time fluorescence quantitative PCR system may be 6 μ M.
The real-time fluorescent quantitative PCR system can be composed of 15 muL TaqMan Gene Expression Master Mix, 0.75 muL primer 1, 0.75 muL primer 2, 0.6 muL probe, 2 muL template and 10.9 muL ddH 2 And (C) O.
The real-time fluorescent quantitative PCR reaction program can be specifically 50 ℃ for 2min,95 ℃ for 10min and 40 cycles (95 ℃ for 15s and 60 ℃ for 1 min).
In any of the above methods, the nucleic acid is genomic DNA.
In any of the above uses or kits or methods, the other mycobacterium may be at least one of the following mycobacteria: mycobacterium marinum, mycobacterium chelonei, mycobacterium tuberculosis, mycobacterium haemophilus.
In order to solve the technical problem, the invention carries out the following experimental processes: specific primers and probes of the mycobacterium fortuitum specific gene ITS are designed by taking the mycobacterium fortuitum specific gene ITS as a target gene. Then, taking the standard strains of the mycobacterium fortuitum as positive control, selecting the culture colonies of the skin tissue samples of 2 definitely diagnosed mycobacterium fortuitum, 100 sea mycobacteria, 1 mycobacterium cheloni, 1 mycobacterium tuberculosis and 2 haemophilus infected patients as experimental groups, setting a blank control group at the same time, and detecting the samples by a fluorescent quantitative PCR technology. The results show that: the cultured colonies of 2 patients infected by mycobacterium fortuitum in the experimental group are all positive, and the accuracy reaches 100%; the rest colonies and the blank control are negative, and the specificity reaches 100 percent. The invention successfully establishes a method for detecting the mycobacterium fortuitum by adopting a real-time fluorescent quantitative PCR technology, and provides an effective technical means for early diagnosis and treatment of patients infected by the mycobacterium fortuitum.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention.
The test methods in the following examples are conventional methods unless otherwise specified.
The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
The quantitative tests in the following examples, all set up three replicates and the results averaged.
The standard strain of Mycobacterium fortuitum in the following examples is the product of ATCC, numbered
Figure BDA0003064107720000042
19709 TM 。
Example 1 primer set for rapidly identifying Mycobacterium fortuitum and method for identifying the same
1. Primer group for rapidly identifying mycobacterium fortuitum and identification method thereof
The specific gene ITS of the mycobacterium fortuitum is taken as a target gene, specific primers and probes of the gene are designed through Primer Express Software for Real-Time PCR Version 3.0 Software, and specific sequences are shown in table 1. The single-stranded DNA molecule shown in the sequence 1, the single-stranded DNA molecule shown in the sequence 2 and the single-stranded DNA molecule shown in the sequence 3 form a primer group for rapidly identifying the mycobacterium fortuitum.
TABLE 1 specific primers and probes
Figure BDA0003064107720000041
2. qPCR method for rapidly identifying mycobacterium fortuitum
1. Experimental equipment
(1) The instrument comprises the following steps: a biological safety cabinet, a carbon dioxide incubator, a 4-degree refrigerator, a-20-degree refrigerator, an-80-degree refrigerator, a micropipettor, a dry type constant temperature metal bath, a centrifuge, a water purifier, ABI StepOnePlus fluorescent quantitative PCR and the like.
(2) Reagent: DNA extracts, primers and probes (invitrogen), taqMan Gene Expression Master Mix (Thermo Fisher, applied BioSystems, 00826956).
(3) Consumable material: spear head, 1.5mL ep tube, eight tubes, etc.
2. Experimental methods
(1) And (3) after the ultraviolet lamp of the biological safety cabinet irradiates for 30 minutes, taking out the cultured bacterial colony of the bacteria to be tested from the carbon dioxide incubator, and placing the bacterial colony in the biological safety cabinet. A small amount of the colonies were scraped off with an inoculating loop and placed in a 1.5mL ep tube.
(2) Add 80. Mu.L of DNA extract to 1.5mL ep tube containing colonies, vortex for 10s, and centrifuge at 10000r/min for 10s to bring colonies and DNA extract to the bottom of the ep tube.
(3) And (5) placing the mixture in a dry constant-temperature metal bath at 100 ℃ for 10min to extract colony DNA.
(4) Taking out the ep tube from the dry constant-temperature metal bath, centrifuging at 10000r/min for 30s, and obtaining supernatant fluid containing the nucleic acid of the bacterial colony. Then storing in a-80 ℃ refrigerator for later use.
