CN111187847A - Probe system, kit and method for identifying mycobacteria - Google Patents
Probe system, kit and method for identifying mycobacteria Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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Abstract
The invention discloses a probe system, a kit and an identification method for identifying mycobacterium. The probe system for identifying the mycobacterium comprises a connecting probe and a fluorescent detection probe. Compared with the traditional mycobacterium identification method, the probe system, the kit and the identification method for identifying the mycobacterium have the advantages of simplicity, convenience, rapidness, high accuracy, good repeatability, no pollution, low cost and the like, and are suitable for being applied to various clinical or scientific research laboratories arranged under medical care, teaching and scientific research institutions and the like.
Description
Technical Field
The invention relates to the field of biochemical detection, in particular to a probe system, a kit and an identification method for identifying mycobacterium.
Background
Mycobacteria include a large group of acid-fast stain-positive bacilli, which are classified into fast-growing mycobacteria and slow-growing mycobacteria according to their growth rate. Among the commonly occurring fast growing mycobacteria are adventitious mycobacteria, mycobacterium cheloniae, mycobacterium abscessus, mycobacterium smegmatis, and the like. The slow-growing mycobacteria are further classified into chromogenic mycobacteria, chromogenic mycobacteria and non-chromogenic mycobacteria according to the characteristics of the produced pigment. The common chromogenes include Mycobacterium kansasii, Mycobacterium marinum, Mycobacterium simian and the like, the common secretogenic mycobacteria include Mycobacterium scrofulaceum, Mycobacterium gordonii, Mycobacterium soulanguis and the like, and the common chromogenes-free mycobacteria include Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium ulcerosa, Mycobacterium terrae and Mycobacterium gastri and the like.
The infection characteristics and drug sensitivity of different mycobacteria are different, so that the identification of mycobacteria has an important role. The identification of mycobacteria includes a phenotypic method and a molecular method, wherein the phenotypic method includes a biochemical identification method, a gas chromatography method, a high performance liquid chromatography method, a serological identification method, a phage lysis method and the like, and the molecular method includes probe hybridization, ordinary PCR, real-time fluorescence PCR, DNA fingerprint, sequencing and the like. Traditionally, both phenotypic methods and molecular methods have disadvantages, such as complex identification process, low accuracy, poor repeatability, easy pollution, and the like.
Disclosure of Invention
Accordingly, there is a need for a probe system, a kit and a method for identifying mycobacteria, which are easy and convenient to operate, highly accurate, highly reproducible and less prone to contamination.
A probe system for identifying mycobacterium comprises a connecting probe and a fluorescence detection probe;
the ligation probe comprises a left ligation probe and a right ligation probe, wherein the left ligation probe is provided with an upstream amplification primer fragment, a filling fragment and a first specific hybridization ligation fragment from the 5 ' end to the 3 ' end in sequence, the right ligation probe is provided with a second specific hybridization ligation fragment and a downstream amplification primer binding fragment from the 5 ' end to the 3 ' end in sequence, and the 3 ' end of the right ligation probe is sealed with a C3 spacer;
the fluorescence detection probe comprises the following components from the 5 'end to the 3' end in sequence: at least 3 ' end of the first specific hybridization connecting segment has a segment with at least 3 ' end partial segment sequence consistent, and at least 5 ' end of the second specific hybridization connecting segment has a segment with at least 5 ' end partial segment sequence consistent, and the 3 ' end of the fluorescence detection probe is connected with a fluorescent group;
the first specific hybridization connecting segment and the second specific hybridization connecting segment respectively correspond to different target segments in the genome DNA of the target mycobacterium;
the first specific hybridization connecting fragment and the second specific hybridization connecting fragment are selected from at least one group of sixteen groups of fragments as follows: corresponding fragments of genomic DNA of Mycobacterium tuberculosis having the sequences shown in SEQ ID NO.1 and SEQ ID NO.2, corresponding fragments of genomic DNA of Mycobacterium gastri having the sequences shown in SEQ ID NO.3 and SEQ ID NO.4, corresponding fragments of genomic DNA of Mycobacterium thuringiensis having the sequences shown in SEQ ID NO.5 and SEQ ID NO.6, corresponding fragments of genomic DNA of Mycobacterium ulcerosa having the sequences shown in SEQ ID NO.7 and SEQ ID NO.8, corresponding fragments of genomic DNA of Mycobacterium abscessus having the sequences shown in SEQ ID NO.9 and SEQ ID NO.10, corresponding fragments of genomic DNA of Mycobacterium avium having the sequences shown in SEQ ID NO.11 and SEQ ID NO.12, corresponding fragments of genomic DNA of Mycobacterium tortoise having the sequences shown in SEQ ID NO.13 and SEQ ID NO.14, corresponding fragments of genomic DNA of Mycobacterium fortuitum having the sequences shown in SEQ ID NO.15 and SEQ ID NO.16, corresponding fragments of the genomic DNA of Mycobacterium gordonae with the sequences shown in SEQ ID NO.17 and SEQ ID NO.18, corresponding fragments of the genomic DNA of Mycobacterium haemolyticus with the sequences shown in SEQ ID NO.19 and SEQ ID NO.20, corresponding fragments of the genomic DNA of Mycobacterium intracellulare with the sequences shown in SEQ ID NO.21 and SEQ ID NO.22, corresponding fragments of the genomic DNA of Mycobacterium kansasii with the sequences shown in SEQ ID NO.23 and SEQ ID NO.24, corresponding fragments of the genomic DNA of Mycobacterium smegmatis with the sequences shown in SEQ ID NO.25 and SEQ ID NO.26, corresponding fragments of the genomic DNA of Mycobacterium terrae with the sequences shown in SEQ ID NO.27 and SEQ ID NO.28, corresponding fragments of the genomic DNA of Mycobacterium minor with the sequences shown in SEQ ID NO.29 and SEQ ID NO.30, and the corresponding fragments of the genomic DNA of Mycobacterium Xenopi with the sequences shown as SEQ ID NO.31 and SEQ ID NO. 32.
