CN112195225A - Method for detecting methicillin-resistant staphylococcus based on drug-resistant gene MecA - Google Patents
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a method for detecting methicillin-resistant staphylococcus based on a drug-resistant gene MecA. The detection method of methicillin-resistant staphylococcus comprises the following steps: s1: designing a primer and a probe; s2: extracting genome DNA in a specimen; s3: detecting by real-time fluorescence PCR; the primers comprise an upstream primer and a downstream primer, and the nucleotide sequences are respectively shown as SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The novel fluorescence PCR method for effectively detecting MRS, which is established by the invention, has higher sensitivity, specificity and repeatability, and the method has economy and can realize the clinical accurate requirement on MRS detection.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for detecting methicillin-resistant staphylococcus based on a drug-resistant gene MecA.
Background
Coagulase Negative Staphylococci (CNS), such as staphylococcus epidermidis, staphylococcus hominis, staphylococcus capitis and the like, belong to the normal flora of human bodies, and can exist on the body surfaces and cavities of human bodies, and generally cause less infection. However, when the organism's immunity is low, CNS flora translocates into abnormal sites, which can become infectious pathogenic bacteria. Because of the wide application of various high-grade antibiotics and the increase of clinical interventional diagnosis and treatment operations, CNS has become an important pathogenic bacterium of nosocomial infection and can cause various invasive infections[1-2]. Its multiple drug resistance brings difficulty to clinical treatment and is also receiving more and more attention from us.
In recent years, the rate of isolation of methicillin-resistant coagulase-negative staphylococci (MRCNS) has continued to increase, and has become a global public health problem. According to the statistical analysis of the national bacterial drug resistance monitoring network (CARSS), the national average detection rate of MRCNS in 2018 is 75.7%, and the national average detection rate of MRCNS reaches 78.2%. The CNS 591 strains were detected in 2019 years in affiliated hospital of Nantong university, wherein the MRCNS separation rate was 72.3%. Methicillin-resistant staphylococcus (MRS) resistance is mainly due to the MecA gene encoding a low affinity penicillin binding protein, resulting in resistance to all drugs of the beta-lactams, beta-lactamase inhibitor complexes, cephalosporins (except ceftaroline) and carbapenems type. Staphylococci carrying mecA gene often carry drug resistance genes linked to other drugs, thus resulting in high resistance rates to MRS. Therefore, the clinical drug resistance and the related drug resistance mechanism of MRS are highly regarded as important points for the drug resistance monitoring network of CARSS and jiangsu province.
The correct control of MRS identification and drug resistance analysis technology has important significance for clinical drug resistance, and can provide accurate data for national bacterial drug resistance monitoring. The MRS conventional detection method comprises a phenotype detection method, a gene detection method and a protein detection method. At present, the clinical microbiology laboratory mostly adopts a cefoxitin paper diffusion method or a oxacillin minimum inhibitory concentration method recommended by the American Clinical Laboratory Standards Institute (CLSI). Naccache et al found that under appropriate conditions, e.g., selectionUnder the conditions of proper antibiotics, culture temperature, bacterial liquid concentration and the like, the MecA gene detected by using a phenotype method for detecting MRSA and a gold standard gene method has higher coincidence rate. However, for CNS, the phenotypical method for MRS detection is falsely enhanced[3]. Therefore, clinical microbiological laboratories need to review this data to ensure the accuracy of the test.
As early as the nineties of the 20 th century, PCR technology was used by a number of scholars to detect the mec A gene. Usually, a primer or a probe can be designed according to a target sequence of the mec A gene, then, the genome DNA of the detection bacteria is extracted, the amplification conditions are searched and optimized, and the products are observed by agarose gel electrophoresis after PCR amplification. PCR has high sensitivity and is often used as a 'gold standard' for MRS detection. With the development of molecular biology technology, researchers are continuously improving the PCR method for detecting mecA gene, such as multiple PCR, real-time fluorescence quantitative PCR and the like for detecting drug-resistant gene of isolate, and the prior art can directly detect mecA gene by using clinical specimen[4-10]. The PCR detection technology is extremely sensitive, false positive can occur in the experimental process, and in order to ensure that the PCR has higher reliability, a laboratory needs to perform probe hybridization or sequencing on an amplification product when establishing a detection method so as to improve the specificity of the method. Meanwhile, under some special conditions, the fact that some mec A genes do not express products and belong to silent genes is also needed to be noticed, and a pseudo-drug resistance conclusion can be obtained. Therefore, in the work of a clinical microbiological laboratory, the PCR technology can be combined with a phenotype detection method, a protein detection method and the like according to laboratory conditions and hospital influenza flow monitoring data, so that the MRS detection accuracy is improved[11]. However, how to establish a fluorescence PCR method for effectively detecting MRS and to perform clinical evaluation on the sensitivity, specificity and repeatability of MRS is a problem which is continuously overcome by the technical personnel in the field.
