CN105567866A - Primers, kit and method for detecting methicillin-resistant Staphalococcus aureus - Google Patents
Primers, kit and method for detecting methicillin-resistant Staphalococcus aureus Download PDFInfo
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- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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Abstract
The invention relates to primers, a kit and a method for detecting methicillin-resistant Staphalococcus aureus, belonging to the field of molecular biology. The primers comprise a forward primer mecA-f and a reverse primer mecA-r; the nucleotide sequence of the forward primer mecA-f is 5'-AATTTCAAACAAGTTTATAAAGATAGC-(THF)-GTTATATTTCTA-biotin-3'; and the nucleotide sequence of the reverse primer mecA-r is 5'-TTGTTGTTTGATATAGTCTTCAGAAATA-(THF)-TTAGTTCTTTA-biotin-3'. A recombinase polymerase amplification method is utilized to detect the methicillin-resistant Staphalococcus aureus. The primers for detecting the methicillin-resistant Staphalococcus aureus have the advantages of high specificity and high amplification efficiency, and can be used for effectively and quickly detecting the methicillin-resistant Staphalococcus aureus.
Description
Technical field
The present invention relates to a kind of primer, test kit and the method that detect for methicillin-resistant staphylococcus aureus, belong to biology field.
Background technology
Streptococcus aureus (Staphalococcusaureus) belongs to gram positive coccus, micrococcaceae, Staphylococcus.It can cause suppurative inflammation, when bacterium invades blood, can cause pathogenic septicemia.
After the forties in last century, penicillin came out, the infectious diseases that streptococcus aureus causes is subject to larger control, but widely using along with penicillin, some streptococcus aureus produces penicillinase, can beta-lactam nucleus be hydrolyzed, show as the resistance to penicillin.Scientist works out a kind of semisynthetic penicillin of new ability penicillinase for this reason, i.e. X-1497 (methicillin).Nineteen fifty-nine be applied to clinical after once effectively controlled the infection that streptococcus aureus produces enzyme strain, but 1961 subsequently have just found methicillin-resistant staphylococcus aureus (MRSA), MRSA infects almost extend over the entire globe so far from discovery, become institute in one of important pathogen of Nosocomial Infections.
After Britain in 1961 finds MRSA, in succession report the ward infection caused by MRSA in American-European and some countries of Asia.From the later stage sixties to the eighties, MRSA infection rate increases greatly.U.S. NNIS reports that 182 hospital MRSA in 1975 account for 2.4% of infection of staphylococcus aureus sum, within 1991, rise to 24.8%, be wherein many with teaching hospitals more than 500 beds and central hospital especially, because the chance that in these hospitals, MRSA infects is more, Resistant strain both can bring hospital into by infected patient, also can produce in hospital because of abuse of antibiotics.The MRSA of Europe 1417 hospital ICU separation in 1993 reaches 60%.And the separation rate of affiliated hospital of Japanese Kansai medical university MRSA reaches 41% in 1993.Domesticly find that there is MRSA in the seventies, the recall rate of MRSA rose year by year in recent years, and Shanghai 1978 MRSA in 200 strain streptococcus aureuses only accounts for 5%, within 1988, rises to 24%, within 1996 years, increases sharply to 72%.MRSA infection is multiple is born in immune deficiency person, large-area burns, patient after major operation, long-term inpatients and gerontal patient, and MRSA very easily causes the popular of infection and breaks out.For the infection of effective control MRSA, and reduce the harm that disease causes, setting up detection method is accurately and rapidly top priority.
The method detecting MRSA conventional clinically at present has bacterial cultivation and PCR method.Wherein, bacterial cultivation is the gold standard detecting MRSA, but the method needs to carry out drug sensitive experiment, and usually need within 3-5 days, just can see final detected result, testing cost is high, sense cycle is long; Utilize PCR method that specific nucleotide sequence copy number geometricprogression can be made at short notice to increase, have very high sensitivity and specificity, but shortcoming needs the test set of specialty, is difficult to promote in Countryside.As can be seen here, the clinical diagnosis that MRSA infects, is badly in need of more succinct, sensitive detection method.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art part and provide that a kind of amplification efficiency is high, the primer detected for methicillin-resistant staphylococcus aureus of high specificity.