Specifically, the first specific hybrid junction fragment and the second specific hybrid junction fragment correspond to different target fragments of 16S rDNA in the genomic DNA of the target mycobacterium, and more specifically are completely identical or completely complementary to different segments of the same strand of 16S rDNA.
In one embodiment, the sequence of the fluorescent detection probe corresponds to the sequence of the ligation probe, and the fluorescent detection probe is selected from at least one of the sixteen fluorescent detection probes as follows: a fluorescence detection probe for Mycobacterium tuberculosis having a sequence shown in SEQ ID NO.33, a fluorescence detection probe for Mycobacterium gastri having a sequence shown in SEQ ID NO.34, a fluorescence detection probe for Mycobacterium thuringiensis having a sequence shown in SEQ ID NO.35, a fluorescence detection probe for Mycobacterium ulcerosa having a sequence shown in SEQ ID NO.36, a fluorescence detection probe for Mycobacterium abscessus having a sequence shown in SEQ ID NO.37, a fluorescence detection probe for Mycobacterium avium having a sequence shown in SEQ ID NO.38, a fluorescence detection probe for Mycobacterium cheloniae having a sequence shown in SEQ ID NO.39, a fluorescence detection probe for Mycobacterium fortuitum having a sequence shown in SEQ ID NO.40, a fluorescence detection probe for Mycobacterium gordonae having a sequence shown in SEQ ID NO.41, a fluorescence detection probe for Mycobacterium haemolyticum having a sequence shown in SEQ ID NO.42, a fluorescence detection probe for Mycobacterium intracellulare having a sequence shown in SEQ ID NO.43, the sequence of the fluorescent detection probe of the mycobacterium kansasii is shown as SEQ ID NO.44, the sequence of the fluorescent detection probe of the mycobacterium smegmatis is shown as SEQ ID NO.45, the sequence of the fluorescent detection probe of the mycobacterium terreus is shown as SEQ ID NO.46, the sequence of the fluorescent detection probe of the mycobacterium minor is shown as SEQ ID NO.47, and the sequence of the fluorescent detection probe of the mycobacterium Xenopi is shown as SEQ ID NO. 48.
In one embodiment, the sequences of the upstream amplification primer fragment and the downstream amplification primer binding fragment are shown as SEQ ID NO.49 and SEQ ID NO.50, respectively.
In one embodiment, the stuffer fragment is selected from one of four fragments having the sequences as shown in SEQ ID NO.51, SEQ ID NO.52, SEQ ID NO.53 and SEQ ID NO. 54.
In one embodiment, the sequence of the stuffer fragment in the left ligation probe corresponding to the mycobacterium tuberculosis, mycobacterium abscessus, mycobacterium gordonii, and mycobacterium smegmatis is shown as SEQ ID No. 51;
the sequences of stuffer fragments in the left ligation probes corresponding to the M.gastri, M.avium, M.haemolyticus and M.terreus are shown in SEQ ID NO. 52;
the sequences of the stuffer fragments in the left ligation probes corresponding to the Mycobacterium thuringiensis, Mycobacterium cheloniae, M.intracellulare and M.sububerculans are shown in SEQ ID NO. 53;
the sequences of stuffer fragments in the left ligation probes corresponding to the M.ulcerosa, the M.fortuitum, the M.kansasii and the M.xenopi are shown in SEQ ID NO. 54.
