CN110938702B

CN110938702B - Method for detecting mycobacterium avium by real-time fluorescent PCR and application thereof

Info

Publication number: CN110938702B
Application number: CN201911189769.1A
Authority: CN
Inventors: 刘洋; 王赛赛; 潘丽萍; 姜广路; 林婷婷; 李自慧; 贾红彦; 孙琦; 张宗德
Original assignee: Beijing Chest Hospital; Beijing Tuberculosis and Thoracic Tumor Research Institute
Current assignee: Beijing Chest Hospital; Beijing Tuberculosis and Thoracic Tumor Research Institute
Priority date: 2019-11-28
Filing date: 2019-11-28
Publication date: 2022-11-01
Anticipated expiration: 2039-11-28
Also published as: CN110938702A

Abstract

The invention discloses a method for detecting mycobacterium avium by real-time fluorescent PCR and application thereof. The invention provides a specific primer pair, and establishes a SYBR Green I fluorescent dye-based real-time fluorescent PCR method for detecting MA. The method has the advantages of high sensitivity, good specificity and good repeatability, and is lower in price and simpler and more convenient to operate compared with methods such as a DNA chip technology and a PCR-fingerprint spectrum; compared with the current 'gold standard' for strain identification, the method for detecting MA has no significant difference, and provides a simple, cheap and high-accuracy molecular biological method for clinical identification of MA. The invention improves the working efficiency of inexperienced experiment technicians, reduces the difficulty of experiment operation, reduces the error probability, and enables the experiment to be carried out after training. Compared with other molecular biological methods, the method has the advantages of reducing cost and experimental expenses under the condition of higher diagnosis accuracy, and is easier to popularize in clinic.

Description

Method for detecting mycobacterium avium by real-time fluorescent PCR and application thereof

Technical Field

The invention relates to a method for detecting mycobacterium avium by real-time fluorescent PCR and application thereof.

Background

Research conducted in different areas of China shows that Mycobacterium Avium (MA) is a non-tuberculous Mycobacterium (NTM) which is mainly prevalent in China. Mycobacterium avium is a zoonotic pathogen and can cause lung, soft tissue, children's lymph nodes, skin infections, etc. in humans. Not only does MA infection lack specificity in clinical presentation and histopathology, but it also has a significant difference in the drug resistance spectrum from M.intracellulare. Therefore, the identification of the species of mycobacterium avium is of great significance to the determination of infection sources, the analysis of transmission ways and the formulation of reasonable treatment schemes.

At present, the traditional strain identification method mainly based on biochemistry is not frequently used due to complex operation and poor accuracy; the method relying on homologous DNA sequence comparison becomes the 'gold standard' for strain identification, but is difficult to be widely applied to clinic due to high price. The QPCR based on the SYBR Green I fluorescent dye not only has the advantages of being simple to operate, shortening the detection time and reducing the cross infection risk, but also is lower in price and simpler and more convenient to operate compared with methods such as a DNA chip technology and a PCR-fingerprint spectrum.

Disclosure of Invention

The invention aims to provide a method for detecting mycobacterium avium by real-time fluorescent PCR and application thereof.

In a first aspect, the invention provides a protective primer pair a or a primer pair B or a primer pair C or a primer pair D;

the primer pair A consists of a primer 3F and a primer 3R;

the primer 3F is (a 1) or (a 2) as follows:

(a1) A single-stranded DNA molecule shown in sequence 5 of the sequence table;

(a2) A DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 5 and has the same function as the sequence 5;

the primer 3R is (a 3) or (a 4) as follows:

(a3) A single-stranded DNA molecule shown in sequence 6 of the sequence table;

(a4) DNA molecules obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 6 and having the same functions as the sequence 6;

the primer pair B consists of a primer 1F and a primer 1R;

the primer 1F is (a 1) or (a 2):

(a1) A single-stranded DNA molecule shown in sequence 1 of a sequence table;

(a2) DNA molecules which are obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 1 and have the same functions as the sequence 1;

the primer 1R is (a 3) or (a 4) as follows:

(a3) A single-stranded DNA molecule shown in a sequence 2 of a sequence table;

(a4) DNA molecules which are obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 2 and have the same functions as the sequence 2;

the primer pair C consists of a primer 2F and a primer 2R;

the primer I2F is (a 1) or (a 2) as follows:

(a1) A single-stranded DNA molecule shown in sequence 3 of the sequence table;

(a2) DNA molecules which are obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 3 and have the same functions as the sequence 3;

the primer 2R is (a 3) or (a 4) as follows:

(a3) A single-stranded DNA molecule shown in a sequence 4 of a sequence table;

(a4) A DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 4 and has the same function as the sequence 4;

the primer pair D consists of a primer 4F and a primer 4R;

the primer 4F is (a 1) or (a 2) as follows:

(a1) A single-stranded DNA molecule shown in sequence 7 of the sequence table;

(a2) DNA molecules obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 7 and having the same functions as the sequence 7;

the primer 4R is (a 3) or (a 4):

(a3) A single-stranded DNA molecule shown in sequence 8 of the sequence table;

(a4) And (b) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 8 and has the same function as the sequence 8.

