CN110079620A - Identify three kinds of nocardial methods simultaneously using high-resolution melting curve method - Google Patents
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Abstract
The present invention provides a kind of methods for identifying Nocard's bacillus strain using high-resolution melting curve method, and the gene magnification primer for this method.Described method includes following steps: (1) using the DNA of sample to be detected as template, expand the sequence of intervals of Nocard's bacillus SecA1 gene, the sequence of intervals is the 181-451bp of Nocardia farcinica SecA1, the 182-452bp of the 181-451bp or nocardia asteroides SecA1 of Gelsenkirchen Nocard's bacillus SecA1；(2) using the high-resolution melting curve of the amplified production of high-resolution melting curve method obtaining step (1)；(3) interpretation is carried out to the melting curve that step (2) obtain.High-resolution melting curve method amplification condition provided by the invention is simple, rapid reaction, has excellent sensitivity and specificity, detection Nocardia farcinica, Gelsenkirchen Nocard's bacillus and nocardia asteroides that can be specific.
Description
Technical field
The present invention relates to high-resolution melting curves in Nocardia farcinica (Nocardia farcinica), Gai Ersen base
Emerging Nocard's bacillus (Nocardia cyriacigeorgica) and nocardia asteroides (Nocardia asteroides) identify
Application in detection, belongs to molecular biology and microbiological art.
Background technique
Nocardia (Nocardia) is widely present in natural environment, is long-term ignored opportunistic pathogenic bacteria,
Its Susceptible population is mainly immunocompromised and immune deficiency crowd.Clinically common Nocard's bacillus is mainly glanders promise card in China
Salmonella (Nocardia farcinica), Gelsenkirchen Nocard's bacillus (Nocardia cyriacigeorgica) and star promise
Cattell bacterium (Nocardia asteroides).These three Nocard's bacillus cause disease to account for about 50% of nocardiasis or more.It should
Pseudomonas mainly causes pulmonary abscess, can also cause brain abscess, dermapostasis or even whole body disseminated disease sometimes.Due to clinical condition
Shape is complicated, and different strain has different pathogenic characteristics and a susceptibility mode, and easily with tuberculosis, other bacterial infection diseases of lung
Disease and nosomycosis are obscured, and diagnosis and treatment difficulty is big, and easy delay treatment causes hospital mortality height.With AIDS patient, organ transplant
The status of patient increasing, clinical immunization inhibitor extensive use and aging of population, concurrent nocardial infection
Chance gradually increases.Therefore, it is badly in need of developing simple, quick, the sensitive and special detection technique of one kind for nocardial inspection
It surveys.
Traditional Nocard's bacillus identification method mainly pass through be separately cultured with biochemical identification method, generally require 5 days or more, and
Process is cumbersome, and accuracy rate is low.With the development of the molecular biological variety identification method based on nucleic acid amplification, some PCR methods
Applied to nocardial identification, such as utilize 16S rRNA, secA1 gene, hsp65 gene, rpoB gene and gyrA gene
Carry out Nocard's bacillus strain idenfication.Wherein secA1 gene has been found that Nocard's bacillus strain can be accurately distinguished, and can be used as identification promise
The reliable target gene of Cattell bacterium.Although nocardial method for identifying molecules is better than traditional isolated culture, regular-PCR method
It is dependent on gel electrophoresis interpretation as a result, time-consuming and laborious, and have a possibility that cross contamination；Conventional Real-time PCR method needs
Expensive probe is bought, testing cost is considerably increased, limits its application clinically.
High-resolution melting curve method (High-resolution melting, HRM) is a kind of poor based on mononucleotide
Different melting temperature is different and forms the genetic analysis new method of different shape melting curve, can carry out quickly, accurately, with high throughput
Strain idenfication.In addition to having many advantages, such as that low cost, sensitivity and specificity are high, HRM method is operated without gel, is truly realized and is closed
Pipe operation, entire detection process only need 2-3 hour.