In one embodiment, the fluorophores attached to the 3' ends of the fluorescent detection probe for mycobacterium tuberculosis, the fluorescent detection probe for mycobacterium gastri, the fluorescent detection probe for mycobacterium suger, the fluorescent detection probe for mycobacterium ulcerans, and the fluorescent detection probe for mycobacterium abscessus are TAMRA;
the fluorescent group connected to the 3' end of the fluorescent detection probe for mycobacterium avium, the fluorescent detection probe for mycobacterium cheloni, the fluorescent detection probe for mycobacterium fortuitum, the fluorescent detection probe for mycobacterium gordonii, the fluorescent detection probe for mycobacterium haemolyticum, and the fluorescent detection probe for mycobacterium intracellularis ROX;
the fluorophore connected to the 3' -end of the M.kansasii fluorescence detection probe, the M.smegmatis fluorescence detection probe, the M.terrae fluorescence detection probe, the M.minor fluorescence detection probe, and the M.xenopi fluorescence detection probe is Cy 5.
A kit for identifying Mycobacterium, comprising the probe system for identifying Mycobacterium described in any one of the above embodiments.
In one embodiment, the kit for identifying mycobacteria further comprises an amplification primer pair, the sequence of the upstream primer in the amplification primer pair is identical to the sequence of the upstream amplification primer fragment, the sequence of the downstream primer in the amplification primer pair is completely complementary to the sequence of the downstream amplification primer binding fragment, and the downstream primer is connected with another fluorescent group.
In one embodiment, the downstream primer has a FAM fluorophore attached to a T base 4nt away from the A base at the 3' end.
In one embodiment, the kit for identifying mycobacteria further comprises at least one of Taq DNA polymerase, Taq DNA ligase, DNA extraction reagent, and PCR reaction reagent.
A method for identifying a Mycobacterium using the probe system for identifying a Mycobacterium described in any one of the above embodiments, the method comprising the steps of:
extracting the genome DNA of the bacteria to be detected;
taking the extracted genome DNA as a template, adding the connecting probe, the fluorescent detection probe and an upstream primer and a downstream primer which have the same sequences as the upstream amplification primer fragment and are completely complementary to the sequence of the downstream amplification primer binding fragment respectively for connection, PCR amplification and melting curve continuous reaction, wherein the downstream primer is connected with a fluorescent group;
and detecting the fluorescent signal generated by the reaction system in real time by using a fluorescence monitoring instrument.
In one embodiment, the extracting the genomic DNA of the bacteria to be tested is extracting the genomic DNA by using a boiling lysis method. The temperature and time for the cleavage can be, but are not limited to, 100 ℃ for 15 min.
Compared with the traditional mycobacterium identification method, the probe system, the kit and the identification method for identifying the mycobacterium have the advantages that:
(1) simple and quick: the DNA can be directly used for subsequent amplification reaction after being extracted, the result can be directly interpreted after the amplification is finished, the whole detection process can be finished within 4h, and the manual operation part is less;
(2) the accuracy is high: the amplification process and the melting curve stage are respectively monitored, so that the accuracy is high, and particularly when probe systems of multiple mycobacteria are combined for use, target Tm values of the various mycobacteria are far apart, and the various mycobacteria are very easy to distinguish in melting curve analysis, so that the common 16 mycobacteria can be identified accurately at most;
(3) the repeatability is good: the three processes of connection, amplification and melting curve analysis are continuously completed in the same PCR reaction tube, and the DNA extraction method is simple and easy to implement, so that the method is less in influenced factors and has good repeatability;
(4) no pollution: the PCR reaction tube does not need to be repeatedly opened and closed in the whole amplification reaction process, and the diffusion and pollution of the amplification product can be effectively prevented;
(5) the cost is low: the method can adopt a single tube to identify 16 common mycobacteria at most once, so that the cost is higher and the cost is lower by adopting a multiple-tube molecular biology method;
(6) the identification spectrum is wide: the method can identify 16 common mycobacteria at most, can identify more than two mixed cultures, and has wider identification spectrum compared with common PCR and real-time PCR.
Generally, the probe system, the kit and the identification method for identifying the mycobacterium have the advantages of simplicity, convenience, rapidness, high accuracy, good repeatability, no pollution, low cost and the like, and are suitable for being applied to various clinical or scientific research laboratories arranged under institutions (hospitals, disease control centers, scientific research institutions and the like) in medical health, teaching and scientific research and the like.
Drawings
FIG. 1 is a schematic diagram of the detection of the method for identifying Mycobacterium of the present invention.
FIG. 2 is a graph showing the results of clinical strain identification, wherein the abscissa is Temperature (Temperature), the ordinate is Fluorescence intensity (Fluorescence), the unit is d/dT, the short broad melting peak is a specific peak of Mycobacterium tuberculosis (Tm value is in the vicinity of 78 ℃), and the high broad melting peak is a specific peak of Mycobacterium avium (Tm value is in the vicinity of 77 ℃).