(5) A PCR system (30. Mu.L) was prepared according to the recipe shown in Table 2, and the final concentrations of the primer F, the primer R and the probe in the PCR system were 7.5. Mu.M, 7.5. Mu.M and 6. Mu.M, respectively. Setting a negative control and a positive control at the same time, wherein the negative control comprises the following steps: ddH 2 O, positive control: mycobacterium fortuitum Standard Strain (A)
Figure BDA0003064107720000051
19709 TM )。
TABLE 2 reaction System
Components 1×(μL)
TaqMan Gene Expression Master Mix 15
F 0.75
R 0.75
probe 0.6
ddH 2 O 10.9
DNA template 2
(6) Detection was carried out using an ABI StepOnePlus fluorescent quantitative PCR instrument with the reaction program of 50 ℃ for 2min,95 ℃ for 10min, and 40 cycles (95 ℃ for 15s, 60 ℃ for 1 min).
3. Determination of results
If the bacteria to be detected have the S-type amplification curve and the CT value is less than 35, the bacteria to be detected are sporadic mycobacteria, otherwise, the bacteria to be detected are not the sporadic mycobacteria, on the premise that the negative control is qualified (the negative control has no amplification curve or the CT value is more than 35, and the positive control has the S-type amplification curve and the CT value is less than 35).
Example 2 sensitivity test
1. Experimental materials
Will branch sporadically Bacillus standard strain (A)
Figure BDA0003064107720000052
19709 TM ) The genomic DNA solution of (1) was diluted in a successive double ratio to give a concentration of 103.4ng/L and 103.4X 10, respectively -1 ng/L、103.4×10 -2 ng/L、103.4×10 -3 ng/L、103.4×10 -4 ng/L、103.4×10 -5 ng/L、103.4×10 -6 ng/L、103.4×10 -7 ng/L、103.4×10 -8 ng/L、103.4×10 -9 ng/L、103.4×10 -10 ng/L of Mycobacterium fortuitum DNA solution.
2. Experimental methods and results
Respectively taking DNA solutions of mycobacterium fortuitum with different concentrations as templates, and adopting the method in the second step of the embodiment 1 to carry out fluorescence quantitative PCR and result judgment.
The results show that: the sensitivity of the detection method can reach 103.4 multiplied by 10 -9 ng/L。
Example 3 specificity test
1. Experimental Material
Experimental materials: cultured colonies of skin tissue samples from patients infected with mycobacteria. The mycobacterial patients were as follows: 2 patients with Mycobacterium fortuitum infection, 100 patients with Mycobacterium marinum infection, 1 patient with Mycobacterium cheloni infection, 1 patient with Mycobacterium tuberculosis infection, and 2 patients with Mycobacterium haemophilus infection, all of which were clinically confirmed and informed.
2. Experimental methods and results
Genomic DNA was extracted from each of the test materials in the first step, and fluorescent quantitative PCR and result determination were performed by the method in the second step of example 1 using the extracted DNA solution as a template. Simultaneously with ddH 2 O is blank control.
The results show that: the detection results of 2 accidental mycobacteria colonies are positive, the accuracy reaches 100%, the other colonies and blank control are negative, and the specificity reaches 100%.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, it is possible to make various improvements and modifications without departing from the technical principle of the present invention, and these improvements and modifications should also be considered as the protection scope of the present invention.
Sequence listing
<110> university of Shandong first medical university affiliated dermatological Hospital (institute for prevention and treatment of skin disease and STD in Shandong province, hospital for skin disease in Shandong province)
<120> rapid identification kit for mycobacterium fortuitum and application thereof
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 1
cctgtagtgg gcacggtttg 20
<210> 2
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<212> DNA
<213> Artificial Sequence
<400> 2
ccaatagtgt gtctggcagt caa 23
<210> 3
<211> 15
<212> DNA
<213> Artificial Sequence
<400> 3
tgcacaacaa acttt 15

Claims (10)

1. The primer group for identifying or assisting in identifying the mycobacterium fortuitum consists of a primer 1, a primer 2 and a probe;
the primer 1 is a 1) or a 2) as follows:
a1 A single-stranded DNA molecule shown as a sequence 1 in a sequence table;
a2 A single-stranded DNA molecule obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 1 and having the same function as the sequence 1;
the primer 2 is a 3) or a 4) as follows:
a3 A single-stranded DNA molecule shown in a sequence 2 in a sequence table;
a4 A single-stranded DNA molecule obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 2 and having the same function as the sequence 2;
the probe is a 5) or a 6) as follows:
a5 A single-stranded DNA molecule shown in a sequence 3 in a sequence table;
a6 Single-stranded DNA molecule obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 3 and having the same function as the sequence 3.