Novel saturable dye (EvaGreen) is added in reaction system by HRM method before PCR reaction, which can be with
It is inserted into DNA double chain ditch, by real-time monitoring temperature-rise period double center chain DNA unwinding, fluorescent dye falls off, acquires and analyze and is glimmering
The process of change in optical signal can generate the melting curve of different shape according to fluorescence intensity and Tm value.It is bent according to different meltings
Line can distinguish single base difference, to distinguish different strain.
In view of being separately cultured, time-consuming, accuracy is low for the methods of biochemical identification, common molecular identification method testing cost
High, the problems such as limited resource is more, by HRM method be used for it is nocardial quickly detect it is very necessary.
Summary of the invention
The purpose of the invention is to make up the deficiencies in the prior art, for clinical common Nocard's bacillus, using high pass
Melting curve analysis method is measured, quick, high sensitivity and high specific detection of nucleic acids system is established, to carry out glanders promise card
Salmonella, Gelsenkirchen Nocard's bacillus and nocardia asteroides Rapid identification.
Based on foregoing invention purpose, identify promise Cattell using high-resolution melting curve method present invention firstly provides a kind of
The method of bacterium strain, the described method comprises the following steps:
(1) using the DNA of sample to be detected as template, the sequence of intervals of SecA1 gene is expanded；The sequence of intervals is glanders
The 181-451bp of Nocard's bacillus SecA1, the 181-451bp or nocardia asteroides of Gelsenkirchen Nocard's bacillus SecA1
The 182-452bp of SecA1；
(2) using the high-resolution melting curve of the amplified production of high-resolution melting curve method obtaining step (1)；
(3) interpretation is carried out to the melting curve that step (2) obtain.
In a preferred embodiment, the gene magnification is fluorescence quantitative PCR method amplification.
In a highly preferred embodiment, the upstream and downstream primer sequence of the PCR amplification is respectively SEQ ID
Shown in NO.1 and SEQ ID NO.2.
It is further preferable that the reaction condition of the PCR is, 95 DEG C of initial denaturation 10min, 95 DEG C of denaturation 15s, 62 DEG C are annealed
30s, 72 DEG C of extension 30s, 35 circulations, 1.6 DEG C/s of warming and cooling rate.
In another preferred embodiment, the reaction condition of the high-resolution melting curve method are as follows: 95 DEG C of 10s,
60 DEG C of 1min, 0.025 DEG C/s are warming up to 95 DEG C of 15s, 60 DEG C of 15s, with 1.6 DEG C/s rate heating and cooling.
It is further preferable that the fluorescent dye used in the high-resolution melting curve method is Evagreen, it is suitable for
Fluorescent dye of the invention further includes but is not limited to LCPLUS dyestuff, ResoLight dyestuff, 9 dyestuff of SYTO.
In still another preferred embodiment, the Nocard's bacillus kind includes Nocardia farcinica, Gelsenkirchen promise
Cattell bacterium and nocardia asteroides.
It is further preferable that the Tm value of Nocardia farcinica is 90.692 DEG C, Gai Ersen base in step (3) described interpretation
Emerging Nocard's bacillus Tm value is 90.098 DEG C, and nocardia asteroides Tm value is 90.233 DEG C.
Secondly, identifying gene magnification in Nocard's bacillus strain for high-resolution melting curve method the present invention provides one group
The nucleic acid molecule of primer, the sequence of the nucleic acid molecule are respectively shown in SEQ ID NO.1 and SEQ ID NO.2.
Identify promise using above-mentioned nucleic acid molecule and with high-resolution melting curve method finally, the present invention also provides a kind of
The method of Cattell bacterium strain.
The present invention can be used for Nocardia farcinica in clinical samples, Gelsenkirchen Nocard's bacillus and nocardia asteroides
Detection, provides technical support for disease surveillance.