Detailed Description
To facilitate an understanding of the invention, the invention will now be described more fully with reference to specific embodiments and accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
As shown in fig. 1, the detection principle of the present embodiment is: the first step is as follows: hybridizing, specifically, when the target DNA is present in the sample, hybridizing the left ligation probe (the sequence comprising the upstream amplification primer segment, the stuffer segment and the first specific hybridization ligation segment) and the right ligation probe (the sequence comprising the second specific hybridization ligation segment and the downstream amplification primer binding segment) with the target DNA; the second step is that: connecting, namely connecting a left connecting probe and a right connecting probe under the action of DNA ligase; the third step: amplifying, specifically, in the presence of DNA polymerase, performing amplification by using the fragment connected in the second step as a template by using an upstream primer and a downstream primer (the upstream primer is identical to the upstream amplification primer fragment, the downstream primer is completely complementary to the downstream amplification primer binding fragment of the right ligation probe, and the 3' end of the downstream amplification primer is labeled with a fluorescent group); a fourth step of: and analyzing, specifically, hybridizing the fluorescence detection probe with the downstream primer extension chain, and generating fluorescence with another wavelength under the excitation of the fluorescence emitted by the fluorescence group on the downstream primer extension chain by the fluorescence group marked at the 3' end of the fluorescence detection probe. Because of the Tm value specific to the fluorescent detection probe and the emission wavelength specific to the fluorescein to be labeled, it is possible to determine whether the sample is genomic DNA of a certain mycobacterium or not by melting curve analysis.
Design of probe system and amplification primer
The probe system designed in this example includes a ligation probe and a fluorescence detection probe. The ligation probe comprises a left ligation probe and a right ligation probe, wherein the left ligation probe is provided with an upstream amplification primer segment, a filling segment and a first specific hybridization connection segment from the 5 ' end to the 3 ' end in sequence, the right ligation probe is provided with a second specific hybridization connection segment and a downstream amplification primer binding segment from the 5 ' end to the 3 ' end in sequence, and the 3 ' end of the right ligation probe is sealed with a C3 spacer. The fluorescence detection probe comprises the following components from the 5 'end to the 3' end in sequence: at least 3 ' end of the first specific hybridization connecting segment has a segment with at least 3 ' end partial segment sequence consistent, and at least 5 ' end of the second specific hybridization connecting segment has a segment with at least 5 ' end partial segment sequence consistent, and the 3 ' end of the fluorescence detection probe is connected with a fluorescent group. The first specific hybridization junction fragment and the second specific hybridization junction fragment correspond to different target fragments in the genomic DNA of the target mycobacterium.
Specifically, referring to table 1, table 1 shows the sequences of the first specific hybrid junction fragment and the second specific hybrid junction fragment designed for different mycobacteria.
TABLE 1
The sequences of the upstream amplification primer fragment and the downstream amplification primer binding fragment are respectively as follows:
(SEQ ID NO.49)；
(SEQ ID NO.50)。
correspondingly, the sequence of the upstream primer in the amplification primer pair for PCR amplification is identical to the sequence of the upstream amplification primer fragment and is also shown as SEQ ID NO.49, and the sequence of the downstream primer in the amplification primer pair is completely complementary to the sequence of the downstream amplification primer binding fragment and is as follows: (SEQ ID NO. 55). The downstream primer is connected with a fluorescent group, preferably, the downstream primer is connected with a fluorescent group on a T base which is 4nt away from the A base at the 3' end, and the fluorescent group is preferably a FAM group.
The stuffer fragment is selected from one of the following four sequence fragments:
(SEQ ID NO.51)；
(SEQ ID NO.52)；
(SEQ ID NO. 53); and
(SEQ ID NO.54)。
specifically, the sequences of stuffer fragments in the left ligation probes corresponding to Mycobacterium tuberculosis, Mycobacterium abscessus, Mycobacterium gordonii and Mycobacterium smegmatis are shown as SEQ ID No. 51; the sequence of the stuffer fragment in the left ligation probe corresponding to M.gastri, M.avium, M.haemolyticus and M.terreus is shown as SEQ ID NO. 52; the sequence of the stuffer fragment in the left ligation probe corresponding to Mycobacterium fortunei, Mycobacterium cheloniae, M.intracellulare and M.subordinatensis is shown in SEQ ID NO. 53; the sequence of the stuffer fragment in the left ligation probe corresponding to Mycobacterium ulcerosa, Mycobacterium fortuitum, Mycobacterium kansasii and Mycobacterium Xenopi is shown in SEQ ID NO. 54.
More specifically, the fluorescent group attached to the 3' -end of the fluorescent detection probe for Mycobacterium tuberculosis, the fluorescent detection probe for Mycobacterium gastri, the fluorescent detection probe for Mycobacterium suljianensis, the fluorescent detection probe for Mycobacterium ulcerans, and the fluorescent detection probe for Mycobacterium abscessus is TAMRA; the fluorescent group connected with the 3' end of the fluorescent detection probe for mycobacterium avium, the fluorescent detection probe for mycobacterium cheloni, the fluorescent detection probe for mycobacterium fortuitum, the fluorescent detection probe for mycobacterium gordonii, the fluorescent detection probe for mycobacterium haemolyticum and the fluorescent detection probe for mycobacterium intracellulare is ROX; the fluorophore attached to the 3' -end of the fluorescent detection probe for M.kansasii, M.smegmatis, M.terrae, M.sububerii, and M.xenopi was Cy 5.