Meanwhile, present invention also offers a kind of test kit for methicillin-resistant staphylococcus aureus detection and method.
For achieving the above object, the technical scheme that the present invention takes is: a kind of primer detected for methicillin-resistant staphylococcus aureus, it comprises upstream primer mecA-f and downstream primer mecA-r, and the nucleotides sequence of upstream primer mecA-f is classified as: 5 '-AATTTCAAACAAGTTTATAAAGATAGC-(THF)-GTTATATTTCTA-biotin-3 '; The nucleotides sequence of downstream primer mecA-r is classified as: 5 '-TTGTTGTTTGATATAGTCTTCAGAAATA-(THF)-TTAGTTCTTTA-biotin-3 ';
Wherein, THF is tetrahydrofuran (THF), and biotin is vitamin H.
Above-mentioned upstream primer mecA-f and downstream primer mecA-r, its 3 ' terminal hydroxy group is all closed by vitamin H., all can play sealing effect with the covalently bound group of probe 3 ' hydroxyl arbitrarily herein, so place closes the 3 ' hydroxyl held be not limited to vitamin H.Separately, for upstream primer mecA-f, what wherein THF replaced is that (sequence of conventional upstream primer is conventional upstream primer: " T " 5 '-AATTTCAAACAAGTTTATAAAGATAGCTGTTATATTTCTA-biotin-3 ').
Primer of the present invention is the gene order that contriver contrasts a large amount of MRSA (methicillin-resistant staphylococcus aureus) and MSSA (the non-resistant Staphylococcus aureus in X-1497) bacterial strain, select the distinctive gene mecA of MRSA as detection target spot, and obtain for this shot design, synthesis upstream and downstream primer.Detect target spot for mecA, contriver's design and synthesis many primers, but experiment shows, primer specificity of the present invention is strong, amplification efficiency is high, can detect methicillin-resistant staphylococcus aureus effectively and quickly.
Because 3 ' end of primer of the present invention is closed, therefore need first to cut THF site with endonuclease, form 3 ' end freely, could amplified reaction be started.Thus, specificity and the preciseness of amplified reaction is also improved.
As the preferred implementation of the primer for methicillin-resistant staphylococcus aureus detection of the present invention, in the nucleotide sequence of described upstream primer mecA-f or downstream primer, there are the replacement of less than 10 bases, interpolation or disappearance.
As the preferred implementation of the primer for methicillin-resistant staphylococcus aureus detection of the present invention, in described upstream primer mecA-f and downstream primer mecA-r, at least one primer is marked with reporter group.As the more preferably embodiment of the primer for methicillin-resistant staphylococcus aureus detection of the present invention, described reporter group is fluorophor.Adopt reporter group labeled primer, can be subsequent detection and provide convenience.
In addition, present invention also offers a kind of test kit detected for methicillin-resistant staphylococcus aureus containing primer described above.
As the preferred implementation of the test kit for methicillin-resistant staphylococcus aureus detection of the present invention, described test kit also comprises recombinase, polysaccharase, DBP, buffered soln and endonuclease.