In the primer pair A or the primer pair B or the primer pair C or the primer pair D, the molar ratio of each primer is the same.

The application of the primer pair A or the primer pair B or the primer pair C or the primer pair D is (B1) or (B2) or (B3) or (B4) as follows:

(b1) Identifying or assisting in identifying whether the bacteria to be detected is mycobacterium avium;

(b2) Preparing a kit for identifying or assisting in identifying whether the bacteria to be detected are mycobacterium avium;

(b3) Detecting whether the biological sample to be detected contains the mycobacterium avium or not;

(b4) Preparing a kit for detecting whether the biological sample to be detected contains the mycobacterium avium.

In a second aspect, the invention provides the use of primer pair a or primer pair B or primer pair C or primer pair D as follows (B1) or (B2) or (B3) or (B4):

(b3) Detecting whether the biological sample to be detected contains mycobacterium avium or not;

The use is non-disease diagnostic and therapeutic.

In a third aspect, the invention protects a kit containing the primer pair A or B or C or D; the application of the kit is (c 1) or (c 2):

(c1) Identifying or assisting in identifying whether the bacteria to be detected is mycobacterium avium;

(c2) And detecting whether the biological sample to be detected contains the mycobacterium avium.

Further, the invention protects the preparation method of the kit, and the preparation method comprises the step of packaging each primer separately.

In a fourth aspect, the invention provides a method.

The invention provides a method for identifying or assisting in identifying whether a bacterium to be detected is mycobacterium avium, which comprises the following steps:

(1) Extracting the genome DNA of the bacteria to be detected;

(2) And (2) taking the genomic DNA extracted in the step (1) as a template, and carrying out real-time fluorescence PCR detection by adopting the primer pair A or the primer pair B or the primer pair C or the primer pair D, wherein if a positive amplification result can be obtained, the bacteria to be detected is or is a candidate of mycobacterium avium, and if the positive amplification result cannot be obtained, the bacteria to be detected is non-mycobacterium avium.

detecting whether the genome DNA of the bacteria to be detected contains a specific DNA fragment, if so, determining that the bacteria to be detected is or is a candidate for mycobacterium avium, and if not, determining that the bacteria to be detected is non-mycobacterium avium; the specific DNA fragment is a target sequence of the primer pair A or the primer pair B or the primer pair C or the primer pair D in the genome of the mycobacterium avium.

The invention provides a method for detecting whether a biological sample to be detected contains mycobacterium avium, which comprises the following steps:

(1) Extracting the total DNA of a biological sample to be detected;

(2) And (2) taking the total DNA extracted in the step (1) as a template, and carrying out real-time fluorescence PCR detection by adopting the primer pair A or the primer pair B or the primer pair C or the primer pair D, wherein if a positive amplification result can be obtained, the biological sample to be detected contains or is candidate to contain the mycobacterium avium, and if the positive amplification result cannot be obtained, the biological sample to be detected does not contain the mycobacterium avium.

detecting whether the total DNA of the biological sample to be detected contains a specific DNA fragment, if so, determining that the biological sample to be detected contains or is candidate to contain the mycobacterium avium, and if not, determining that the biological sample to be detected does not contain the mycobacterium avium; the specific DNA fragment is a target sequence of the primer pair A or the primer pair B or the primer pair C or the primer pair D in the genome of the mycobacterium avium.

In any of the above methods, the reaction system of the real-time fluorescence PCR may specifically be: 2 XSuperReal Premix Plus 10. Mu.l, each primer 0.6. Mu.l (concentration in reaction system 0.3. Mu. Mol), DNA 2. Mu.l, 50 XRox Reference Dye^△0.4μl、ddH₂O6.4. Mu.l. In the reaction system, the minimum quality of the DNA is 1.12 x 10^-1ng。

In any of the above methods, the reaction conditions of the real-time fluorescence PCR may specifically be: pre-denaturation at 95 deg.C for 5min, amplifying at 95 deg.C for 10s and 60 deg.C (collecting fluorescent signal) for 32s for 40 cycles, and completing the melting curve at 1.6 deg.C/s for 95 deg.C for 15s, 60 deg.C for 1min and 95 deg.C (collecting fluorescent signal) for 15 s.