The detailed sequence information of the finally constructed ligation probe is as follows:
left ligation probe 1:
right ligation probe 1:
left ligation probe 2:
right ligation probe 2:
left ligation probe 3:
right ligation probe 3:
left ligation probe 4:
right ligation probe 4:
left ligation probe 5:
right ligation probe 5:
left ligation probe 6:
right ligation probe 6:
left ligation probe 7:
right ligation probe 7:
left ligation probe 8:
right ligation probe 8:
left ligation probe 9:
right ligation probe 9:
left ligation probe 10:
right connection probe 10:
left ligation probe 11:
right connection probe 11:
left ligation probe 12:
right ligation probe 12:
left ligation probe 13:
right ligation probe 13:
left ligation probe 14:
right ligation probe 14:
left ligation probe 15:
right ligation probe 15:
left ligation probe 16:
right ligation probe 16:
the detailed sequence information of the finally constructed fluorescence detection probe is as follows:
fluorescent detection probe 1(SEQ ID NO. 33): 5 '- (mark TAMRA) -3'; tm: 78 ℃;
fluorescent detection probe 2(SEQ ID NO. 34): 5 '- (marker TAMRA) -3', Tm: 73 ℃;
fluorescent detection probe 3(SEQ ID NO. 35): (marker TAMRA) -3', Tm: at 66 ℃;
fluorescent detection probe 4(SEQ ID NO. 36): (marker TAMRA) -3', Tm: 60 ℃;
fluorescent detection probe 5(SEQ ID NO. 37): (marker TAMRA) -3', Tm: 70 ℃;
fluorescent detection probe 6(SEQ ID NO. 38): (marker ROX) -3', Tm: 77 ℃;
fluorescent detection probe 7(SEQ ID NO. 39): (marker ROX) -3', Tm: 60 ℃;
fluorescent detection probe 8(SEQ ID NO. 40): label ROX) -3', Tm: 74 ℃;
fluorescent detection probe 9(SEQ ID NO.41) (marker ROX) -3', Tm: 70 ℃;
fluorescent detection probe 10(SEQ ID NO. 42): (marker ROX) -3', Tm: 67 deg.C;
fluorescent detection probe 11(SEQ ID NO. 43): (marker ROX) -3', Tm: 63 ℃;
fluorescent detection probe 12(SEQ ID NO. 44): (labeled Cy5) -3', Tm: 83 ℃;
fluorescent detection probe 13(SEQ ID NO. 45): (labeled Cy5) -3', Tm: 64 ℃;
fluorescent detection probe 14(SEQ ID NO. 46): (labeled Cy5) -3', Tm: 76 ℃;
fluorescent detection probe 15(SEQ ID NO. 47): (labeled Cy5) -3', Tm: 72 ℃;
fluorescent detection probe 16(SEQ ID NO. 48): (labeled Cy5) -3', Tm: 67 ℃.
II, identifying strains
1. Collecting the strains
Totally collecting 50 mycobacteria strains, wherein the mycobacteria strains comprise 5 MTC, 10 M.avium complex, 10 M.cheloniae complex, 5 M.kansasii, 3 M.scrofula, 3 M.marinum, 3 M.ulcerosa, 3 G.gordonae, 3 M.fortuitum, 3 M.subordinatensis, 1 M.canogenes and 1 M.terrestris.
2. Extraction of Strain DNA
(1) A small amount of bacterial colonies are scraped from the solid culture medium by an inoculating loop and put into a small amount of sterilized water, then the bacterial colonies are prepared into bacterial suspension after being shaken for a period of time by an oscillator, and then the concentration of the bacterial suspension is adjusted to 1 standard Mycoplasma turbidity by a Mycoplasma turbidimeter.
(2) 1ml of the bacterial suspension was taken into a 1.5ml microcentrifuge tube and centrifuged for 5min under a centrifugal force of 10000 g.
(3) Centrifuging, removing supernatant, adding 40 μ l DNA extractive solution, shaking, mixing, heating at 100 deg.C for 15min, cooling, and centrifuging at 13000g for 10 min. The DNA extract contained 8% sucrose, 50mM EDTA and 50mM Tris-HCl (pH 8.0).
(4) After centrifugation, the supernatant containing the DNA template was transferred to another 1.5ml micro-centrifuge tube without nucleic acid and stored at-20 ℃ until use.
3. Detecting sample DNA
(1) The extracted DNA template is added into a reaction system, and specific reaction components and addition amount are shown in the following table 2.