2. The primer set according to claim 1, wherein: the molar ratio of the primer 1, the primer 2 and the probe is 5.
3. Use of the primer set of claim 1 or 2 in any one of the following b 1) -b 8):
b1 Preparing a product for identifying or assisting in identifying whether the bacteria to be detected are mycobacterium fortuitum;
b2 Identifying or assisting in identifying whether the test bacterium is mycobacterium fortuitum;
b3 Preparing a product for diagnosing or assisting in diagnosing whether a subject is infected with mycobacterium fortuitum;
b4 To diagnose or aid in diagnosing whether a subject is infected with mycobacterium fortuitum;
b5 Preparing a product for detecting or assisting in detecting whether a sample to be detected contains mycobacterium fortuitum;
b6 Detecting or aiding in detecting whether the sample to be detected contains mycobacterium fortuitum;
b7 Preparing a product for identifying or assisting in identifying the mycobacterium fortuitum and other mycobacteria;
b8 ) or to aid in the identification of Mycobacterium fortuitum and other Mycobacteria.
4. A kit containing the primer set according to claim 1 or 2;
the function of the kit is any one of the following c 1) -c 4):
c1 Identifying or assisting in identifying whether the test bacterium is mycobacterium fortuitum;
c2 To diagnose or aid in diagnosing whether a subject is infected with mycobacterium fortuitum;
c3 Detecting or aiding in detecting whether the sample to be detected contains mycobacterium fortuitum;
c4 ) or to aid in the identification of mycobacterium fortuitum from other mycobacteria.
5. The method for preparing the kit according to claim 4, wherein the method is d 1) or d 2):
d1 Separately packaging each primer in the primer set according to claim 1 or 2;
d2 Mixing the respective primers in the primer set according to claim 1 or 2 together in proportion.
6. The method of claim 5, wherein: in the d 2), the primer 1, the primer 2 and the probe in the primer group are mixed together according to a molar ratio of 5.
7. A method for identifying or assisting in identifying whether a test bacterium is mycobacterium fortuitum comprises the following steps: extracting nucleic acid of bacteria to be detected, and carrying out real-time fluorescence quantitative PCR by using the primer group of claim 1 by using the nucleic acid of the bacteria to be detected as a template; and (3) judging whether the bacteria to be detected are mycobacterium fortuitum or not through the amplification curve and the CT value after the reaction is finished: if the bacteria to be detected have an S-type amplification curve and the CT value is less than 35, the bacteria to be detected are or are selected as the accidental mycobacteria; otherwise, the bacteria to be tested is not or is not candidate to be the mycobacterium fortuitum.
8. A method of diagnosing or aiding in the diagnosis of a subject for infection with mycobacterium fortuitum comprising the steps of: extracting nucleic acid of a person to be detected, and carrying out real-time fluorescence quantitative PCR by using the primer group of claim 1 by using the nucleic acid of the person to be detected as a template; and (3) judging whether the person to be detected is infected with mycobacterium fortuitum or not through the amplification curve and the CT value after the reaction is finished: if the person to be tested has an S-type amplification curve and the CT value is less than 35, the person to be tested is infected or candidate infected with mycobacterium fortuitum; otherwise, the tested person is not infected or candidate is not infected with mycobacterium fortuitum.
9. A method for detecting or assisting in detecting whether a sample to be detected contains mycobacterium fortuitum comprises the following steps: extracting nucleic acid of a sample to be detected, and carrying out real-time fluorescent quantitative PCR by using the primer group of claim 1 by using the nucleic acid of the sample to be detected as a template; and (3) judging whether the sample to be detected contains mycobacterium fortuitum or not through the amplification curve and the CT value after the reaction is finished: if the sample to be detected has an S-shaped amplification curve and the CT value is less than 35, the sample to be detected contains or is candidate to contain the mycobacterium fortuitum; otherwise, the sample to be tested does not contain or candidate does not contain mycobacterium fortuitum.
10. A method for identifying or assisting in identifying Mycobacterium fortuitum and other mycobacteria, comprising the steps of: extracting nucleic acid of bacteria to be detected, and carrying out real-time fluorescence quantitative PCR by using the primer group of claim 1 by using the nucleic acid of the bacteria to be detected as a template; and after the reaction is finished, judging whether the bacteria to be detected are the mycobacterium fortuitum or other mycobacteria according to the amplification curve and the CT value: if the bacteria to be detected have an S-type amplification curve and the CT value is less than 35, the bacteria to be detected are or are selected as the accidental mycobacteria; otherwise, the bacteria to be tested are or are selected as other mycobacteria.
CN202110521387.5A 2021-05-13 2021-05-13 Rapid identification kit for mycobacterium fortuitum and application thereof Pending CN115341041A (en)

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