CN110938702A - Method for detecting mycobacterium avium by real-time fluorescent PCR and application thereof - Google Patents

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

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CN110938702A
CN110938702A CN201911189769.1A CN201911189769A CN110938702A CN 110938702 A CN110938702 A CN 110938702A CN 201911189769 A CN201911189769 A CN 201911189769A CN 110938702 A CN110938702 A CN 110938702A
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primer
mycobacterium avium
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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
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Beijing Chest Hospital
Beijing Tuberculosis and Thoracic Tumor Research Institute
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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, good repeatability, lower price and simpler and more convenient operation compared with methods such as a DNA chip technology, a PCR-fingerprint spectrum and the like; 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 infectious pathogen and in humans can cause lung, soft tissue, childhood lymph node, skin infections, and the like. Not only does MA infection lack specificity in clinical presentation and histopathology, but also there is a clear difference in the drug resistance spectrum with 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 simple operation, shortened detection time and reduced cross infection risk, but also has lower price and simpler and more convenient operation compared with methods such as a DNA chip technology, a PCR-fingerprint spectrum and the like.
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 (a1) or (a2) as follows:
(a1) a single-stranded DNA molecule shown in sequence 5 of the sequence table;
(a2) DNA molecules obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 5 and having the same functions as the sequence 5;
the primer 3R is (a3) or (a4) 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 (a1) or (a 2):
(a1) a single-stranded DNA molecule shown in sequence 1 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 1 and have the same functions as the sequence 1;
the primer 1R is (a3) or (a 4):
(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 (a1) or (a 2):
(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 (a3) or (a4) as follows:
(a3) a single-stranded DNA molecule shown in a sequence 4 of the sequence table;
(a4) DNA molecules obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 4 and having the same functions as the sequence 4;
the primer pair D consists of a primer 4F and a primer 4R;
the primer 4F is (a1) or (a2) 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 (a3) or (a4) as follows:
(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 protects the application 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):
(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.
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 use of the kit is as follows (c1) 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.
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:
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 of 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.
The invention provides a method for detecting whether a biological sample to be detected contains mycobacterium avium, which comprises the following steps:
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. mu.l of SuperRealPreMix Plus 10. mu.l, 0.6. mu.l of each primer (concentration in reaction system: 0.3. mu. mol), 2. mu.l of DNA, 50X Roxreference 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 finishing the procedure of melting curve at 1.6 deg.C/s at 95 deg.C for 15s, 60 deg.C for 1min and 95 deg.C (collecting fluorescent signal) for 15 s.
Any of the above methods is a method of non-disease diagnosis and treatment.
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 test.
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 conventional 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. 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
Bacterial strain
21 standard strain of mycobacterium: mycobacterium avium (ATCC25291), Mycobacterium intracellulare (ATCC13950), Mycobacterium abscessus (ATCC19977), Mycobacterium chelonii (ATCC14472), Mycobacterium kansasii (ATCC12478), Mycobacterium gordonae (ATCC14470), Mycobacterium fortuitum (ATCC6481), Mycobacterium phlei (ATCC11758), Mycobacterium aurum (ATCC23366), Mycobacterium scrofulae (ATCC19981), Mycobacterium flavum (ATCC43909), Mycobacterium smegmatis (ATCC19420), Mycobacterium tuberculosis (ATCC27294), Mycobacterium marmorum (ATCC29571), Mycobacterium simian (ATCC25275), Mycobacterium thuringiensis (ATCC35799), Mycobacterium suis (ATCC33776), Mycobacterium terrestris (ATCC15755), Mycobacterium microflavus (ATCC14474), Mycobacterium dietzia (ATCC19340), Mycobacterium schchensiei (ATCC 27962).
5 common pathogenic bacteria: staphylococcus aureus (ATCC29213), Escherichia coli (ATCC25922), Pseudomonas aeruginosa (ATCC27853), Micrococcus luteus (CMCC28001), Staphylococcus saprophyticus (ATCC BAA 750).
Secondly, analyzing the accuracy of the strain
(1)21 standard strain of mycobacterium: amplifying the DNA of the mycobacterium standard strain by adopting universal primers Hsp65 (F: 5'-ACCAACGATGGTGTGTCCAT-3'; R: 5'-CTTGTCGAACCGCATACCCT-3') and 16Sr RNA (F: 5'-AGAGTTTGATCCTGGCTCAG-3'; R: 5'-CCCCGTCAATTCATTTGAGTTT-3'); 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 show that after the amplification products of the 21 standard strains are sequenced, the consistency of the amplification products and the DNA comparison 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 by the invention meet the requirements. 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 (merriella, 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 meets the requirement.
Example 2 obtaining of primers for detection of Mycobacterium avium
First, 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, as shown in table 1.
TABLE 1 four pairs of primers for identifying M.avium
Figure BDA0002293273180000061
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, 0.6. mu.l of each primer (concentration in reaction system 0.3. mu. mol), 2. mu.l of sample DNA (total genomic DNA mass 11.2ng), 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 finishing the procedure of melting curve at 1.6 deg.C/s at 95 deg.C for 15s, 60 deg.C for 1min and 95 deg.C (collecting fluorescent signal) for 15 s.
Set up using ddH2O replaces the negative control 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. mu.l of SuperReal Premix Plus, 0.6. mu.l of each primer (concentration in reaction system 0.3. mu. mol), 2. mu.l of sample DNA, 50X Rox 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 finishing the procedure of melting curve at 1.6 deg.C/s at 95 deg.C for 15s, 60 deg.C for 1min and 95 deg.C (collecting fluorescent signal) for 15 s.
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- 10ng/. mu.l of the dilution was used as the amplification result for the template and the negative control.
As a result, although the reaction systems 1 to 5 obtained by detecting the amplification of four pairs of M.avium primers in the amplification graph all gave positive results, 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 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 chest hospital, Beijing, affiliated to the university of capital medical science.
1. Amplifying the DNA of the strain to be detected by adopting a universal primer Hsp65 (F: 5'-ACCAACGATGGTGTGTCCAT-3'; R: 5'-CTTGTCGAACCGCATACCCT-3') and 16SrRNA (F: 5'-AGAGTTTGATCCTGGCTCAG-3'; R: 5'-CCCCGTCAATTCATTTGAGTTT-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 reaction system: 2 XSuperReal Premix Plus 10. mu.l, 0.6. mu.l of each primer (concentration in reaction system 0.3. mu. mol), 2. mu.l of sample DNA (total genomic DNA mass 11.2ng), 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 finishing the procedure of melting curve at 1.6 deg.C/s at 95 deg.C for 15s, 60 deg.C for 1min and 95 deg.C (collecting fluorescent signal) for 15 s.
Set up using ddH2O replaces the negative control 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), 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(12/12), 100% (15/15) and 100% (14/14), the negative predictive value is 97.9% (184/188), 97.9% (184/188), 99.5% (184/185) and 98.9% (184/186), and the coincidence rate is 98% (196/200), 98% (196/200), 99.5% (199/200) and 99% (198/200). The difference between the result of respectively detecting the MA by the four pairs of primers and the sequencing result is not statistically significant (P is more than 0.05); the sequence has high consistency with the sequencing result, 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
Figure BDA0002293273180000081
Figure BDA0002293273180000091
TABLE 3 comparison of four pairs of primers and sequencing to detect 200 clinical strains respectively
Figure BDA0002293273180000092
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.
QPCR reaction system: 2 XSuperReal Premix Plus 10. mu.l, 0.6. mu.l of each primer (concentration in reaction system 0.3. mu. mol), 2. mu.l of sample DNA (total genomic DNA mass 11.2ng), 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 finishing the procedure of melting curve at 1.6 deg.C/s at 95 deg.C for 15s, 60 deg.C for 1min and 95 deg.C (collecting fluorescent signal) for 15 s.
Set up using ddH2O replaces the negative control of template DNA.
The detection variation coefficient in the detection reaction group (based on the data of 6 repeated detections in each reaction) and the detection variation coefficient between groups (based on the average value of CT values obtained by 6 repeated detections in each reaction, the variation coefficient of 3 reactions) were calculated for 2 samples.
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
Figure BDA0002293273180000101
Sequence listing
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<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 (8)

