阅读说明：本技术 耐药性金黄色葡萄球菌MecA和ErmA基因的多重荧光RPA检测方法及试剂盒 (Multiplex fluorescence RPA detection method and kit for drug-resistant staphylococcus aureus MecA and ErmA genes ) 是由 朱灿灿 朱灵 时忠林 胡安中 杨柯 刘勇 于 2021-09-26 设计创作，主要内容包括：本发明公开同时检测耐药性金黄色葡萄球菌MecA和ErmA耐药基因的引物探针,涉及耐药基因检测技术领域,本发明还公开包含引物探针的试剂盒、多重荧光RPA检测方法。本发明的有益效果在于：采用本发明中引物探针的多重荧光RPA检测方法具有很强的特异性、较好的抗干扰性和较高的敏感性。(The invention discloses a primer probe for simultaneously detecting drug-resistant genes MecA and ErmA of drug-resistant staphylococcus aureus, relates to the technical field of drug-resistant gene detection, and further discloses a kit containing the primer probe and a multiplex fluorescence RPA detection method. The invention has the beneficial effects that: the multiplex fluorescence RPA detection method adopting the primer probe has strong specificity, better anti-interference performance and higher sensitivity.)

1. The primer probe for simultaneously detecting drug-resistant genes MecA and ErmA of drug-resistant staphylococcus aureus is characterized in that: the primer pair nucleotide sequence of the MecA drug resistance gene is as follows:

M-F:CCAAAGAATGTATCTAAAAAAGATTATAAAGCAATC；

M-R:AACGGTTTTAAGTGGAACGAAGGTATCATC；

the primer pair nucleotide sequence of the ErmA drug resistance gene is as follows:

E-F:TCAGTAAACAAGACAACGTAATAGAAATCGGATC；

E-R:TATCCGTTTGAATCACTTTTATATTCTCAG；

the specific probe nucleotide sequence of the MecA drug resistance gene is as follows:

M-Probe：ACTAAGTATTTCTGAAGACTATATCAAACAACAAA(FAMdT)C(THF)A(BHQ1-dT)CAAAATTGGGTACAA[3’-block]；

the nucleotide sequence of the specific probe of the ErmA drug resistance gene is as follows:

E-Probe：AGAGCTAGTCAAAATGAGTCGATCAGTTAC(HEXdT)G(THF)TA(BHQ1-dT)AGAAATTGATGGAGGC[3’-block]。

2. the kit comprising the primer probe for simultaneously detecting the MecA and ErmA drug resistance genes of the drug-resistant staphylococcus aureus as claimed in claim 1.

3. The kit for simultaneously detecting the primer probe of the drug-resistant genes MecA and ErmA of the drug-resistant staphylococcus aureus according to claim 2, which is characterized in that: the kit also comprises hydrolysis buffer solution, MgOAC and ddH₂O。

4. The kit for simultaneously detecting the primer probe of the drug-resistant genes MecA and ErmA of the drug-resistant staphylococcus aureus according to claim 2, which is characterized in that: the concentration of the MecA resistance gene primer pair, the concentration of the ErmA resistance gene primer pair, the concentration of the MecA resistance gene specific probe and the concentration of the ErmA resistance gene specific probe are all 10 mu M.

5. According to claim2 the kit for simultaneously detecting the primer probes of drug-resistant genes MecA and ErmA of the drug-resistant staphylococcus aureus is characterized in that: the system of the kit for RPA detection comprises the following components in 20 μ L: 11.8. mu.l of hydrolysis buffer, 0.85. mu.l of 10. mu.M MecA gene upstream primer, 0.85. mu.l of 10. mu.M MecA gene downstream primer, 0.24. mu.l of 10. mu.M MecA gene probe, 0.85. mu.l of 10. mu.M ErmA gene upstream primer, 0.85. mu.l of 10. mu.M ErmA gene downstream primer, 0.24. mu.l of 10. mu.M ErmA gene probe, 1. mu.l of template, and the rest ddH₂O was replenished and 2.5. mu.L of OAC was added to initiate the test before the start of the experiment.

6. A multiplex fluorescence RPA detection method for detecting drug-resistant genes MecA and ErmA of drug-resistant staphylococcus aureus using the primer probe of claim 1, characterized in that: the method comprises the following steps:

(1) extracting DNA of a sample to be detected;

(2) and (3) taking the extracted DNA as a template, preparing an RPA reaction system by adopting the primer probe, carrying out multiple RPA amplification reactions under isothermal condition, and detecting a fluorescent signal corresponding to the probe.