As the more preferably embodiment of the test kit for methicillin-resistant staphylococcus aureus detection of the present invention, described recombinase is T4uvsX/uvsY, and described polysaccharase is BsuDNA polysaccharase, and described DBP is T4gp32; Described buffered soln contains following component: Tris, potassium acetate, magnesium acetate, dithiothreitol (DTT), Carbowax20M, dNTPs, ATP, phosphocreatine, creatine kinase and reversed transcriptive enzyme; Described endonuclease is E. coli endonuclease IV.Further, described buffered soln contains following component: 50mmo/LTris-HCl damping fluid, 100mmo/L potassium acetate, 14mmo/L magnesium acetate, 2mmo/L dithiothreitol (DTT), 5%Carbowax20M, 200 μm of o/LdNTPs, 3mmo/LATP, 50mmo/L phosphocreatines, 100ng/L creatine kinase and 100ng/L reversed transcriptive enzymes; Wherein, the pH value of Tris-HCl damping fluid is 7.9.Described uvsX/uvsY is two kinds of enzymes of conversion running balance mutually in reaction system.
As the preferred implementation of the test kit for methicillin-resistant staphylococcus aureus detection of the present invention, described test kit also comprises ddH
2o.
Finally, present invention also offers a kind of method adopting mentioned reagent box to detect methicillin-resistant staphylococcus aureus, described method is restructuring enzymatic polymerization enzymatic amplification method, and it comprises the following steps:
(1) sample rna is extracted;
(2) reaction system is configured: mixing sample RNA, upstream primer mecA-f, downstream primer mecA-r, recombinase, polysaccharase, DBP, buffered soln and ddH
2o, obtains reaction system;
(3) after the reaction system that concussion mixing step (2) is obtained, add endonuclease, concussion mixes again, and isothermal reaction at 37 ~ 42 DEG C, carries out RPA amplification;
(4) product after RPA amplification is detected.
Recombinase polymeric enzymatic amplification (RecombinasePolymeraseAmplification, RPA) is a kind of novel nucleic acid amplification detection technique.Compared with the pcr amplification (having to pass through sex change, annealing, extension three steps) of routine, RPA reaction only needs 37 ~ 42 DEG C of isothermal reactions about 30 minutes, namely can by the nucleic acid-templated amplification 10 of trace
12doubly, reach the level that can detect, and RPA amplification does not need temperature control device, really can realize portable Rapid nucleic acid and detect.RPA technology depends on three kinds of enzymes: can in conjunction with the recombinase of single-chain nucleic acid, single-stranded DNA binding protein and strand displacement archaeal dna polymerase.Recombinase is combined the Protein-DNA mixtures formed with primer, can find homologous sequence in double-stranded DNA, once primer located homologous sequence, Exchange reaction of chain will occur and formed and start DNA synthesis, carry out exponential amplification to the target area in template.The DNA chain be replaced is combined with DBP, prevents further replacement.In this individual system, by two relative initial compound events of primer.Whole process is carried out quickly, generally can obtain the amplified production that can detect level within 30 minutes.
The mixture of three kinds of enzymes needed for RPA reaction just has activity at normal temperatures, and often in mixing process, amplified reaction just starts, and causes reaction background higher, even has non-specific amplification to occur.The present invention uses 3 ' of primer end closure to fall, and amplified reaction can not be started, and after having mixed, then cuts THF site with endonuclease, forms 3 ' end freely, start amplified reaction, improve specificity and the preciseness of amplified reaction.
As the preferred implementation of the method for detection methicillin-resistant staphylococcus aureus of the present invention, in described step (1), adopt Trizol or RNA to extract test kit and extract sample rna; In described step (4), the product after adopting the methods such as electrophoretic method, test strip method, turbidimetry or fluorescent quantitation to detect RPA amplification.
Beneficial effect of the present invention is: the invention provides a kind of primer detected for methicillin-resistant staphylococcus aureus, this primer specificity is strong, amplification efficiency is high, can detect methicillin-resistant staphylococcus aureus effectively and quickly.The present invention also establishes the reaction system of stable RPA augmentation detection MRSA, overcomes the problems that MRSA Present clinical detects, and the diagnosis, the prevention and corntrol that infect for MRSA provide new detection method.
The present invention adopts recombinase polymeric enzymatic amplification technology for detection methicillin-resistant staphylococcus aureus, and highly sensitive, high specificity, detection speed are fast and without the need to specialized equipment.