Disclosure of Invention
The invention aims to design a primer by taking a MecA gene as a target sequence, establish a fluorescence PCR method for effectively detecting MRS by using a probe technology, and carry out clinical evaluation on sensitivity, specificity and repeatability to obtain a method with higher sensitivity, specificity and economy, thereby meeting the clinical accurate requirement on MRS detection.
Specifically, the technical scheme of the invention is as follows:
the invention discloses a method for detecting methicillin-resistant staphylococcus (MRS), which comprises the following steps:
s1: designing a primer and a probe;
s2: extracting genome DNA in a specimen;
s3: detecting by real-time fluorescence PCR;
the primers comprise an upstream primer and a downstream primer, and the nucleotide sequences are respectively shown as SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
It should be understood that the method of the present invention is not limited to the above steps, and those skilled in the art may add other additional steps before S1, between S1 and S2, between S2 and S3, and after S3, as needed, and all of which are within the protection scope of the present invention.
It is understood that, in comparison to SEQ ID NO: 1 and SEQ ID NO: 2 sequences have more than 90% (e.g., 92%, 95%, 98%, etc.) homology, and functionally identical sequences are within the scope of the invention.
Preferably, the nucleotide sequence of the probe is as shown in SEQ ID NO: 3 is shown in the specification; preferably, the probe is a TaqMan fluorescent probe.
It is understood that, in comparison to SEQ ID NO: 3 sequences have more than 90% (e.g., 92%, 95%, 98%, etc.) homology, and functionally identical sequences are within the scope of the invention.
Preferably, the probe is labeled with a fluorescence reporter FAM at the 5 'end and a fluorescence quencher TAMRA at the 3' end.
Preferably, the primer concentration is 0.25 to 1 μ M/L; more preferably, the primer concentration is 0.5. mu.M/L.
Preferably, the probe concentration is 100-300 nmol/L; more preferably, the probe concentration is 200 nmol/L.
Preferably, in S3, the amount of Taq enzyme used in the PCR system is 5.0U Taq enzyme.
Preferably, in S3, the annealing temperature in the PCR system is 55 ℃ and the annealing time is 30S.
Preferably, in S3, the PCR reaction conditions: sequentially performing pre-denaturation at 94 ℃ for 5 min; performing 35 cycles of amplification; extension at 72 ℃ for 7 minutes;
wherein the cycling amplification conditions are: denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 90 s.
In a second aspect, the invention discloses the use of the above method in clinical bacterial species detection.
Preferably, the bacterial species is methicillin-resistant coagulase-negative staphylococci (MRCNS).
On the basis of the common general knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily without departing from the concept and the protection scope of the invention.
Compared with the prior art, the invention has the following remarkable advantages and effects:
according to the invention, a novel fluorescent PCR method for effectively detecting MRS is established by using the MecA gene as a target sequence and applying a probe technology, and clinical evaluation is carried out on the sensitivity, specificity and repeatability of the fluorescent PCR method, so that the method is proved to have higher sensitivity, specificity and repeatability, and the method is economical and can realize the clinical accurate requirement on MRS detection.
Drawings
FIG. 1 is an agarose gel electrophoresis image of a PCR product according to an example of the present invention.
FIG. 2 is a diagram of bidirectional sequencing of PCR products (BioEdit Sequence Alignment Editor software analysis of fragmented sections) in an embodiment of the present invention.
FIG. 3 is a graph showing PCR amplification of MecA gene in the examples of the present invention.
FIG. 4 is a PCR lysis curve of MecA gene in the example of the present invention.
FIG. 5 is a diagram showing the results of the sensitivity of real-time fluorescent PCR detection of MecA gene in the example of the present invention.