TABLE 2
Note: the components and concentrations of the reaction solution were as follows: 5mM MgCl2、20mM Tris·HCl、0.4mM NAD、1.6mMdNTP；
The concentration of the connection probe is 3 nmol/L;
the concentration of the amplification primer is 3 mu mol/L;
the concentration of the fluorescence detection probe is 3 mu mol/L;
taq DNA polymerase and Taq DNA ligase are hot-start DNA polymerase and heat-resistant DNA ligase produced by NEB company, and the concentrations are 5U/mul and 5U/mul respectively;
the concentration of the DNA template was 50 ng/. mu.l.
The reaction system was placed on a fluorescent quantitative PCR instrument (Roche diagnostics, LightCycler 480) for multiplex PCR detection.
(2) Reaction conditions are as follows: 95 ℃, 3min- >45 ℃, 15min- >95 ℃, 15s, 60 ℃, 15s (during which fluorescence signals are detected), 72 ℃, 30s, 30 cycles- >72 ℃, 2min- >95 ℃, 15s- >50 ℃ renaturation for 1min, then heating to 95 ℃ at the speed of 0.05 ℃/s and maintaining for 15s (during which fluorescence signals are detected), and finally 50 ℃ renaturation for 15 s.
(3) And (4) interpretation of results: firstly, determining a main melting curve analysis channel according to the amplification curve conditions of four channels, and then determining which mycobacterium a strain to be detected belongs to according to the Tm value of the melting curve; when more than two channels have obvious amplification curves or more than two characteristic peaks appear in melting curves, the mixed existence of two or more mycobacteria needs to be considered.
As shown in FIG. 2, the results show that the probe system and the amplification primer constructed above can accurately identify the above-mentioned common mycobacteria, and thus have a good mycobacteria identification effect.
Third, analysis of specificity evaluation
The analytical specificity of the method is evaluated by detecting standard or clinical strains of 20 common mycobacteria and 10 common bacteria, including specifically Mycobacterium tuberculosis, Mycobacterium gastri, Mycobacterium sulgardii, Mycobacterium ulcerosa, Mycobacterium abscessus, Mycobacterium avium, Mycobacterium chelonii, Mycobacterium fortuitum, Mycobacterium gordonae, Mycobacterium haemolyticum, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium smegmatis, Mycobacterium terrae, Mycobacterium minor, Mycobacterium mycobacter xenopii, Mycobacterium bovis, Mycobacterium aurum, Mycobacterium neoaurum, Mycobacterium parafortuitum, Staphylococcus aureus, Escherichia coli, Candida albicans, Streptococcus pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa, Klebsiella pneumoniae, enterococcus faecalis, Bacillus thermophilus, Neisseria gonorrhoeae.
The result shows that the probe system and the amplification primer designed by the method can accurately identify 16 common mycobacteria, so that the method has good analysis specificity.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. A probe system for identifying mycobacterium is characterized by comprising a connecting probe and a fluorescence detection probe;
the ligation probe comprises a left ligation probe and a right ligation probe, wherein the left ligation probe is sequentially provided with an upstream amplification primer fragment, a filling fragment and a first specific hybridization ligation fragment from the 5 ' end to the 3 ' end, the right ligation probe is sequentially provided with a second specific hybridization ligation fragment and a downstream amplification primer binding fragment from the 5 ' end to the 3 ' end, and the 3 ' end of the right ligation probe is sealed with a C3 spacer;
the fluorescence detection probe comprises the following components from the 5 'end to the 3' end in sequence: at least 3 ' end of the first specific hybridization connecting segment has a segment with at least 3 ' end partial segment sequence consistent, and at least 5 ' end of the second specific hybridization connecting segment has a segment with at least 5 ' end partial segment sequence consistent, and the 3 ' end of the fluorescence detection probe is connected with a fluorescent group;
the first specific hybridization connecting segment and the second specific hybridization connecting segment respectively correspond to different target segments in the genome DNA of the target mycobacterium;
the first specific hybridization connecting fragment and the second specific hybridization connecting fragment are selected from at least one group of sixteen groups of fragments as follows: corresponding fragments of genomic DNA of Mycobacterium tuberculosis having the sequences shown in SEQ ID NO.1 and SEQ ID NO.2, corresponding fragments of genomic DNA of Mycobacterium gastri having the sequences shown in SEQ ID NO.3 and SEQ ID NO.4, corresponding fragments of genomic DNA of Mycobacterium thuringiensis having the sequences shown in SEQ ID NO.5 and SEQ ID NO.6, corresponding fragments of genomic DNA of Mycobacterium ulcerosa having the sequences shown in SEQ ID NO.7 and SEQ ID NO.8, corresponding fragments of genomic DNA of Mycobacterium abscessus having the sequences shown in SEQ ID NO.9 and SEQ ID NO.10, corresponding fragments of genomic DNA of Mycobacterium avium having the sequences shown in SEQ ID NO.11 and SEQ ID NO.12, corresponding fragments of genomic DNA of Mycobacterium chelonii having the sequences shown in SEQ ID NO.13 and SEQ ID NO.14, corresponding fragments of genomic DNA of Mycobacterium fortuitum having the sequences shown in SEQ ID NO.15 and SEQ ID NO.16, corresponding fragments of the genomic DNA of Mycobacterium gordonae with the sequences shown in SEQ ID NO.17 and SEQ ID NO.18, corresponding fragments of the genomic DNA of Mycobacterium haemolyticus with the sequences shown in SEQ ID NO.19 and SEQ ID NO.20, corresponding fragments of the genomic DNA of Mycobacterium intracellulare with the sequences shown in SEQ ID NO.21 and SEQ ID NO.22, corresponding fragments of the genomic DNA of Mycobacterium kansasii with the sequences shown in SEQ ID NO.23 and SEQ ID NO.24, corresponding fragments of the genomic DNA of Mycobacterium smegmatis with the sequences shown in SEQ ID NO.25 and SEQ ID NO.26, corresponding fragments of the genomic DNA of Mycobacterium terrae with the sequences shown in SEQ ID NO.27 and SEQ ID NO.28, corresponding fragments of the genomic DNA of Mycobacterium minor with the sequences shown in SEQ ID NO.29 and SEQ ID NO.30, and the corresponding fragments of the genomic DNA of Mycobacterium Xenopi with the sequences shown as SEQ ID NO.31 and SEQ ID NO. 32.