In addition, the sample that the present invention detects is extensive, comprises blood, body fluid, fester, urine, food sample and the sample from soil, empty G&W, and their culture etc.
Accompanying drawing explanation
Fig. 1 is the result figure adopting test kit of the present invention to carry out recombinase polymeric enzymatic amplification and pcr amplification method detection methicillin-resistant staphylococcus aureus.
In figure, M represents DNAmarker, and 1 represents 10
7mRSA, 2 represent 10
6mRSA, 3 represent 10
5mRSA, 4 represent 10
4mRSA, 5 represent 10
3mRSA, 6 represent 10
2mRSA ,-represent 10
7mSSA.
Embodiment
For better the object, technical solutions and advantages of the present invention being described, below in conjunction with the drawings and specific embodiments, the invention will be further described.
In embodiment, RPA represents recombinase polymeric enzymatic amplification, and PCR represents polymerase chain reaction, and MRSA represents methicillin-resistant staphylococcus aureus, and MSSA represents the non-resistant Staphylococcus aureus in X-1497.Embodiment 1: primer and test kit
A kind of embodiment of the primer for methicillin-resistant staphylococcus aureus detection of the present invention, the primer detected for methicillin-resistant staphylococcus aureus described in the present embodiment comprises upstream primer mecA-f and downstream primer mecA-r, and the nucleotides sequence of upstream primer mecA-f is classified as: 5 '-AATTTCAAACAAGTTTATAAAGATAGC-(THF)-GTTATATTTCTA-biotin-3 '; The nucleotides sequence of downstream primer mecA-r is classified as: 5 '-TTGTTGTTTGATATAGTCTTCAGAAATA-(THF)-TTAGTTCTTTA-biotin-3 ';
Wherein, THF is tetrahydrofuran (THF), and biotin is vitamin H.
The test kit detected for methicillin-resistant staphylococcus aureus described in the present embodiment comprises above-mentioned upstream primer mecA-f and downstream primer mecA-r, and described test kit is also containing recombinase, polysaccharase, DBP, buffered soln, endonuclease and ddH
2o; Described recombinase is T4uvsX/uvsY, and described polysaccharase is BsuDNA polysaccharase, and described DBP is T4gp32; Described buffered soln contains following component: 50mmo/LTris-HCl damping fluid, 100mmo/L potassium acetate, 14mmo/L magnesium acetate, 2mmo/L dithiothreitol (DTT), 5%Carbowax20M, 200 μm of o/LdNTPs, 3mmo/LATP, 50mmo/L phosphocreatines, 100ng/L creatine kinase and 100ng/L reversed transcriptive enzymes, wherein, the pH value of Tris-HCl damping fluid is 7.9; Described endonuclease is E. coli endonuclease IV.
Further, in above-mentioned upstream primer mecA-f and downstream primer mecA-r, reporter group can be marked with at least one primer.Wherein, reporter group can preferred fluorophor.
Embodiment 2:RPA detects MRSA
The present embodiment adopts test kit described in embodiment 1 to detect MRSA, and detecting the method adopted is restructuring enzymatic polymerization enzymatic amplification method, and concrete steps are as follows:
A, sample prepare
(1) get the methicillin-resistant staphylococcus aureus (MRSA) of cultivation, adopt turbidimetry quantitative, get 10
7bacterium;
(2) by 10 times of gradient dilutions, 10 are obtained
6, 10
5, 10
4, 10
3, 10
2mRSA;
(3) with 10
7rNA is extracted as negative control in X-1497 non-resistant Staphylococcus aureus (MSSA).
