CN116676405A - Primer group for detecting nocardia bacteria and application thereof - Google Patents
Primer group for detecting nocardia bacteria and application thereof Download PDFInfo
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Abstract
The invention relates to a primer group for detecting nocardia bacteria and application thereof. The primer set for detecting nocardia bacteria includes: an upstream primer F shown as SEQ ID NO. 1, a downstream primer R shown as SEQ ID NO. 2 and a probe P shown as SEQ ID NO. 3. The primer group can be used for rapidly and efficiently detecting 12 nocardia of nocardia with high sensitivity and high specificity, so that the detection range of nocardia bacteria is greatly improved, and compared with the traditional separation culture technology, the detection efficiency is improved.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a primer group for detecting nocardia bacteria and application thereof.
Background
Nocardia (Nocardia) is a class of actinomycetes widely present in air, soil, and sea water. Nocardia is an acute or chronic suppurative or granulomatous lesion, wherein the crowd with low immunity is most susceptible to infection, and nocardia infection parts mainly occur in the lung, skin soft tissues, brain and the like of the human body. Because the clinical manifestation of the nocardia pneumoconiosis patient is not specific, the misdiagnosis of tuberculosis, fungal infection, bacterial abscess and other infectious diseases is easy, so that the optimal treatment time is shortened, and the death rate is improved. With the wide use of clinical immunosuppressants and the increasing number of immunodeficient patients such as organ transplantation, aids, and systemic lupus erythematosus, there is a need to develop a simple and rapid detection technique for assisting clinical diagnosis of nocardia.
According to domestic and foreign reports, nocardia pathogenic to human is found to be mainly: nocardia stellate (n.asteroides), nocardia guinea pig (n.otitidissaviarum), nocardia bassinesis (n.brasiliensis), nocardia melitensis (n.farcinica), and the like. However, the conventional identification methods, such as isolated culture and phenotype identification, generally need 5-7 days in period, and the operation process is complicated, so that the disease is easily delayed, and the current clinical diagnosis requirements are difficult to meet. With the development of molecular biology identification technology, there are some PCR methods for identifying nocardia, such as nested PCR and multiplex PCR, but they rely on gel electrophoresis to interpret results, and there is a possibility of cross contamination. However, the real-time fluorescence quantitative PCR detection method has the advantages of high speed, high efficiency, high flux, high sensitivity and specificity, and has important significance in assisting the clinical diagnosis of nocardia.
Disclosure of Invention
In order to solve the problems, the invention provides a primer group for detecting nocardia bacteria, which can rapidly and efficiently detect 12 nocardia bacteria of nocardia with high sensitivity and high specificity, greatly improves the detection range of nocardia bacteria, and improves the detection efficiency compared with the traditional separation culture technology.
In order to achieve the above object, the present invention provides a primer set for detecting nocardia bacteria, comprising:
upstream primer F: AGCGAACAGGATTAGATACC (SEQ ID NO: 1);
the downstream primer R: TACACCGACCACAAGGGG (SEQ ID NO: 2);
probe P: TTCCTTCCACGGGATC (SEQ ID NO: 3).
Considering that a plurality of kinds of nocardia are detected by amplification of a pair of primers, and considering that the 16S rRNA gene is very similar to the 16S rRNA gene of some pathogenic bacteria in Mycobacterium genus, and ensuring that no false positive or false negative occurs, the inventors have found out through a large number of experiments that the above primer set is finally obtained. The upstream primer, the downstream primer and the probe in the primer set are designed for the conserved region of the 16S rRNA genes of 12 nocardia, and the nocardia bacteria can be detected rapidly, efficiently and highly sensitively by adopting the upstream primer and the downstream primer, and the detection range of the nocardia bacteria is greatly improved by covering the detection of 12 nocardia bacteria; the probe is matched with the upstream and downstream primers, so that nocardia bacteria can be detected with high specificity, and are distinguished from mycobacteria bacteria, and false positive results are avoided; the primer group is used for detecting nocardia bacteria, so that detection can be completed in a short time, and compared with the traditional separation culture technology which requires more than 3 days, the detection efficiency is improved.
In one embodiment, the probe P uses FAM as a fluorescent group and MGB as a fluorescence quenching group.
In one embodiment, the nocardia bacteria include: at least 1 of nocardia brasiliensis, nocardia melitensis, nocardia caviae, nocardia guinea pig otitis, nocardia arthritides, nocardia pseudobaziensis, nocardia grace, nocardia guerbensis, nocardia abscessus, nocardia terpene, nocardia tenuifolia, and nocardia waldens.