2. The probe system for mycobacterial identification according to claim 1, wherein the sequence of said fluorescent detection probe corresponds to the sequence of said ligation probe, and said fluorescent detection probe is selected from at least one of the sixteen following fluorescent detection probes: a fluorescence detection probe for Mycobacterium tuberculosis having a sequence shown in SEQ ID NO.33, a fluorescence detection probe for Mycobacterium gastri having a sequence shown in SEQ ID NO.34, a fluorescence detection probe for Mycobacterium thuringiensis having a sequence shown in SEQ ID NO.35, a fluorescence detection probe for Mycobacterium ulcerosa having a sequence shown in SEQ ID NO.36, a fluorescence detection probe for Mycobacterium abscessus having a sequence shown in SEQ ID NO.37, a fluorescence detection probe for Mycobacterium avium having a sequence shown in SEQ ID NO.38, a fluorescence detection probe for Mycobacterium cheloniae having a sequence shown in SEQ ID NO.39, a fluorescence detection probe for Mycobacterium fortuitum having a sequence shown in SEQ ID NO.40, a fluorescence detection probe for Mycobacterium gordonae having a sequence shown in SEQ ID NO.41, a fluorescence detection probe for Mycobacterium haemolyticum having a sequence shown in SEQ ID NO.42, a fluorescence detection probe for Mycobacterium intracellulare having a sequence shown in SEQ ID NO.43, the sequence of the fluorescent detection probe of the mycobacterium kansasii is shown as SEQ ID NO.44, the sequence of the fluorescent detection probe of the mycobacterium smegmatis is shown as SEQ ID NO.45, the sequence of the fluorescent detection probe of the mycobacterium terreus is shown as SEQ ID NO.46, the sequence of the fluorescent detection probe of the mycobacterium minor is shown as SEQ ID NO.47, and the sequence of the fluorescent detection probe of the mycobacterium Xenopi is shown as SEQ ID NO. 48.
3. The probe system for identifying mycobacteria according to claim 1 or 2, wherein the sequences of the upstream amplification primer fragment and the downstream amplification primer binding fragment are shown in SEQ ID No.49 and SEQ ID No.50, respectively.
4. The probe system for the identification of mycobacteria according to claim 3, wherein said stuffer fragment is selected from one of the four fragments having the sequences shown in SEQ ID No.51, SEQ ID No.52, SEQ ID No.53 and SEQ ID No. 54.
5. The probe system for Mycobacterium identification according to claim 4, wherein the sequences of stuffer fragments in left ligation probes corresponding to said Mycobacterium tuberculosis, said Mycobacterium abscessus, said Mycobacterium gordonii and said Mycobacterium smegmatis are represented by SEQ ID No. 51;
the sequences of stuffer fragments in the left ligation probes corresponding to the M.gastri, M.avium, M.haemolyticus and M.terreus are shown in SEQ ID NO. 52;
the sequences of the stuffer fragments in the left ligation probes corresponding to the Mycobacterium thuringiensis, Mycobacterium cheloniae, M.intracellulare and M.sububerculans are shown in SEQ ID NO. 53;
the sequences of stuffer fragments in the left ligation probes corresponding to the M.ulcerosa, the M.fortuitum, the M.kansasii and the M.xenopi are shown in SEQ ID NO. 54.