B, employing RPA method detect MRSA
(1) extract test kit with Trizol or RNA and extract sample rna;
(2) by following proportional arrangement RPA reaction system:
| Sample rna | 2μg |
| MecA-f and mecA-r | Each 1 μ L |
| Buffered soln | 5μL |
| T4uvsX/uvsY，130ng/μL | 1μL |
| Bsu archaeal dna polymerase, 30ng/ μ L | 1μL |
| T4gp32，900ng/μL | 1μL |
| ddH2O | Complement to 50 μ L |
(3), after the reaction system that concussion mixing step (2) is obtained, 200ng/ μ L E. coli endonuclease IV is added;
(4) after concussion mixing, 42 DEG C of isothermal reactions 20 minutes;
(5) the DNA product after electrophoresis detection RPA amplification.
The present embodiment adopts recombinase polymeric enzymatic amplification to detect the result of methicillin-resistant staphylococcus aureus as shown in Figure 1.
Comparative example 1:PCR method detects MRSA
In this comparative example, the step that sample prepares is with embodiment 2, and it adopts the step of PCR method detection MRSA as follows:
(1) extract test kit (HYQ) with bacteria RNA and extract bacteria RNA in sample;
(2) with reverse transcription reagents with carry out reverse transcription to RNA and obtain cDNA;
(3) detect, pcr amplification condition with MRSA detection kit (PCR method, Thymopetidum Injection thing): 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C extend 1 minute;
(4) electrophoresis detection pcr amplification product.
This comparative example adopts pcr amplification method to detect the result of methicillin-resistant staphylococcus aureus as shown in Figure 1.
As seen from Figure 1: PCR and RPA (the inventive method) all effectively can detect 10
3and above MRSA, but RPA effectively can detect and is low to moderate 10
2mRSA, for 10
2the detection of MRSA, RPA accounts for clear superiority, and its expanding effect is better than PCR.Meanwhile, PCR and RPA can avoid up to 10 effectively
7the interference of MSSA.RPA with PCR is the same, and not by the interference of MSSA, but compared with PCR method, RPA, without the need to special reverse transcription step, does not need special instrument yet, and detection speed is faster, accurately, more more economical.
Finally to should be noted that; above embodiment is only in order to illustrate technical scheme of the present invention but not limiting the scope of the invention; although be explained in detail the present invention with reference to preferred embodiment; those of ordinary skill in the art is to be understood that; can modify to technical scheme of the present invention or equivalent replacement, and not depart from essence and the scope of technical solution of the present invention.
Claims (10)
1. the primer detected for methicillin-resistant staphylococcus aureus, it is characterized in that: described primer comprises upstream primer mecA-f and downstream primer mecA-r, and the nucleotides sequence of upstream primer mecA-f is classified as: 5 '-AATTTCAAACAAGTTTATAAAGATAGC-(THF)-GTTATATTTCTA-biotin-3 '; The nucleotides sequence of downstream primer mecA-r is classified as: 5 '-TTGTTGTTTGATATAGTCTTCAGAAATA-(THF)-TTAGTTCTTTA-biotin-3 '.
2., as claimed in claim 1 for the primer that methicillin-resistant staphylococcus aureus detects, it is characterized in that: in the nucleotide sequence of described upstream primer mecA-f or downstream primer, have the replacement of less than 10 bases, interpolation or disappearance.
3., as claimed in claim 1 or 2 for the primer that methicillin-resistant staphylococcus aureus detects, it is characterized in that: in described upstream primer mecA-f and downstream primer mecA-r, at least one primer is marked with reporter group.
4., as claimed in claim 3 for the primer that methicillin-resistant staphylococcus aureus detects, it is characterized in that: described reporter group is fluorophor.
5. the test kit detected for methicillin-resistant staphylococcus aureus containing, for example primer described in any one of Claims 1 to 4.
6., as claimed in claim 5 for the test kit that methicillin-resistant staphylococcus aureus detects, it is characterized in that: described test kit also comprises recombinase, polysaccharase, DBP, buffered soln and endonuclease.