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 (a1) or (a2) as follows:
(a1) a single-stranded DNA molecule shown in sequence 5 of the sequence table;
(a2) DNA molecules obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 5 and having the same functions as the sequence 5;
the primer 3R is (a3) or (a4) 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 (a1) or (a 2):
(a1) a single-stranded DNA molecule shown in sequence 1 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 1 and have the same functions as the sequence 1;
the primer 1R is (a3) or (a 4):
(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 2F is (a1) or (a2) 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 (a3) or (a4) as follows:
(a3) a single-stranded DNA molecule shown in a sequence 4 of the sequence table;
(a4) DNA molecules obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 4 and having the same functions as the sequence 4;
the primer pair D consists of a primer 4F and a primer 4R;
the primer 4F is (a1) or (a2) 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 (a3) or (a4) as follows:
(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.
2. The primer pair of claim 1, which is used as (b1), (b2), (b3) or (b4):
(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.
3. A kit containing the primer pair of claim 1; the use of the kit is as follows (c1) 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.
4. A method for preparing the kit according to claim 3, comprising the step of packaging each primer individually.
5. 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 selected as mycobacterium avium, and if the positive amplification result cannot be obtained, the bacteria to be detected is non-mycobacterium avium.
6. A method for identifying or assisting in identifying whether a bacterium to be tested is Mycobacterium avium, comprising the steps of:
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 of 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 set according to claim 1 in the genome of Mycobacterium avium.
7. 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 the mycobacterium avium, and if the positive amplification result cannot be obtained, the biological sample to be detected does not contain the mycobacterium avium.
8. A method for detecting whether a biological sample to be detected contains mycobacterium avium comprises the following steps:
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 set according to claim 1 in the genome of Mycobacterium avium.
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CN108866216A (en) * 2018-07-11 2018-11-23 首都医科大学附属北京胸科医院 It is a kind of for identifying the primer combination of probe of mycobacterium abscessus and Marseille mycobacteria

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
CN105219882A (en) * 2015-11-19 2016-01-06 北京利德曼生化股份有限公司 Mycobacterium avium-intracellulare compound group detects by primer and probe and its detection method
CN108866216A (en) * 2018-07-11 2018-11-23 首都医科大学附属北京胸科医院 It is a kind of for identifying the primer combination of probe of mycobacterium abscessus and Marseille mycobacteria

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JIM等: "A real time PCR assay for the detection and quantitation of Mycobacterium avium subsp. paratuberculosis using SYBR Green and the Light Cycler", 《JOURNAL OF MICROBIOLOGICAL METHODS》 *
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