7. The multiplex fluorescence RPA detection method for detecting drug-resistant genes MecA and ErmA in staphylococcus aureus according to claim 6, wherein the multiplex fluorescence RPA detection method comprises the following steps: the RPA reaction system comprises the following components in 20 mu L: 11.8. mu.l of hydrolysis buffer, 0.85. mu.l of 10. mu.M MecA gene upstream primer, 0.85. mu.l of 10. mu.M MecA gene downstream primer, 0.24. mu.l of 10. mu.M MecA gene probe, 0.85. mu.l of 10. mu.M ErmA gene upstream primer, 0.85. mu.l of 10. mu.M ErmA gene downstream primer, 0.24. mu.l of 10. mu.M ErmA gene probe, 1. mu.l of template, and the rest ddH₂O was replenished and 2.5. mu.L of OAC was added to initiate the test before the start of the experiment.

8. The multiplex fluorescence RPA detection method for detecting drug-resistant genes MecA and ErmA in staphylococcus aureus according to claim 7, wherein the multiplex fluorescence RPA detection method comprises the following steps: the reaction temperature is 39 ℃, and the reaction time is 30 min.

Technical Field

The invention relates to the technical field of drug-resistant gene detection, in particular to a multiple fluorescence RPA detection method and a kit for drug-resistant staphylococcus aureus MecA and ErmA genes.

Background

Staphylococcus aureus (Staphylococcus aureus) belongs to the genus Staphylococcus and is an important pathogen causing infectious diseases in humans and animals. Drug-resistant staphylococcus aureus is widely distributed in nature and is the most common pathogen of pyogenic infections of humans.

In clinic, compared with other drug-resistant pathogenic bacteria such as acinetobacter baumannii, klebsiella pneumoniae, pseudomonas aeruginosa and the like, the drug-resistant staphylococcus aureus accounts for a more remarkable proportion in infected people, and reaches 27.60%; at the same time, the medicine can cause wound suppurative infection, mastitis and other local infections of human and livestock, and can also cause general infection. Clinical investigation shows that the drug-resistant staphylococcus aureus has drug resistance to various drugs, mainly comprising beta-lactams, macrolides, aminoglycosides and tetracyclines. The drug-resistant staphylococcus aureus has the highest drug resistance rate to beta-lactams and macrocyclic lipids, because the drug-resistant staphylococcus aureus generally contains two drug resistance genes of MecA and ermA.

At present, the clinical detection of the drug-resistant staphylococcus aureus mainly comprises the traditional separation and identification method, Pulse Field Gel Electrophoresis (PFGE), multi-site sequence typing (MLST), enzyme-linked immunosorbent assay (ELISA), Polymerase Chain Reaction (PCR) and the like. These methods are complex to design, time consuming and professional to operate. When epidemiological investigation and diagnosis are needed for emergencies of public health, it is a challenge to meet the demand for rapid and accurate diagnosis in the field. For example, patent application with publication number CN106222248A discloses a primer, a probe, a method and a kit for detecting drug resistance genes of methicillin-resistant staphylococcus aureus, but the minimum detection limit is higher and is 10³copies/mL, therefore, there is a strong need for a rapid, accurate, unique, sensitive, and drug-resistant staphylococcus aureus detection technique to assist medical personnel in making an early diagnosis.

Recombinase polymeraseSynthase Amplification (RPA) is an isothermal nucleic acid Amplification technique, and realizes rapid Amplification of template DNA by specific recognition of complementary sequences of template DNA by a Recombinase-primer complex. By adding in amplification systemsThe Exo probe can realize real-time fluorescence monitoring of an amplification product. The RPA method has the advantages of high sensitivity, short detection time, high specificity and normal temperature amplification (37-42 ℃), and is very suitable for rapid detection of clinical pathogenic microorganisms and drug-resistant genes. However, there are some disadvantages: due to the complex design of the RPA probe and the interference between primers, the research of multiple-target detection by adopting multiple fluorescent RPA is less.

Disclosure of Invention

The invention aims to solve the technical problems that the lowest detection limit of staphylococcus aureus drug resistance gene detection in the prior art is higher, and provides a primer probe, a kit and a multiple fluorescence RPA detection method for rapidly, simply and sensitively detecting drug resistance staphylococcus aureus MecA and ErmA drug resistance genes at the same time.