7., as claimed in claim 6 for the test kit that methicillin-resistant staphylococcus aureus detects, it is characterized in that: described recombinase is T4uvsX/uvsY, described polysaccharase is BsuDNA polysaccharase, and described DBP is T4gp32; Described buffered soln contains following component: Tris, potassium acetate, magnesium acetate, dithiothreitol (DTT), Carbowax20M, dNTPs, ATP, phosphocreatine, creatine kinase and reversed transcriptive enzyme; Described endonuclease is E. coli endonuclease IV.
8., as claimed in claim 7 for the test kit that methicillin-resistant staphylococcus aureus detects, it is characterized in that: described buffered soln contains following component: 50mmo/LTris-HCl damping fluid, 100mmo/L potassium acetate, 14mmo/L magnesium acetate, 2mmo/L dithiothreitol (DTT), 5%Carbowax20M, 200 μm of o/LdNTPs, 3mmo/LATP, 50mmo/L phosphocreatines, 100ng/L creatine kinase and 100ng/L reversed transcriptive enzymes; Wherein, the pH value of Tris-HCl damping fluid is 7.9.
9., as claimed in claim 6 for the test kit that methicillin-resistant staphylococcus aureus detects, it is characterized in that: described test kit also comprises ddH
2o.
10. adopt test kit as claimed in claim 6 to detect a method for methicillin-resistant staphylococcus aureus, it is characterized in that: described method is restructuring enzymatic polymerization enzymatic amplification method, and it comprises the following steps:
(1) sample rna is extracted;
(2) reaction system is configured: mixing sample RNA, upstream primer mecA-f, downstream primer mecA-r, recombinase, polysaccharase, DBP, buffered soln and ddH
2o, obtains reaction system;
(3) after the reaction system that concussion mixing step (2) is obtained, add endonuclease, concussion mixes again, and isothermal reaction at 37 ~ 42 DEG C, carries out RPA amplification;
(4) product after RPA amplification is detected.
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| CN107058513A (en) * | 2017-02-23 | 2017-08-18 | 浙江工商大学 | Primer and its application of the pseudomonas aeruginosa of quinolones drug resistant gene are carried for RPA quick detections |
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| WO2018014799A1 (en) * | 2016-07-22 | 2018-01-25 | 广州康昕瑞基因健康科技有限公司 | Recombinase polymerase amplification reagent kit, amplification method, and amplification reagent |
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| CN107058513A (en) * | 2017-02-23 | 2017-08-18 | 浙江工商大学 | Primer and its application of the pseudomonas aeruginosa of quinolones drug resistant gene are carried for RPA quick detections |
| CN107058513B (en) * | 2017-02-23 | 2023-01-17 | 浙江工商大学 | Primer for RPA (reverse transcriptase polymerase chain reaction) rapid detection of pseudomonas aeruginosa carrying quinolone drug-resistant genes |
| CN108165611A (en) * | 2017-12-26 | 2018-06-15 | 天津科技大学 | A kind of methods and applications of recombinase polymerase constant-temperature amplification combination ELISA test strip staphylococcus aureus |
| CN113699259A (en) * | 2021-09-23 | 2021-11-26 | 中国人民解放军军事科学院军事医学研究院 | Exo-RPA technology-based staphylococcus aureus detection method and complete set of reagents thereof |
| CN113699259B (en) * | 2021-09-23 | 2022-06-28 | 中国人民解放军军事科学院军事医学研究院 | Exo-RPA technology-based staphylococcus aureus detection method and complete set of reagents thereof |
| CN113755616A (en) * | 2021-09-26 | 2021-12-07 | 合肥中科易康达生物医学有限公司 | Multiplex fluorescence RPA detection method and kit for drug-resistant staphylococcus aureus MecA and ErmA genes |
| CN113755616B (en) * | 2021-09-26 | 2024-02-13 | 安徽中科易康达生物科技有限公司 | Multiplex fluorescence RPA detection method and kit for drug-resistant staphylococcus aureus MecA and ErmA genes |
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