Any of the methods described above are non-disease diagnostic and therapeutic methods.

In each of the above aspects, the bacterium may be a mycobacterium, and more specifically may be mycobacterium avium, mycobacterium intracellulare, mycobacterium abscessus, mycobacterium cheloni, mycobacterium kansasii, mycobacterium gordonae, mycobacterium fortuitum, mycobacterium phlei, mycobacterium aureofaciens, mycobacterium scrofulaceum, mycobacterium flavum, mycobacterium smegmatis, mycobacterium tuberculosis, mycobacterium marmor, mycobacterium simian, mycobacterium thuringiensis, mycobacterium suis, mycobacterium terrestris, mycobacterium microflavus, mycobacterium dychii, or mycobacterium schutlii. The mycobacterium may be a standard strain or a clinically isolated strain. The bacterium can also be staphylococcus aureus, escherichia coli, pseudomonas aeruginosa, micrococcus luteus, or staphylococcus saprophyticus.

The invention provides a specific primer pair and establishes a real-time fluorescent PCR method for detecting MA based on SYBR Green I fluorescent dye. The sensitivity of the method for detecting MA is 93.8% (15/16), the specificity is 100% (184/184), the positive predictive value is 100% (15/15), the negative predictive value is 99.5% (184/185), and the coincidence rate is 99.5% (199/200); the intra-group variation coefficient is less than 1 percent, the inter-group variation coefficient is less than 3.2 percent, and the repeatability is better. Compared with methods such as DNA chip technology, PCR-fingerprint spectrum and the like, the price is lower, and the operation is simpler and more convenient; compared with the existing 'gold standard' -method relying on homologous DNA sequence comparison for strain identification, the method for detecting MA has no significant difference, and provides a simple, cheap and high-accuracy molecular biological method for clinical identification of MA. The invention improves the working efficiency of inexperienced experiment technicians, reduces the difficulty of experiment operation, reduces the error probability, and enables the experiment to be carried out after training. Compared with other molecular biological methods, the method has the advantages of reducing cost and experimental expenses under the condition of higher diagnosis accuracy, and is easier to popularize in clinic.

Drawings

FIG. 1 shows the results of the primer specificity experiment.

FIG. 2 shows the results of the primer sensitivity experiment.

FIG. 3 shows the results of the primer reproducibility test.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are all conventional ones unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. In the quantitative tests in the following examples, three replicates were set up and the results averaged. In the qualitative tests in the following examples, three repeated tests are set, and when the results are inconsistent, two same results are taken as final results.

Example 1, strains and their accuracy analysis

1. Bacterial strains

21 standard strain of mycobacterium: mycobacterium avium (ATCC 25291), mycobacterium intracellulare (ATCC 13950), mycobacterium abscessus (ATCC 19977), mycobacterium chelonii (ATCC 14472), mycobacterium kansasii (ATCC 12478), mycobacterium gordonae (ATCC 14470), mycobacterium fortuitum (ATCC 6481), mycobacterium phlei (ATCC 11758), mycobacterium aurum (ATCC 23366), mycobacterium scrofulae (ATCC 19981), mycobacterium pale flavum (ATCC 43909), mycobacterium smegmatis (ATCC 19420), mycobacterium tuberculosis (ATCC 27294), mycobacterium marmorgani (ATCC 29571), mycobacterium simian (ATCC 25275), mycobacterium thuringiensis (ATCC 35799), mycobacterium suis (ATCC 77633776339), mycobacterium terrestris (ATCC 15755), mycobacterium microflavus (ATCC 14474), mycobacterium dietitian (ATCC 19340), and Mycobacterium schneisseria (ATCC 27962).

5 common pathogenic bacteria: staphylococcus aureus (ATCC 29213), escherichia coli (ATCC 25922), pseudomonas aeruginosa (ATCC 27853), micrococcus luteus (CMCC 28001), staphylococcus saprophyticus (ATCC BAA 750).