Present invention application high-resolution melting curve analysis method is applied to three kinds of nocardial detections, compared to common
PCR and quantitative fluorescent PCR have the advantages that significant.Firstly, HRM method amplification condition provided by the invention is simple, rapid reaction,
It is detected using totally-enclosed reaction tube, without carrying out subsequent PCR product electrophoresis detection, avoids the cross contamination of sample and environment.The
Two, without designing specific probe, testing cost is reduced, is easy to carry out in laboratories.Third, it is full using Eva Green
And dyestuff, can be saturated in the double-strand ditch for being incorporated into PCR reaction product, will not suppression PCR reaction, tool fluorescence signal is stable, real
Test the features such as reproducible.
HRM method susceptibility provided by the invention is high, and standard peak shape is distinguished obviously, and the remolding sensitivity of amplified reaction is common
PCR method is 100 times high.Meanwhile amplification method provided by the invention has very high specificity.Under the conditions of standard reaction, with knot
28 kinds of common pathogenic bacteria such as core mycobacteria, streptococcus pneumonia, Legionella pneumonia, Pseudomonas aeruginosa, C. striatum and item
Part DNA of pathogenic is template, does not occur specific amplification using the detection architecture, illustrates that the specificity of the detection architecture is good,
Detection Nocardia farcinica, Gelsenkirchen Nocard's bacillus and nocardia asteroides that can be specific.
Detailed description of the invention
Tri- kinds of Fig. 1 nocardial high-resolution melt peak value and identify map (one)；
Tri- kinds of Fig. 2 nocardial high-resolution melt peak value and identify map (two)；
The sensitivity evaluation result figure of Fig. 3 high-resolution melting curve.
Specific embodiment
The invention will now be further described with reference to specific embodiments, the advantages and features of the present invention will be with description and
It is apparent.But examples are merely exemplary for these, does not constitute any restrictions to protection scope of the present invention.
Main agents used in the present invention:
DNA extraction kit (QIAamp DNA minikits) is purchased from German Qiagen company, the mixing of PCR reaction system
Object (2 × Taqman PCR MasterMix) is purchased from Thermo Fisher Scientific company, the U.S..Saturable dye
(Evagreen, 20 × in Water) is purchased from Biotium company, the U.S..Remaining reagent is conventional commercial reagent.
Key instrument used in the present invention:
NanoDrop ND-1000 spectrophotometric is calculated as U.S.'s Thermo Products.Fluorescence quantitative PCR instrument is ABI
QuantStudio 6flex。
Reference culture used in the present invention and clinical samples:
The reference culture of Nocardia farcinica, Gelsenkirchen Nocard's bacillus and nocardia asteroides is purchased from German microorganism
Culture Collection Center.Nocard's bacillus clinical separation strain collected from 2013 to 2018 Beijing, Sichuan, Chongqing, Shenzhen, Ningxia,
15 provinces and cities in the whole nation such as Guangxi, Hainan, Hebei, Hunan.Other bacterium sources are in Chinese Center for Disease Control and Prevention infectious disease
The preservation of prevention and control institute, specifying information are shown in Table 3.
HRM primer design:
Above-mentioned three kinds of Nocard's bacillus conserved sequence secA1 gene is chosen, downloads target-gene sequence in GenBank.It uses
Seqman in DNAStar software carries out homology analysis, intercepts concensus sequence, carries out primer using software CmSuite v8 and sets
Meter.Primer is evaluated to avoid the formation of primer dimer, while carrying out BLAST comparison via NCBI, verifies its specificity.
Primer sequence are as follows:
F:CGACGAGGTCGACTCCATCC (SEQ ID NO.1)；
R:GGCCTTGATGGCGTTGTTCAG (SEQ ID NO.2).
The foundation of embodiment 1:HRM method detection architecture
1. extracting genome: using the DNA extraction kit of Qiagen company, extracting Nocardia farcinica reference culture
IFM 10152, nocardia asteroides reference culture DSM 43757 and Gelsenkirchen Nocard's bacillus reference culture DSM 44484
Genome.DNA purity and concentration are detected using NanoDrop ND-1000 spectrophotometer.After genomic DNA dispenses on a small quantity,
- 20 DEG C are placed in save backup.