The primer group provided by the invention can cover detection of 12 nocardia, including nocardia bassiana (N.brasiliensis), nocardia melitensis (N.farcinica), nocardia sinica (N.concava), nocardia guinea-pig N.ototidis cavium, nocardia arthritides (N.arthridis), nocardia pseudobassiana (N.pseudorasiliensis), nocardia grace (N.elegans), nocardia guerbensis (N.cyriaceica), nocardia abscess (N.abscessus), nocardia terpene (N.terntica), nocardia tennesis (N.amamiensis), and nocardia warriopsis (N.wallaceae). The detection range of nocardia bacteria is greatly improved, and meanwhile, compared with the traditional separation culture technology, the detection efficiency is improved, and the detection range of nocardia bacteria is greatly improved.
The invention also provides application of the primer group in preparation of a detection reagent for nocardia bacteria.
The invention also provides a kit for detecting nocardia bacteria, which comprises the primer group.
In one embodiment, the kit further comprises: premix, ddH 2 O, and a reference dye.
The invention also provides a method for detecting nocardia bacteria with non-diagnostic purposes, which comprises the following steps: extracting nucleic acid of a sample to be detected, and carrying out PCR reaction by adopting the kit.
In one embodiment, the conditions of the PCR reaction are: pollution digestion for 2min at 37 ℃; pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 15sec;55℃extends for 45sec,40 cycles.
By adopting the temperature in the extension/annealing step, compared with the conventional 60 ℃, the CT value in certain nocardia can be improved, and the false negative result can be reduced for the sample to be tested with low loading.
In one embodiment, the PCR reaction collects fluorescent signals at 55℃for 45sec.
Compared with the prior art, the invention has the following beneficial effects:
the primer group for detecting nocardia bacteria and the application thereof can rapidly and efficiently detect nocardia bacteria with high sensitivity and high specificity, greatly improve the detection range of nocardia bacteria, and improve the detection efficiency compared with the traditional separation culture technology. The primer group only needs to design a pair of specific upstream and downstream primers and a specific probe aiming at 12 nocardia, so that the cost is greatly saved, the detection time is short, the detection can be completed within about 80 minutes, and the clinical diagnosis efficiency is accelerated.
Drawings
FIG. 1 is a diagram showing the result of DNAMAN sequence comparison summarized in the probe determination process in the embodiment, wherein the highlighted area in the middle of the diagram is the probe sequence of the invention.
Detailed Description
In order that the invention may be readily understood, a more complete description of the invention will be rendered by reference to the appended 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 herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
Definition:
reference dye: refers to a solution for normalizing fluorescent reporter gene signals in real-time quantitative PCR or RT-PCR.
The reagents, materials and equipment used in the examples are all commercially available sources unless otherwise specified; the test methods are conventional in the art unless otherwise specified.
Examples
A kit for detecting nocardia bacteria and a detection method thereof.
1. Materials and methods.
1. And (5) experimental samples.
In this example, a positive clinical sample of nocardia was detected from the metagenome of the pathogenic microorganism of this unit.
2. And (3) an instrument.
The detection is carried out by using an ABI7500 fluorescent quantitative PCR instrument, and is a product of Siemens Feishul technology.
3. And (3) designing and synthesizing a primer.
(1) The pathogen gene reference sequences were downloaded in the NCBI (National Center for Biotechnology Information ) website as follows: nocardia brazii, nocardia melitensis, nocardia caviae, nocardia guinea pig otitis, nocardia arthritides, nocardia pseudobaziensis, nocardia grace, nocardia guerbensis, nocardia abscessus, nocardia terpene, nocardia tenuifolia, nocardia waldens 16S rRNA genes; and simultaneously downloading genes of Mycobacterium tuberculosis complex, legionella pneumophila, mycobacterium abscess, mycobacterium fortuitum, mycobacterium Qimei, mycobacterium malassa, haemophilus influenzae, klebsiella pneumoniae and cytomegalovirus.
(2) The nucleotide sequences are subjected to sequence alignment by using DNAMAN software, primers and probes are designed by searching for a more conserved sequence of nocardia bacteria, and the primers and probes are designed by using Primer Premier 5 software, and the following conditions are required to be met:
(1) tm value: the Tm value of the probe is 8-10 ℃ higher than that of the primer, wherein the Tm value of the probe is about 65 ℃;
(2) GC content: between 40% and 70%;
(3) the absence of primer dimer and hairpin structure;
(4) the amplified fragment size is typically less than 200bp.