Detailed Description
The technical solutions of the present invention are described in detail below with reference to the drawings and the embodiments, but the present invention is not limited to the scope of the embodiments.
The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions. The reagents and starting materials used in the present invention are commercially available.
Example 1
The design idea of the embodiment is as follows:
firstly, synthesizing a primer and a probe according to a MecA gene sequence and a probe design principle, and establishing a real-time fluorescent quantitative PCR method for effectively detecting MRS strains;
secondly, optimizing the established PCR method (a PCR reaction system and reaction conditions);
and thirdly, carrying out clinical application evaluation (sensitivity and repeatability tests) on the established method.
Specifically, the technical scheme of the invention is as follows:
1. design of primers and probes
Searching a MecA gene sequence from Genebank, analyzing the sequence by using MegAlign software, designing primers and probes (shown in table 1), wherein 5 'ends of TaqMan fluorescent probes are both marked with a fluorescent reporter group FAM, and 3' ends of the TaqMan fluorescent probes are both marked with a fluorescent quencher group TAMRA. Primers and probes were synthesized by Invitrogen corporation.
TABLE 1 primer and Probe sequences
2. Extraction of genomic DNA
Total 40 MRCNS clinical isolates separated from various specimens of the subsidiary hospital of Nantong university in 2018 were collected, ATCC 25923 was set as a negative control, and ATCC 43300 was set as a positive control. Taking activated isolated strain 4 region, streaking, inoculating on blood plate, supplementing 5% CO at 35 deg.C2After 24 hours of the atmospheric culture, colonies were collected, and genomic DNA was extracted according to the instructions of the bacterial genomic DNA extraction kit of Omega Bio-Tek.
3. Real-time fluorescent PCR detection
And (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 5min, followed by 35 cycles of amplification (denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 90s), followed by extension at 72 ℃ for 7 min, ABI 7500 collecting fluorescence at each cycle annealing temperature, and data was analyzed by the instrument-mounted software after the run procedure was completed.
4. Optimization of PCR systems and conditions
4.1 primer concentration
In the PCR reaction system, the primer is the key of the PCR specific reaction. The yield of PCR amplification is reduced due to the fact that the concentration of the primer is too low; whereas, when the concentration of the primer is too high, a mismatch is induced, and thus non-specific amplification occurs. Therefore, 4 concentrations of 0.25, 0.5, 0.75 and 1.0. mu.M/L were set for optimization, and the concentrations of the upstream and downstream primers were identical.
4.2 concentration of fluorescent Probe
The detection sensitivity can be obviously improved by proper probe concentration, and the research sets 4 concentrations of 100, 150, 200 and 300nmol/L for optimization.
4.3 amount of Taq enzyme
The PCR amplification efficiency can be improved by properly increasing the amount of Taq enzyme, but the generation of non-specific bands can be caused by too high amount. The amplification efficiencies were compared at 1.0, 2.0, 2.5 and 5.0U/system, and the optimum concentration was selected.
4.4 PCR annealing temperature
And after denaturation, rapidly cooling to 40-60 ℃ to enable the primer and the template to be combined. The annealing temperature generally depends on the length of the primer, the base composition and its concentration, and the length of the target base sequence. Annealing temperature is a major factor affecting the specificity of PCR. Therefore, the annealing temperature was selected in the vicinity of the Tm value, and the experiments were carried out while setting 4 temperatures of 56, 55, 54 and 53 ℃.
4.5 PCR annealing time
The annealing time has a great influence on the stability of the PCR product, so that 4 times of 10 s, 20 s, 30s and 40s are selected for comparison to determine the optimal annealing time.
5. Evaluation of the established fluorescent quantitative PCR method
5.1 sensitivity test
MecA gene recombinant plasmid is constructed by taking ATCC 43300 genome DNA as a template, diluted by TE multiple ratio and prepared into 10-concentration3-107copying/mL reference substance, taking 3 mu L as template, PCR amplifying, detecting sensitivity of fluorescent quantitative PCR
5.2 repeatability test
Selection of 1X 105And 1X 107And performing PCR amplification on the copied/mL two reference substances, measuring 10 tubes of each reference substance for 3 times, and calculating the coefficient of variation according to the Ct value.
6. Clinical sample testing
PCR amplification was performed on 40 collected clinical isolates to detect the MecA gene. Positive, negative and no template controls were set for each experiment.