6. The probe system for Mycobacterium species identification according to claim 5, wherein the fluorophore attached to the 3' end of said Mycobacterium tuberculosis fluorescent detection probe, said Mycobacterium gastri fluorescent detection probe, said Mycobacterium sulgata fluorescent detection probe, said Mycobacterium ulcerans fluorescent detection probe, and said Mycobacterium abscessus fluorescent detection probe is TAMRA;
the fluorescent group connected to the 3' end of the fluorescent detection probe for mycobacterium avium, the fluorescent detection probe for mycobacterium cheloni, the fluorescent detection probe for mycobacterium fortuitum, the fluorescent detection probe for mycobacterium gordonii, the fluorescent detection probe for mycobacterium haemolyticum, and the fluorescent detection probe for mycobacterium intracellularis ROX;
the fluorophore connected to the 3' -end of the M.kansasii fluorescence detection probe, the M.smegmatis fluorescence detection probe, the M.terrae fluorescence detection probe, the M.minor fluorescence detection probe, and the M.xenopi fluorescence detection probe is Cy 5.
7. A kit for identifying mycobacteria, comprising the probe system for identifying mycobacteria according to any one of claims 1 to 6.
8. The kit for mycobacterial identification according to claim 7, further comprising an amplification primer pair, wherein the sequence of the upstream primer in said amplification primer pair is identical to the sequence of said upstream amplification primer fragment, the sequence of the downstream primer in said amplification primer pair is fully complementary to the sequence of said downstream amplification primer binding fragment, and said downstream primer is linked to another fluorophore.
9. The kit for the identification of Mycobacterium according to claim 8, wherein said downstream primer has a FAM fluorophore linked to a T base 4nt away from the A base at the 3' end.
10. A method for identifying a Mycobacterium using the probe system for identifying a Mycobacterium according to any one of claims 1 to 6, the method comprising the steps of:
extracting the genome DNA of the bacteria to be detected;
taking the extracted genome DNA as a template, adding the connecting probe, the fluorescent detection probe and an upstream primer and a downstream primer which have the same sequences as the upstream amplification primer fragment and are completely complementary to the sequence of the downstream amplification primer binding fragment respectively for connection, PCR amplification and melting curve continuous reaction, wherein the downstream primer is connected with a fluorescent group;
and detecting the fluorescent signal generated by the reaction system in real time by using a fluorescence monitoring instrument.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100297630A1 (en) * | 2007-07-11 | 2010-11-25 | Pathofinder B.V. | Method for the simultaneous detection of multiple nucleic acid sequences in a sample |
| CN102229999A (en) * | 2011-06-01 | 2011-11-02 | 亚能生物技术（深圳）有限公司 | Kit for identifying Mycobacterium tuberculosis and nontuberculous mycobacteria and application method thereof |
| CN102634575A (en) * | 2012-03-26 | 2012-08-15 | 中国人民解放军第三O九医院 | Rapid identification method and kit of novel mycobacterium strain |
| CN103290115A (en) * | 2013-05-16 | 2013-09-11 | 武汉缉熙生物科技有限公司 | Method for detecting serotyping of streptococcus pneumoniae |
| CN106520923A (en) * | 2016-10-11 | 2017-03-22 | 深圳市疾病预防控制中心 | Kit and method for simultaneously detecting Staphylococcus aureus and five enterotoxins thereof |
-
2018
- 2018-11-15 CN CN201811359784.1A patent/CN111187847A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100297630A1 (en) * | 2007-07-11 | 2010-11-25 | Pathofinder B.V. | Method for the simultaneous detection of multiple nucleic acid sequences in a sample |
| CN102229999A (en) * | 2011-06-01 | 2011-11-02 | 亚能生物技术（深圳）有限公司 | Kit for identifying Mycobacterium tuberculosis and nontuberculous mycobacteria and application method thereof |
| CN102634575A (en) * | 2012-03-26 | 2012-08-15 | 中国人民解放军第三O九医院 | Rapid identification method and kit of novel mycobacterium strain |
| CN103290115A (en) * | 2013-05-16 | 2013-09-11 | 武汉缉熙生物科技有限公司 | Method for detecting serotyping of streptococcus pneumoniae |
| CN106520923A (en) * | 2016-10-11 | 2017-03-22 | 深圳市疾病预防控制中心 | Kit and method for simultaneously detecting Staphylococcus aureus and five enterotoxins thereof |
Non-Patent Citations (2)
| Title |
|---|
| INDRA BERGVAL ET AL.: ""Combined Species Identification, Genotyping, and Drug Resistance Detection of Mycobacterium tuberculosis Cultures by MLPA on a Bead-Based Array"" * |
| LIJUAN WU ET AL.: ""Practical Prediction of Ten Common Streptococcus pneumoniae Serotypes/ Serogroups in One PCR Reaction by Multiplex Ligation-Dependent Probe Amplification and Melting Curve (MLPA-MC) Assay in Shenzhen, China"" * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115927686A (en) * | 2022-12-06 | 2023-04-07 | 潍坊医学院 | Primer probe composition for detecting mycobacterium tuberculosis and nontuberculous mycobacteria, kit and detection method |
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