2. three kinds of Nocard's bacillus (Nocardia farcinica, nocardia asteroides and Gelsenkirchen Nocard's bacillus) SecA1 bases
Because of sequence, it is shown in Table 1.PCR amplification is carried out using above-mentioned primer, PCR product length is 271bp.
1 three kinds of Nocard's bacillus SecA1 Gene Partial sequences of table
3. reaction system: total system is 30 μ l, includes 2 × Mix Taqman PCR Master 15ul, 0.9 μ of primers F
L, 0.9 μ l of primer R (primer concentration is 10 μM), Rox Reference Dye II (100 ×) 0.3ul, Evagreen, 20
1.5 μ l of × in Water, 2 μ l of DNA profiling to be measured, 9.4 μ l of sterile deionized water.Negative control is with the mycobacterium tuberculosis of 2 μ l
Genome replaces nucleic acid as template, blank control with the aseptic deionized water of 2 μ l.
4. amplification condition: it is reacted on ABI QuantStudio 6flex, PCR response procedures such as table 2:
2 PCR response procedures of table
5. result interpretation: utilizing QuantStudioTMReal-Time PCR software carries out interpretation of result, bent based on melting
Line offset and curve shape variation judge Nocard's bacillus strain.
Embodiment 2:HRM detection architecture sensitivity evaluation
Using deionized water to three kinds of Nocard's bacillus (Nocardia farcinica, nocardia asteroides and Gelsenkirchen promise card
Salmonella) genomic DNA progress doubling dilution: 100pg/ μ l, 10pg/ μ l, 1 pg/ μ l, 100fg/ μ l, 10fg/ μ l.It is with 10 times
Arranging diluted three kinds of nocardial standard DNAs is template, and blank control is carried out with the aseptic deionized water of 2 μ l instead of nucleic acid
HRM is expanded and is analyzed its standard peak shape.The result shows that this method is to the Monitoring lower-cut of three kinds of Nocard's bacillus DNA profilings
100fg/ μ l, is shown in Fig. 3.
Embodiment 3:HRM detection architecture Evaluation on specificity
With common pathogenic bacteria and conditioned pathogen (mycobacterium tuberculosis, streptococcus pneumonia, Legionella pneumonia, green pus bar
Bacterium, C. striatum etc.) DNA be template, evaluate HRM reaction system specificity.HRM method detect 28 kinds of common pathogens and
Do not occur specific amplification when opportunist, shows that the specificity of the detection architecture is good, strain background is shown in Table 3.
To be located away from the 24 plants of Nocardia farcinicas, 19 plants of Gelsenkirchen Nocard's bacillus and 10 plants of stars of 15 provinces and cities, China
Shape Nocard's bacillus clinical separation strain DNA is template, identifies three kinds of Nocard's bacillus, detection knot using high-resolution melting curve method
Fruit is consistent with traditional isolated culture and 16S rRNA identification method, shows that the method for the present invention can be effectively used to three kinds of promise cards
The differentiation of Salmonella is identified.The Tm value of Nocardia farcinica is 90.692 DEG C as shown in Figure 1:；Gelsenkirchen Nocard's bacillus Tm value
It is 90.098 DEG C；Nocardia asteroides Tm value is 90.233 DEG C.In order to become apparent from the differentiation of three strains, with Gelsenkirchen
Nocard's bacillus is baseline, constructs high-resolution melting curve disparity map (as shown in Figure 2), can obviously distinguish three strains.
Strain background information of the table 3 for detection
Note: DSM: Germany Microbiological Culture Collection Center
ICDC: Inst of Infection Disease Prevention and Control, Chinese Diseases Prevention an.