2. Analysis of Strain accuracy

(1) 21 standard strain of mycobacterium: amplifying the mycobacterium standard strain DNA by adopting universal primers Hsp65 (F: 5) -; 10 mul of the amplification product was taken and subjected to 2% agarose gel, and the amplification product was sequenced, the sequencing results were compared with the gene amplification sequences of each standard bacterium in the GenBank database using BLAST, and the accuracy of the standard mycobacteria used in this study was analyzed and evaluated.

The results show that after sequencing the amplification products of 21 standard strains, the consistency of the amplification products with the DNA of corresponding strains in a GenBank database (http:// www.ncbi.nlm.nih.gov) is more than 99 percent, which indicates that the standard strains used in the invention meet the requirements. Wherein, the sequencing result of the mycobacterium avium is compared with AF281650.1 in a GenBank database, the consistency reaches 100 percent, and the mycobacterium avium standard bacteria used by the invention meet the requirements of standard bacteria.

(2) 5 common pathogenic bacteria: the 5 bacteria were identified using the automated rapid microbiological identification intelligent analysis system VITEK2-compact (Merria, france) with reference to the 2019 Clinical Laboratory Standards Institute (CLSI) standard.

The detection result of the VITEK2-compact system is consistent with that of 5 bacterial strains, and the requirements are met.

Example 2 obtaining of primers for detection of Mycobacterium avium

1. Primer design, screening and preparation

A plurality of primers for identifying the mycobacterium avium are obtained by carrying out a large amount of sequence analysis and alignment. And performing a preliminary experiment on each primer, and comparing the performances such as sensitivity, specificity, repeatability and the like to finally obtain four pairs of primers for identifying the mycobacterium avium, wherein the four pairs of primers are shown in table 1.

TABLE 1 four pairs of primers for identifying M.avium

Note: f represents a forward primer, and R represents a reverse primer.

Example 3 accuracy

Amplifying the standard strain genome DNA of the mycobacterium avium by respectively adopting four pairs of primers in the table 1; 10 mul of amplification product is taken to carry out 2% agarose gel, the amplification product is sequenced at the same time, the sequencing result is compared with the gene amplification sequence of each standard bacterium in the GenBank database by BLAST, and the accuracy of the mycobacterium standard bacterium used in the research is analyzed and evaluated.

The results showed that the sequencing results of the amplification products of primer pairs 1, 2, 3 and 4 aligned with the target gene sequences in the GenBank database, and their identities were 100%, 100% and 99.32%, respectively. The four pairs of primers of the mycobacterium avium designed by the invention have higher diagnosis accuracy on the mycobacterium avium.

Example 4 specificity

The strains to be detected are as follows: the 21 standard strains and 5 common pathogenic bacteria in example 1.

And extracting the genome DNA of the strain to be detected, and respectively adopting four pairs of primers in the table 1 to carry out QPCR.

QPCR reaction system: 2 XSuperReal Premix Plus 10. Mu.l, each primer 0.6. Mu.l (concentration in reaction system 0.3. Mu. Mol), sample DNA (total genomic DNA mass 11.2 ng) 2. Mu.l, 50 XRox Reference Dye^△0.4μl、ddH₂O 6.4μl。

QPCR amplification conditions: pre-denaturation at 95 deg.C for 5min, amplifying at 95 deg.C for 10s and 60 deg.C (collecting fluorescent signal) for 32s for 40 cycles, and completing the melting curve at 1.6 deg.C/s for 95 deg.C for 15s, 60 deg.C for 1min and 95 deg.C (collecting fluorescent signal) for 15 s.

Set up using ddH₂O negative control instead of template DNA.

The results are shown in FIG. 1. In FIG. 1, 1 is the result of amplification using M.avium as a template; and 2, taking other 20 mycobacterium standard strains, 5 common pathogenic bacteria and negative control as templates for amplification. The results show that the four pairs of M.avium primers respectively have amplification curves only with M.avium standard strains in 30 cycles, have no amplification curve with Q-PCR of other 20 M.avium standard strains and 5 common pathogenic bacteria, and have the specificity of 100%.

Example 5 sensitivity

1. Genomic DNA of a standard strain of Mycobacterium avium was extracted to obtain a DNA solution having a DNA concentration of 5.6 ng/. Mu.L.

2. By ddH₂The mixed solution obtained in step 1 was subjected to O10-fold gradient dilution to obtain each dilution (5.6 ng/. Mu.L, 5.6X 10)^-1ng/μl、5.6×10^-2ng/μl、5.6×10^-3ng/μl、5.6×10^-4ng/μl、5.6×10^-5ng/μl、5.6×10^- ⁶ng/μl、5.6×10^-7ng/μl、5.6×10^-8ng/μl、5.6×10^-9ng/μl、5.6×10^-10ng/μl)。

3. And (3) performing QPCR by respectively adopting four pairs of primers in the table 1 by taking the diluent obtained in the step 2 as a template.