7. Evaluation of results
7.1 optimization of PCR systems and conditions
When the concentration of the primer is 0.25. mu.M/L, the amplification efficiency is deteriorated, and when the concentration of the primer is 0.5. mu.M/L, the amplification efficiency is stable and there is no non-specific amplification; when the concentration of the TaqMan probe is 200nmol/L, the PCR amplification fluorescence intensity value is the highest, and the Ct value is smaller; the using amount of the Taq enzyme has great influence on the amplification efficiency of the PCR, and when 5.0U of Taq enzyme is added into each system, the amplification efficiency is high; the annealing temperature is 55 ℃, the annealing time is 30s, the PCR amplification efficiency is high, and the result is stable. The PCR product was subjected to agarose gel electrophoresis, and the amplified product was confirmed by sequencing to be the target gene (the experimental results are shown in FIGS. 1 to 4).
7.2 sensitivity analysis
All 5 diluted concentration reference samples showed positive reaction, and 10 in the reference sample5-107In the dilution range of copy/mL concentration, the Ct value increases correspondingly with the decrease of the concentration, and the two are in a linear relationship (r is 1.00, P<0.01), the results are shown in fig. 5.
7.3 repeatability analysis
1×105And 1X 107The copy/mL two reference products have intra-batch variation coefficients of 3.10% and 2.30% respectively, and inter-batch variation coefficients of 3.23% and 2.75%, both of which are less than 5.00%, and the method has good repeatability and meets the requirements of clinical detection.
7.4 clinical sample testing
MecA genotype tests are carried out on 40 CNS strains collected clinically, wherein all the CNS strains are identified as MRCNS strains by using a phenotypic method oxacillin minimum inhibitory concentration method (VITEK 2COMPACT determination), and only 21 MecA genes are positive.
As described above, in recent years, MRCNS is one of the important pathogenic bacteria causing hospital-related infections, and the isolation rate is high, and serious nosocomial infections caused by MRCNS are reported all over the world one after another in an extremely severe form. MRCNS often carries drug-resistant genes to other drugs in a linkage manner besides containing MecA drug-resistant genes, so that drug resistance to various antibacterial drugs is caused, and severe pressure is brought to clinical treatment[12-15]. MRS is difficult to detect due to inhomogeneous drug resistance of MRS, and MRCNS (central nervous system) is falsely increased by detecting MRS by a cefoxitin paper diffusion method or a oxacillin minimum inhibitory concentration method which is frequently adopted in laboratories. The detection needs to be rechecked in a clinical microorganism laboratory, so that an accurate drug sensitive result is provided for a clinician, and induction of drug resistance is avoided. The time for detecting MRS by the phenotypic method is long, incubation is required for 24 hours, and the phenotypic method is influenced by various factors such as incubation temperature, culture medium quality and bacterial liquid concentration. The fluorescence PCR method established by the invention only needs 3-4 hours from nucleic acid extraction to amplification, can directly detect the MecA gene of CNS, is rapid and sensitive, has high specificity through sequencing verification, has high repeatability, and can be applied to clinical detection.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Reference to the literature
[1]C Heilmann,W Ziebuhr,K Becker.Are Coagulase-Negative Staphylococci VirulentClin Microbiol Infect.2019；25(9):1071-1080.
[2]Xavier Argemi1,Yves Hansmann,Kevin Prola,et al.Coagulase-Negative Staphylococci Pathogenomics.Int J Mol Sci.2019；20(5):1215.
[3]Naccache SN,Callan K,Burnham CA,et al.Evaluation of Oxacillin and Cefoxitin Disk Diffusion and Microbroth Dilution Methods for Detecting mecA-Mediatedβ-Lactam Resistance in Contemporary Staphylococcus epidermidis Isolates.J Clin Microbiol.2019；57(12):e00961-19.
[4]Ferreira RB,Nunes AP,Kokis VM,et al.Simultaneous detection of the mecA and ileS-2 genes in coagulase-negative staphylococci isolated from Brazilian hospitals by multiplex PCR.DiagnMicrobiol Infect Dis.2002；42(3):205-212.
[5]Bogado I,Limansky A,Sutich E,et al.Molecular characterization of methicillin-resistant coagulase-negative staphylococci from a neonatal intensive care unit.Infect Control Hosp Epidemiol.2002；23(8):447-451.