(3) Based on the recommended primer of the software, after manual screening, the primer pair is subjected to specific detection at NCBI blast interface, and the condition that the primer pair does not match with the human genome sequence is taken as the reference.
(4) The primer pairs determined preliminarily were synthesized by Beijing qing Biotech Co., ltd.
TABLE 1 preliminary screening primers
2. Experimental procedure and results analysis.
1. Preparation of sample nucleic acids.
Taking 400 mu L of experimental sample, and extracting the nucleic acid according to the specification of a general DNA/RNA extraction kit for the environmental sample of the North Ammonia Biotechnology Co., ltd.
2. Preference of the primer.
The specificity of the designed primers was detected using SYBR Green dye reagent purchased from Nanjinouzan Biotechnology Co., ltd.
3. SYBR Green dye method amplification system.
TABLE 2SYBR Green dye method amplification System
4. SYBR Green dye method amplification procedure.
PCR procedure: 30sec (1 cycle) at 95 ℃;95℃10sec,60℃30sec (40 cycles). Dissolution profile procedure: 95℃15sec,60℃60sec,95℃15sec. The invention uses ABI7500 for detection.
TABLE 3 preferred results for primers
According to the detection conditions of different nocardia, finally, a primer pair 4 is selected:
16S rRNA-4-F：AGCGAACAGGATTAGATACC(SEQ ID NO:1)；
16S rRNA-4-R：TACACCGACCACAAGGGG(SEQ ID NO:2)。
5. and (5) determining a probe.
According to the primer set 4 preferably obtained, the inventors found that there was also amplification of a sample of Mycobacterium bacteria by SYBR Green dye method, so that ordinary dye method cannot distinguish Nocardia bacteria from Mycobacterium bacteria by simply relying on primers, and therefore the inventors designed MGB probes based on the sequences of Nocardia bacteria and Mycobacterium bacteria. The inventor selects a section of very conservative area of nocardia bacteria through research and experimental screening, finds a section with 2 bases different than mycobacteria bacteria, designs a plurality of probes for screening according to the principle of primer design, and finally determines the sequence of the probes. The DNAMAN sequence alignment result is shown in fig. 1, and the middle highlight region is the probe sequence of the present invention, specifically: TTCCTTCCACGGGATC (SEQ ID NO: 3).
6. The probe method is used for amplifying the system.
2 XAceQ U+Probe Master Mix and 50X Rox Reference Dye1 (available from Nanjinozan Biotechnology Co., ltd., product No. Q113-02/03) were prepared according to the following reaction system.
TABLE 4 Probe method amplification System
In this example, the premix is AceQ U+Probe Master Mix, and the DNA in the above table refers to the DNA extracted from the sample (respiratory tract sample, etc.) of the patient.
7. Probe method amplification procedure.
2min (1 cycle) at 37 ℃;95℃for 5min (1 cycle); 95℃15sec,55℃45sec (40 cycles). In the parameter setting of the fluorescent quantitative PCR instrument, ROX is taken as Passive reference, a reporting group (namely a fluorescent group) is provided with FAM, a fluorescent quenching group is provided with MGB, and a probe P (Nocardia-P) connected with the reporting group and the fluorescent quenching group is provided with: 5'FAM-TTCCTTCCACGGGATC-3' MGB; the quencher fluorescent signal was collected at 55℃for 45sec. The invention uses ABI7500 for detection.
8. And judging the result.
When the amplification curve in the FAM fluorescent channel is S-shaped and Ct is less than or equal to 38, judging that the sample is nocardia positive;
and when Ct is more than 38 or amplification is not carried out in the FAM fluorescence channel, judging that the sample is nocardia negative.
9. And (5) analyzing results.
(1) And (5) verifying correctness.
By adopting the primer pair 4 and the probe which are preferred by the invention and the amplification program by the probe method, 20 positive samples which are clinically diagnosed as nocardia infection are detected, the results are positive, and the positive coincidence rate is 100 percent.