The invention solves the technical problems through the following technical means:

the primer probe for simultaneously detecting drug-resistant genes MecA and ErmA of the drug-resistant staphylococcus aureus is characterized in that the primer pair nucleotide sequence of the MecA drug-resistant gene is as follows:

M-F:CCAAAGAATGTATCTAAAAAAGATTATAAAGCAATC；

M-R:AACGGTTTTAAGTGGAACGAAGGTATCATC；

the primer pair nucleotide sequence of the ErmA drug resistance gene is as follows:

E-F:TCAGTAAACAAGACAACGTAATAGAAATCGGATC；

E-R:TATCCGTTTGAATCACTTTTATATTCTCAG；

the specific probe nucleotide sequence of the MecA drug resistance gene is as follows:

M-Probe：ACTAAGTATTTCTGAAGACTATATCAAACAACAAA(FAMdT)C(THF)A(BHQ1-dT)CAAAATTGGGTACAA[3’-block]；

the nucleotide sequence of the specific probe of the ErmA drug resistance gene is as follows:

E-Probe：AGAGCTAGTCAAAATGAGTCGATCAGTTAC(HEXdT)G(THF)TA(BHQ1-dT)AGAAATTGATGGAGGC[3’-block]。

has the advantages that: 2 specific probes and corresponding specific primers are designed according to conserved sequences of MecA and ErmA resistance genes of the drug-resistant staphylococcus aureus, wherein the fluorescent probe corresponding to the MecA resistance gene is marked with FAM fluorescein, and the fluorescent probe corresponding to the ErmA resistance gene is marked with HEX fluorescein. Experiments prove that the primers of the invention have no interference and strong specificity, and the primer probe of the invention has strong specificity, better anti-interference performance and higher sensitivity, thereby realizing the simultaneous detection of genes and achieving the purpose of rapid, efficient and parallel detection of drug-resistant genes.

A kit comprising the primer probe.

Has the advantages that: the multiple fluorescence RPA detection method adopting the kit containing the primer probe has strong specificity, good anti-interference performance and high sensitivity.

Preferably, the kit further comprises a hydrolysis buffer, MgOAC, ddH₂O。

Preferably, the concentration of the MecA resistance gene primer pair, the concentration of the ErmA resistance gene primer pair, the concentration of the MecA resistance gene specific probe and the concentration of the ErmA resistance gene specific probe are all 10 μ M.

Preferably, the system for the kit to perform the RPA detection comprises the following components in 20 μ L: 11.8. mu.l of hydrolysis buffer, 0.85. mu.l of 10. mu.M MecA gene upstream primer, 0.85. mu.l of 10. mu.M MecA gene downstream primer, 0.24. mu.l of 10. mu.M MecA gene probe, 0.85. mu.l of 10. mu.M ErmA gene upstream primer, 0.85. mu.l of 10. mu.M ErmA gene downstream primer, 0.24. mu.l of 10. mu.M ErmA gene probe, 1. mu.l of template, and the rest ddH₂O was replenished and 2.5. mu.L of OAC was added to initiate the test before the start of the experiment.

A multiplex fluorescence RPA detection method for detecting drug resistance genes MecA and ErmA of drug resistance staphylococcus aureus by adopting the primer probe comprises the following steps:

(1) extracting DNA of a sample to be detected;

(2) and (3) taking the extracted DNA as a template, preparing an RPA reaction system by adopting the primer probe, carrying out multiple RPA amplification reactions under isothermal condition, and detecting a fluorescent signal corresponding to the probe.

Has the advantages that: the multiplex fluorescence RPA detection method adopting the primer probe has strong specificity, better anti-interference performance and higher sensitivity, and the lowest detectable copy number is 2 x 10¹copies/μl。

Primarily screening out proper primers and probes in a single amplification system by repeatedly optimizing the primers, probe sequences and the amplification system; secondly, adding suitable primers and probes of the two drug-resistant genes into the same amplification system, developing an optimization experiment, and finally screening the suitable primers and probes by observing whether specific fragments can be simultaneously and efficiently amplified. Establishes a multiple fluorescence RPA method of drug-resistant genes (MecA and ErmA) in drug-resistant staphylococcus aureus, and realizes the rapid, efficient and parallel detection of the two drug-resistant genes.