[6]Stegger M,Andersen PS,Kearns A,et al.Rapid detection,differentiation and typing of methicillin-resistant Staphylococcus aureus harbouring either mecA or the new mecA homologue mecA(LGA251).Clin Microbiol Infect.2012；18(4):395-400.
[7]Wang HY,Kim S,Kim J,et al.Multiplex real-time PCR assay for rapid detection of methicillin-resistant staphylococci directly from positive blood cultures.J Clin Microbiol.2014；52(6):1911-1920.
[8]Weidner J,Cassens U,
W,et al.A new triplex real time PCR which distinguishes between MRSA,MSSA,and mecA coagulase negative strains by means of melting point analysis using SYTO 9.Clin Lab.2013；59(7-8):795-804.
[9]Nijjar CK,Smith MH,Eltringham IJ.Adjunctive mecA PCR for routine detection of methicillin susceptibility in clinical isolates of coagulase-negative staphylococci.J Clin Microbiol.2014；52(5):1678-1681.
[10]Okolie CE.Real-Time PCR to Identify Staphylococci and Assay for Virulence from Blood.Methods Mol Biol.2017；1616:183-207.
[11]Siripornmongcolchai T,Chomvarin C,Chaicumpar K,et al.Evaluation of different primers for detecting mecA gene by PCR in comparison with phenotypic methods for discrimination of methicillin-resistant Staphylococcus aureus.Southeast Asian J Trop Med Public Health.2002；33(4):758-763.
[12]Maria Miragaia.Factors Contributing to the Evolution of mecA-Mediatedβ-Lactam Resistance in Staphylococci:Update and New Insights From Whole Genome Sequencing(WGS).Front Microbiol.2018；9:2723.
[13]Nguyen DT,Yeh E,Perry S,et al.Real-time PCR testing for mecA reduces vancomycin usage and length of hospitalization for patients infected with methicillin-sensitive staphylococci.J Clin Microbiol.2010；48(3):785-90.
[14]Pedroso SHSP,Sandes SHC,Filho RAT,et al.Coagulase-Negative Staphylococci Isolated from Human Bloodstream Infections Showed Multidrug Resistance Profile.Microb Drug Resist.2018；24(5):635-647.
[15]Ho YC,Chang SC,Lin SR,et al.High levels of mecA DNA detected by a quantitative real-time PCR assay are associated with mortality in patients with methicillin-resistant Staphylococcus aureus bacteremia.J Clin Microbiol.2009 May；47(5):1443-1451.
Claims (10)
1. A method for detecting methicillin-resistant staphylococcus is characterized by comprising the following steps:
s1: designing a primer and a probe;
s2: extracting genome DNA in a specimen;
s3: detecting by real-time fluorescence PCR;
the primers comprise an upstream primer and a downstream primer, and the nucleotide sequences are respectively shown as SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
2. The method of claim 1, wherein the nucleotide sequence of the probe is as set forth in SEQ ID NO: 3 is shown in the specification; preferably, the probe is a TaqMan fluorescent probe.
3. The method of claim 1, wherein the probe is labeled with a fluorescence reporter FAM at the 5 'end and a fluorescence quencher TAMRA at the 3' end.
4. The method of claim 1, wherein the primer concentration is 0.25 μ Μ/L to 1 μ Μ/L; preferably, the primer concentration is 0.5. mu.M/L.
5. The method as claimed in claim 1, wherein the probe concentration is 100-300 nmol/L; preferably, the probe concentration is 200 nmol/L.
6. The method according to claim 1, wherein the amount of Taq enzyme used in the PCR system is 5.0U Taq enzyme in S3.
7. The method of claim 1, wherein the annealing temperature in the PCR system is 55 ℃ and the annealing time is 30S in S3.
8. The method of claim 1, wherein in S3, the PCR reaction conditions: sequentially performing pre-denaturation at 94 ℃ for 5 min; performing 35 cycles of amplification; extension at 72 ℃ for 7 minutes;
wherein the cycling amplification conditions are: denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 90 s.
9. Use of the method of any one of claims 1 to 8 for clinical species detection.
10. The use as claimed in claim 9, wherein the bacterial species is methicillin-resistant coagulase-negative staphylococci.
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