Table 5 correctness checking table
| Numbering device | Macrogene detection results | qPCR detection result (Ct value) |
| Clinical positive sample 1 | Nocardia caving | 29.80 |
| Clinical positive sample 2 | Gelson-Xinghuoka bacterium | 33.69 |
| Clinical positive sample 3 | Nocardia of guinea pig ear inflammation | 23.78 |
| Clinical positive sample 4 | Tianminobac | 36.95 |
| Clinical positive sample 5 | Nocardia set for treating melitema | 23.00 |
| Clinical positive sample 6 | Nocardia brazil | 32.10 |
| Clinical positive sample 7 | North China Ka fungus | 37.34 |
| Clinical positive sample 8 | Pseudobacinacalcet bacteria | 33.06 |
| Clinical positive sample 9 | Nocardia arthritic | 33.49 |
| Clinical Positive sample 10 | Nocardia grace | 37.21 |
| Clinical positive sample 11 | Gelson-Xinghuoka bacterium | 35.38 |
| Clinical positive sample 12 | Nocardia abscess | 20.93 |
| Clinical positive sample 13 | Nocardia set for treating melitema | 21.61 |
| Clinical positive sample 14 | Nocardia terpene | 26.63 |
| Clinical positive sample 15 | Nocardia of guinea pig ear inflammation | 30.76 |
| Clinical positive sample 16 | Nocardia of guinea pig ear inflammation | 29.88 |
| Clinical positive sample 17 | Nocardia terpene | 31.78 |
| Clinical positive sample 18 | Nocardia terpene | 36.40 |
| Clinical positive sample 19 | Nocardia abscess | 28.85 |
| Clinical positive sample 20 | Nocardia grace | 37.92 |
(2) And (5) specificity verification.
10 positive samples which are clinically diagnosed as other infections are detected, 10 pathogenic bacteria such as mycobacterium tuberculosis, legionella pneumophila, haemophilus influenzae and Klebsiella pneumoniae are detected by adopting the preferable primer pair 4 and the probe and the amplification program by the probe method, and the detection results are negative and the coincidence rate is 100%.
TABLE 6 specificity verification Table
| Numbering device | Macrogene detection results | qPCR detection result (Ct value) |
| Clinical positive sample 21 | Mycobacterium tuberculosis complex | Negative of |
| Clinical positive sample 22 | Legionella pneumophila (L.) Mey | Negative of |
| Clinical positive sample 23 | Mycobacterium abscessum | Negative of |
| Clinical positive sample 24 | Mycobacterium fortuitum | Negative of |
| Clinical positive sample 25 | Mycobacterium zimeriae | Negative of |
| Clinical positive sample 26 | Mycobacterium malase | Negative of |
| Clinical positive sample 27 | Haemophilus influenzae | Negative of |
| Clinical positive sample 28 | Klebsiella pneumoniae | Negative of |
| Clinical positive sample 29 | Cytomegalovirus | Negative of |
| Clinical positive sample 30 | Cytomegalovirus | Negative of |
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (9)
1. A primer set for detecting nocardia bacteria, comprising:
upstream primer F: AGCGAACAGGATTAGATACC (SEQ ID NO: 1);
the downstream primer R: TACACCGACCACAAGGGG (SEQ ID NO: 2);
probe P: TTCCTTCCACGGGATC (SEQ ID NO: 3).
2. The primer set of claim 1, wherein the probe P employs FAM as a fluorescent group and MGB as a fluorescence quenching group.
3. The primer set according to claim 1-2, wherein the nocardia bacterium comprises: at least 1 of nocardia brasiliensis, nocardia melitensis, nocardia caviae, nocardia guinea pig otitis, nocardia arthritides, nocardia pseudobaziensis, nocardia grace, nocardia guerbensis, nocardia abscessus, nocardia terpene, nocardia tenuifolia, and nocardia waldens.
4. Use of a primer set according to any one of claims 1 to 3 for the preparation of a reagent for nocardia bacteria detection.
5. A kit for detecting nocardia bacteria, comprising the primer set of any one of claims 1-3.
6. The kit of claim 5, further comprising: premix, ddH 2 O, and a reference dye.
7. A method for detecting nocardia bacteria for non-diagnostic purposes, comprising the steps of: extracting nucleic acid of a sample to be tested, and performing PCR reaction by using the kit according to any one of claims 5 to 6.
8. The method according to claim 7, wherein the PCR reaction conditions are: pollution digestion for 2min at 37 ℃; pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 15sec;55℃extends for 45sec,40 cycles.
9. The method of claim 8, wherein the PCR reaction collects fluorescent signals at 55 ℃ for 45sec.
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| CN202310752269.4A CN116676405A (en) | 2023-06-25 | 2023-06-25 | Primer group for detecting nocardia bacteria and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310752269.4A CN116676405A (en) | 2023-06-25 | 2023-06-25 | Primer group for detecting nocardia bacteria and application thereof |
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| CN116676405A true CN116676405A (en) | 2023-09-01 |
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| CN202310752269.4A Pending CN116676405A (en) | 2023-06-25 | 2023-06-25 | Primer group for detecting nocardia bacteria and application thereof |
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| CN (1) | CN116676405A (en) |
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