The method is high in reliability and strong in practicability, can be used for detecting samples such as tissues, sputum and saliva, is suitable for any laboratory, and has great application prospect and potential in resource shortage and on-site rapid detection.

Preferably, the RPA reaction system, in 20 μ L, comprises the following components: 11.8. mu.l of hydrolysis buffer, 0.85. mu.l of 10. mu.M MecA gene upstream primer, 0.85. mu.l of 10. mu.M MecA gene downstream primer, 0.24. mu.l of 10. mu.M MecA gene probe, 0.85. mu.l of 10. mu.M ErmA gene upstream primer, 0.85. mu.l of 10. mu.M ErmA gene downstream primer, 0.24. mu.l of 10. mu.M ErmA gene probe, 1. mu.l of template, and the rest ddH₂O was replenished and 2.5. mu.L of OAC was added to initiate the test before the start of the experiment.

Preferably, the reaction temperature is 39 ℃ and the reaction time is 30 min.

The invention has the advantages that: 2 specific probes and corresponding specific primers are designed according to conserved sequences of MecA and ErmA resistance genes of the drug-resistant staphylococcus aureus, wherein the fluorescent probe corresponding to the MecA resistance gene is marked with FAM fluorescein, and the fluorescent probe corresponding to the ErmA resistance gene is marked with HEX fluorescein. Experiments prove that the multiple fluorescence RPA detection method adopting the primer probe has strong specificity, better anti-interference performance and higher sensitivity, realizes the simultaneous detection of genes, and achieves the aim of rapid, efficient and parallel detection of drug-resistant genes.

The multiple fluorescence RPA detection method adopting the kit containing the primer probe has strong specificity, better anti-interference performance and higher sensitivity, and the lowest detectable copy number is 2 x 10¹copies/μl。

The method is high in reliability and strong in practicability, can be used for detecting samples such as tissues, sputum and saliva, is suitable for any laboratory, and has great application prospect and potential in resource shortage and on-site rapid detection.

Drawings

FIG. 1 is a schematic diagram of the sensitivity curve of the multiplex fluorescent RPA in example 3 of the present invention, in which the solid line is MecA resistance gene, and the corresponding fluorescent probe is labeled with FAM fluorescein; the dotted line is an ErmA drug resistance gene, and a corresponding fluorescent probe marks HEX fluorescein;

FIG. 2 is a schematic diagram of the multiple fluorescent RPA specificity curve in example 3 of the present invention, in which the solid line is MecA resistance gene, and the corresponding fluorescent probe is labeled FAM fluorescein; the dotted line is an ErmA drug resistance gene, and a corresponding fluorescent probe marks HEX fluorescein;

FIG. 3 is a diagram showing the results of the measurement of the suitability of the multiplex RPA assay for practical samples in example 5 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Test materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The specific techniques or conditions not specified in the examples can be performed according to the techniques or conditions described in the literature in the field or according to the product specification.

The RPA kit Exo kit used in the examples below was purchased from twist dx Inc, england; wherein, the recombinase capable of binding single-stranded nucleic acid (oligonucleotide primer), single-stranded DNA binding protein (SSB) and strand displacement DNA polymerase are present in the RPA reaction tube provided by the kit in a lyophilized powder state, and are directly diluted with a reaction buffer carried by the kit when in use. The entire RPA reaction was carried out in an RPA reaction tube.

Example 1

Selecting conserved sequences of drug-resistant staphylococcus aureus MecA and ErmA genes, and designing M-F/M-R and E-F/E-R two groups of primers and M-Probe and E-Probe specific probes according to a fluorescent RPA primer design principle; wherein, the fluorescent probe corresponding to MecA gene is marked with FAM fluorescein, and the fluorescent probe corresponding to ErmA gene is marked with HEX fluorescein.

M-F:CCAAAGAATGTATCTAAAAAAGATTATAAAGCAATC；

M-R:AACGGTTTTAAGTGGAACGAAGGTATCATC；

E-F:TCAGTAAACAAGACAACGTAATAGAAATCGGATC；

E-R:TATCCGTTTGAATCACTTTTATATTCTCAG；

M-Probe：ACTAAGTATTTCTGAAGACTATATCAAACAACAAA(FAMdT)C(THF)A(BHQ1-dT)CAAAATTGGGTACAA[3’-block]；

E-Probe：AGAGCTAGTCAAAATGAGTCGATCAGTTAC(HEXdT)G(THF)TA(BHQ1-dT)AGAAATTGATGGAGGC[3’-block]。

On the basis, a kit is designed, and the kit comprises 10 mu M of the 2 primer pairs, 10 mu M of the 2 probes and a hydrolysis buffer solution.