QPCR reaction system: 2 × SuperReal Premix Plus1Mu.l, 0.6. Mu.l of each primer (concentration 0.3. Mu. Mol in the reaction system), 2. Mu.l of sample DNA, 50 XRox Reference Dye^△0.4μl、ddH₂O 6.4μl。

Set up using ddH₂O replaces the negative control of template DNA.

The results are shown in FIG. 2. In FIG. 2, 1 to 5 are concentrations of 5.6 ng/. Mu.L and 5.6X 10 in this order^-1ng/μl、5.6×10^- ²ng/μl、5.6×10^-3ng/μl、5.6×10^-4The amplification result of the template in ng/ul of the dilution; 6 is at a concentration of 5.6X 10^-5ng/μl、5.6×10^-6ng/μl、5.6×10^-7ng/μl、5.6×10^-8ng/μl、5.6×10^-9ng/μl、5.6×10^- ¹⁰The result of amplification was expressed in ng/. Mu.l of the diluted solution as a template and a negative control.

As a result, the results of the amplification graph showed that the positive results were obtained in all of the reaction systems 1 to 5 obtained by detecting the amplification of four pairs of M.avium primers, but the results of the melting curves of the

reaction systems

4 and 5 were poor, and therefore the template content in the reaction system 3 was set as the minimum detection limit, i.e., the detection limits of the four pairs of M.avium primers were all set to 1.12X 10^-1ng。

Example 6 clinical Strain testing

The strains to be detected are as follows: 200 clinical strains of mycobacterium from national tuberculosis clinical laboratory of the chest Hospital, beijing, affiliated to the university of capital medicine.

1. Amplifying the DNA of the strain to be detected by using universal primers Hsp65 (F: 5) -ACCAACGATGTGTGTGTCCAT-3'; R:5' -CTTGTCGAACCGCATACCCT-3'), 16SrRNA (F: 5' -AGTTTGATCCTGGCTCGCAGG-3'; R:5' -CCCCCCGTTCAATTCATTTGAGTT-3'); 10 μ l of the amplification product was applied to 2% agarose gel and the amplification product was sequenced, and the sequencing results were compared with the gene amplification sequences of each standard cell in the GenBank database using BLAST.

The results show that 200 clinical strains are identified as follows after Hsp65 and 16Sr RNA amplification sequencing: mycobacterium avium 16 strain, mycobacterium intracellulare 100 strain, mycobacterium abscessus 30 strain, mycobacterium cheloniae 2 strain, mycobacterium kansasii 10 strain, mycobacterium gordonii 6 strain, mycobacterium paracogonii 2 strain, mycobacterium fortuitum 2 strain, mycobacterium tuberculosis 24 strain, mycobacterium mosaicae 4 strain, and other mycobacteria (Mycobacterium paracluohonii, mycobacterium marmor, mycobacterium schnei, and Mycobacterium suis 1 strain each) 4 strain.

2. And extracting the genome DNA of the strain to be detected, and respectively adopting four pairs of primers in the table 1 to carry out QPCR.

QPCR amplification conditions: pre-denaturing at 95 deg.c for 5min, amplifying at 95 deg.c for 10s and 60 deg.c for 32s for 40 cycles, and completing the fusion curve at 1.6 deg.c/s for 15s, 1min and 95 deg.c for 15 s.

Set up using ddH₂O negative control instead of template DNA.

The results show that in 30 cycles, 12 clinical strains of the primer pair 1 and the primer pair 2 have amplification curves, 15 clinical strains of the primer pair 3 have amplification curves, 14 clinical strains of the primer pair 4 have amplification curves, all the strains are mycobacterium avium through sequencing, and the rest strains have no amplification curves.

Therefore, the sensitivity of four pairs of primers for detecting MA is 75% (12/16), 93.8% (15/16) and 87.5% (14/16), the specificity is 100% (184/184), the positive predictive value is 100 (12/12), 100% (15/15) and 100% (14/14), the negative predictive value is 97.9% (184/188), 99.5% (184/185) and 98.9% (184/186), and the coincidence rate is 98% (196/200), 99.5% (199/200) and 99% (198/200). The difference of the results of the four pairs of primers for respectively detecting MA compared with the sequencing result has no statistical significance (P is more than 0.05); the consistency with the sequencing result is higher, the Kappa value is minimum 0.847 and is more than 0.75, and P is less than 0.01. See in particular tables 2 and 3.