Sequence table
<110>Inst of Infection Disease Prevention and Control, Chinese Diseases Prevention an
<120>identify three kinds of nocardial methods simultaneously using high-resolution melting curve method
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213> Nocardia aerocolonigenes
<400> 1
cgacgaggtc gactccatcc 20
<210> 2
<211> 21
<212> DNA
<213> Nocardia aerocolonigenes
<400> 2
ggccttgatg gcgttgttca g 21
Claims (10)
1. a kind of method for being identified Nocard's bacillus strain using high-resolution melting curve method, the described method comprises the following steps:
(1) using the DNA of sample to be detected as template, the sequence of intervals of Nocard's bacillus SecA1 gene is expanded, the sequence of intervals is
The 181-451bp or star promise card of the 181-451bp of Nocardia farcinica SecA1, Gelsenkirchen Nocard's bacillus SecA1
The 182-452bp of Salmonella SecA1；
(2) using the high-resolution melting curve of the amplified production of high-resolution melting curve method obtaining step (1)；
(3) interpretation is carried out to the melting curve that step (2) obtain.
2. the method according to claim 1, wherein the gene magnification is fluorescent quantitative PCR.
3. according to the method described in claim 2, it is characterized in that, the upstream and downstream primer sequence of the PCR amplification is respectively SEQ
Shown in ID NO.1 and SEQ ID NO.2.
4. according to the method described in claim 3, it is characterized in that, the reaction condition of the PCR are as follows: 95 DEG C of initial denaturation 10min,
95 DEG C of denaturation 15s, 62 DEG C of annealing 30s, 72 DEG C of extension 30s, 35 recycle, 1.6 DEG C/s of warming and cooling rate.
5. the method according to claim 1, wherein the reaction condition of the high-resolution melting curve method are as follows:
95 DEG C of 10s, 60 DEG C of 1min, 0.025 DEG C/s are warming up to 95 DEG C of 15s, 60 DEG C of 15s, with 1.6 DEG C/s rate heating and cooling.
6. according to the method described in claim 5, it is characterized in that, the fluorescence used in the high-resolution melting curve method
Dyestuff is Evagreen.
7. the method according to claim 1, wherein the Nocard's bacillus kind includes Nocardia farcinica, Gai Er
Sen Jixing Nocard's bacillus and nocardia asteroides.
8. the method according to the description of claim 7 is characterized in that in step (3) described interpretation, the Tm of Nocardia farcinica
Value is 90.692 DEG C, and Gelsenkirchen Nocard's bacillus Tm value is 90.098 DEG C, and nocardia asteroides Tm value is 90.233 DEG C.
9. one group of nucleic acid molecule for gene magnification primer in high-resolution melting curve method identification Nocard's bacillus strain,
It is characterized in that, the sequence of the nucleic acid molecule is respectively shown in SEQ ID NO.1 and SEQ ID NO.2.
10. a kind of identify Nocard's bacillus strain using nucleic acid molecule described in claim 9 and with high-resolution melting curve method
Detection method.
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| CN110408720A (en) * | 2019-08-07 | 2019-11-05 | 中国医学科学院北京协和医院 | A kind of method of high-resolution melting curve identification ear candida albicans |
| CN110512013A (en) * | 2019-09-04 | 2019-11-29 | 中国疾病预防控制中心传染病预防控制所 | A method of identifying three kinds of corynebacterias using high-resolution melting curve method |
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| CN110408720A (en) * | 2019-08-07 | 2019-11-05 | 中国医学科学院北京协和医院 | A kind of method of high-resolution melting curve identification ear candida albicans |
| CN110512013A (en) * | 2019-09-04 | 2019-11-29 | 中国疾病预防控制中心传染病预防控制所 | A method of identifying three kinds of corynebacterias using high-resolution melting curve method |
| CN110512013B (en) * | 2019-09-04 | 2022-07-08 | 中国疾病预防控制中心传染病预防控制所 | Method for identifying three corynebacteria by using high-resolution melting curve method |
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Application publication date: 20190802 |