Example 2

Establishment of multiple fluorescence RPA detection method for drug resistance genes MecA and ErmA of drug resistance staphylococcus aureus

(1) Bacterial culture and DNA extraction: culturing about staphylococcus aureus ATCC 4330024 h in LB culture medium, and extracting DNA according to an operation manual provided by a bacterial genome DNA extraction kit; the concentration and purity of the obtained nucleic acid are measured by a NanoDrop spectrophotometer, and the A260/A280 is about 1.8 for later use.

(2) The RPA reaction system is 20 μ l, wherein 11.8 μ l of hydrolysis buffer Primer Free reduction buffer, 0.85 μ l of 10 μ M MecA gene upstream Primer, 0.85 μ l of 10 μ M MecA gene downstream Primer, 0.24 μ l of 10 μ M MecA gene probe, 0.85 μ l of 10 μ M ErmA gene upstream Primer, 0.85 μ l of 10 μ M ErmA gene downstream Primer, 0.24 μ l of 10 μ M ErmA gene probe, 1 μ l of template, and the rest of ddH2O are supplemented, 2.5 μ M MgOAC start-up test is added before the start of the test, the reaction temperature is 39 ℃ for 30min, an isothermal amplification reaction program and a fluorescence signal corresponding to a detection probe (FAM/HEX) are set, and multiple RPA amplification reaction is carried out on a LightCyclerr 96.

The program set therein comprises a two-step process: denaturation at 39 ℃ for 10 s; and (3) amplifying at 39 ℃ for 50 s. For a total of 30 cycles.

(3) In the experiment, a standard control group and a blank group are arranged, and within 30 cycles of amplification, if the template group has a fluorescence signal and the negative group has no fluorescence signal, the template group is judged to be positive.

Example 3

Multiplex fluorescent RPA sensitivity assay

The concentration of the extracted drug-resistant staphylococcus aureus DNA is measured by using NanoDrop, the concentration is calculated according to a standard curve, DNA with different concentrations is respectively used as templates, the detection is carried out under the optimal reaction condition of the RPA detection method in the embodiment 2, the minimum detection amount of the RPA detection method is determined, and the sensitivity of the method is evaluated.

FIG. 1 shows that the designed multiplex fluorescence RPA detection method has the lowest detectable copy number of 2 x 10¹copies/μl。

Example 4

Multiplex fluorescent RPA-specific detection

The method comprises the steps of selecting the three most important and most common pathogenic bacteria in the respiratory tract pathogenic bacteria, namely acinetobacter baumannii, escherichia coli and pseudomonas aeruginosa as control groups, taking DNA of the three pathogenic bacteria as control group templates, taking drug-resistant staphylococcus aureus DNA as an experimental group template, detecting under the same reaction conditions and reaction systems, observing fluorescence signals of the control groups and the experimental groups, and evaluating the specificity of the method.

FIG. 2 shows that the designed multiplex fluorescence RPA detection method has strong specificity, and only the staphylococcus aureus experimental group with drug resistance has fluorescence signal detection.

Example 5

Multiplex fluorescence RPA detection practical sample applicability determination

Clinically staphylococcus aureus is often present in the sputum of patients. Clinical patient sputum samples were used to assess the potential use and applicability of real-time fluorescent RPA detection.

Firstly, enrichment culture is carried out on a clinical sputum specimen to obtain a single bacterial colony; single colonies were selected for 24 hours at 37 ℃ in liquid shaking culture. Secondly, extracting genome DNA, and analyzing the extraction effect according to the PCR amplification result. Finally, the multiplex fluorescent RPA assay was performed using the method in example 2. And evaluating the applicability of the method for detecting the actual sample according to the fluorescence signal amplified by the clinical sample.

FIG. 3 shows that in the designed multiplex fluorescence RPA detection method, among 68 clinical detection samples, 66 positive samples and 2 negative samples are detected by real-time fluorescence quantitative PCR; the detection results of 65 cases are positive, the detection results of 3 cases are negative, and the method has good clinical applicability.

The result shows that the double RPA method is equivalent to the fluorescent PCR in the aspect of sensitivity, but the amplification time is greatly reduced, and the amplification result can be obtained more quickly.

The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.