TABLE 2 comparison of four pairs of primers and sequencing to detect 200 clinical strains respectively

TABLE 3 comparison of four pairs of primers and sequencing to detect 200 clinical strains respectively

Example 7 reproducibility

The strains to be detected are as follows: two clinical strains of M.avium in example 6.

Extracting genome DNA of a strain to be detected, respectively adopting four pairs of primers in the table 1 to carry out three independent QPCR, carrying out 6 repeated detections on each sample, and recording the cycle Count (CT) value of each experiment.

Set up using ddH₂O negative control instead of template DNA.

The detection variation coefficients within the detection reaction groups (based on the data of 6 replicate detections in each reaction) and between the groups (based on the average of the CT values obtained by 6 replicate detections in each reaction, the variation coefficients for 3 reactions) were calculated for 2 samples, respectively.

The results are shown in FIG. 4 and FIG. 3. The results show that the four pairs of primers respectively detect the clinical strains with good repeatability.

TABLE 4 results of four pairs of primers for determining the reproducibility of two samples

Sequence listing

<110> research institute of tuberculosis and breast tumor in Beijing

Beijing Chest Hospital, Capital Medical University

<120> method for detecting mycobacterium avium by real-time fluorescence PCR and application thereof

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 16

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

cacctccacc cgcaaa 16

<210> 2

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

aggaacacat acgggaag 18

<210> 3

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

acctccaccc gcaaa 15

<210> 4

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

aggaacacat acgggaa 17

<210> 5

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gcatctccaa aagcgaggt 19

<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

cgaggaacac atacgggaag 20

<210> 7

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atcactaccg agaggaac 18

<210> 8

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

cgtcagctcc gcatc 15

Claims

1. Primer pair A or B or C or D;

the primer pair A consists of a primer 3F and a primer 3R;

the primer 3F is a single-stranded DNA molecule shown in a sequence 5 of a sequence table;

the primer 3R is a single-stranded DNA molecule shown in a sequence 6 in a sequence table;

the primer pair B consists of a primer 1F and a primer 1R;

the primer 1F is a single-stranded DNA molecule shown in a sequence 1 in a sequence table;

the primer 1R is a single-stranded DNA molecule shown in a sequence 2 of a sequence table;

the primer pair C consists of a primer 2F and a primer 2R;

the primer 2F is a single-stranded DNA molecule shown in a sequence 3 in a sequence table;

the primer 2R is a single-stranded DNA molecule shown in a sequence 4 of a sequence table;

the primer pair D consists of a primer 4F and a primer 4R;

the primer 4F is a single-stranded DNA molecule shown in a sequence 7 of a sequence table;

the primer 4R is a single-stranded DNA molecule shown in a sequence 8 of a sequence table.

2. The primer set according to claim 1, which is used as (b 1), (b 2), (b 3) or (b 4):

(b1) Identifying or assisting in identifying whether the bacteria to be detected is mycobacterium avium; the use is for non-disease diagnostic and therapeutic purposes;

(b3) Detecting whether the biological sample to be detected contains the mycobacterium avium or not; the use is for non-disease diagnostic and therapeutic purposes;

3. A kit containing the primer set according to claim 1; the application of the kit is (c 1) or (c 2):

4. A method for identifying or assisting in identifying whether a bacterium to be tested is Mycobacterium avium, comprising the steps of:

(1) Extracting the genome DNA of the bacteria to be detected;

(2) Performing real-time fluorescence PCR detection by using the genomic DNA extracted in the step (1) as a template and the primer pair of claim 1, wherein if a positive amplification result can be obtained, the bacteria to be detected is or is a candidate for mycobacterium avium, and if the positive amplification result cannot be obtained, the bacteria to be detected is non-mycobacterium avium;

the methods are methods for non-disease diagnostic and therapeutic purposes.

5. A method for detecting whether a biological sample to be detected contains mycobacterium avium comprises the following steps:

(1) Extracting the total DNA of a biological sample to be detected;

(2) Performing real-time fluorescence PCR detection by using the total DNA extracted in the step (1) as a template and the primer pair of claim 1, wherein if a positive amplification result can be obtained, the biological sample to be detected contains or is a candidate for containing mycobacterium avium, and if the positive amplification result cannot be obtained, the biological sample to be detected does not contain mycobacterium avium;

the methods are methods for non-disease diagnostic and therapeutic purposes.