CN111979142B - Methicillin-resistant Staphylococcus aureus carrying drug-resistant genes cfr and lsa(E) at the same time and detection method thereof - Google Patents

Abstract

The invention discloses a methicillin-resistant Staphylococcus aureus carrying drug-resistant genes cfr and lsa(E) at the same time and a detection method thereof. Staphylococcus aureus Sta2868B2, deposit number: GDMCC No: 61037. The invention provides a primer set and a double PCR detection method for simultaneously detecting multiple drug resistance genes cfr and lsa(E). The method has good stability and specificity. Two pairs of optimal primers are selected after comparing multiple sets of primers; the specificity of the reaction can be further improved by adjusting the annealing temperature. The detection has high specificity, no cross-reaction with each other and with drug resistance genes, and also has good repeatability; in addition, the invention can detect two drug resistance genes at one time, and the whole reaction is completed within 2 hours, save time and energy. At the same time, using the present invention does not require an expensive fluorescent PCR instrument, nor does it need to synthesize expensive probes and corresponding reagents, the detection cost is low, the operation is simple, and it is beneficial to the application and popularization of laboratories in hospitals and grass-roots departments.

Description

Methicillin-resistant Staphylococcus aureus carrying drug-resistant genes cfr and lsa(E) at the same time and detection method thereof

technical field

The invention relates to the technical field of biomedicine, in particular to the use of a methicillin-resistant Staphylococcus aureus carrying drug-resistant genes cfr and lsa(E).

Background technique

Staphylococcus aureus is a cause of serious community- and hospital-acquired disease (including skin and soft tissue infections, infective endocarditis, necrotizing pneumonia, life-threatening sepsis, and toxic shock syndrome) pathogens (Diep et al., 2006). In recent years, the problem of drug resistance of Staphylococcus aureus has become increasingly prominent. Among them, Methicillin-resistant S. aureus (MRSA) has become the focus of attention at home and abroad. MRSA is not only resistant to β-lactam antibiotics, but also resistant to many commonly used clinical drugs such as tetracyclines, aminoglycosides and macrolides, and its morbidity and mortality exceed AIDS , SARS and avian influenza (Kluytmans, 2010). At the end of February 2017, WHO even listed it as one of the twelve superbugs in the world. In general, drug resistance in S. aureus can arise through point mutation or horizontal transfer of drug resistance genes mediated by mobile elements (such as plasmids, transposons, or insertion sequence elements, etc.), and these drug resistance genes Can be derived from horizontal transfer from the same species or from other related species (Ferrero et al., 1995; Gao et al., 2012; Tsiodras et al., 2001; Weigel et al., 2003; Weigel et al., 2007; Yan et al., 2007; al., 2016). Therefore, the emergence of drug resistance genes is the main reason for bacterial drug resistance.

cfr is a kind of plasmid-mediated multidrug resistance gene, which was obtained from a strain of Staphylococcus squirrels isolated from the nasal cavity of calves with respiratory tract infection by Professor Stefan Schwarz of the Institute of Animal Breeding of the German Federal Agricultural Center in 2000. . The results of the drug susceptibility test at that time showed that the strain was resistant to tetracycline, erythromycin, kanamycin, chloramphenicol and florfenicol. Plasmid analysis showed that the strain carried 6 plasmids ranging in size from 1.5kb to 16.5kb. The electrotransformation test was carried out and a 16.5kb plasmid pSCFS1 was obtained after screening with florfenicol. After cloning and digestion experiments, an opening encoding 349 amino acid sequences was obtained that could mediate resistance to chloramphenicol and florfenicol. The reading frame, a fragment of size 2 kb, was named chloramphenicol-florfenicol resistance, abbreviated as cfr (Schwarzet al., 2000). Further research found that the protein encoded by the cfr gene belongs to the rRNA methyltransferase, which acts on the A2503 and C2498 nucleotides of 23S rRNA, and also inhibits C2498 while performing the methylation of A2503. methylation. Due to the partial overlap of the chloramphenicol binding site and the lincomycin binding site in the 23S rRNA of Staphylococcus, and the methylation of the A2503 position leads to the above-mentioned chloramphenicol and lincomycin in the 23S rRNA. Changes in the binding site configuration of these two classes of antibiotics have resulted in resistance to chloramphenicol and lincomycin in staphylococci (Kehrenberg et al., 2005). In addition, since pleuromutilins, oxazolidinones and streptavidin A drugs all act on the transpeptidation center of 23S rRNA in Gram-positive bacteria, and the A2503 position is adjacent to the transpeptidation center, cfr The presence of genes also mediates resistance to these three classes of drugs. In 2006, Long et al. reported that cfr increased the MIC of tiamulin (a pleuromutilin antibiotic) by 128-fold against E. coli and 2048-fold against Staphylococcus aureus (Longet al., 2006). It can be seen that the cfr gene has a profound impact on bacterial resistance.

The lsa(E) gene, belonging to the ABC transporter gene, encodes an ABC transporter, which can simultaneously mediate the resistance of three classes of antibiotics with different chemical structures (linkamides, pleuromutilins, and streptavidin A) These three types of antibacterial drugs are very important anti-infective drugs in human and veterinary clinics. It is worth noting that the lsa(E) gene often exists in the multidrug resistance cluster aadE-spc-lsa(E)-lnu(B)-tnp, which usually contains multiple drug resistance genes, which are mediated by amino acids. Glycoside antibiotic resistance genes aadE and spc, lincosamide-pleuromutilin-streptanocin A resistance gene lsa (E), lincosamide resistance gene lnu (B) and Transposase tnp. In 2013, the multidrug resistance gene lsa(E) was first detected in European human-derived MRSA and MSSA (Wendlandt et al. 2013). In the same year, Li et al. detected the lsa(E) gene in pig-derived MRSA. Sequence analysis confirmed that the gene was located in the multidrug-resistant gene cluster. Subsequently, the gene was found in a variety of bacteria, including: Enterococcus faecalis, Enterococcus faecium, Staphylococcus epidermidis, Streptococcus agalactiae, Erysipelas suis, etc. Sarah Wendlandt et al. studied the lsa(E) gene in porcine methicillin-resistant Staphylococcus aureus and found that the gene could mediate pleuromutilin resistance by MIC detection of tiamulin and vonimulin. After extracting the plasmid from the lsa(E) positive strain and transforming it into Staphylococcus aureus RN4220 by electric shock, it was found that the gene was located on a 41kb plasmid, and sequence analysis confirmed that the gene was located in the multidrug resistance gene cluster aadE-spc-lsa(E )-lnu(B)-tnp, the plasmid carrying the lsa(E) drug resistance gene spreads horizontally between strains, and the drug resistance problem is serious, and people's attention has begun to be paid.

With the extensive use of antibiotics, the problem of bacterial resistance has become increasingly prominent. The emergence of drug resistance genes is the main reason for the emergence of drug resistance in bacteria. For example, the horizontal transfer of vanA drug resistance genes leads to vancomycin resistance in methicillin-resistant Staphylococcus aureus; the mcr-1 gene is the cause of gram resistance. The main reason for the rapid resistance of polymyxins in the negative bacteria. The emergence of multidrug-resistant bacterial genes can cause bacteria to appear resistant to multiple antibiotics at the same time. How to quickly and accurately discover drug-resistant genes is particularly important for further preventing the spread of drug-resistant strains and deepening the treatment of drug-resistant bacteria. As two very important multidrug resistance genes, at present, related methods can only detect cfr gene or lsa(E) gene alone, and a dual PCR detection technology that can detect cfr and lsa(E) gene at the same time has not been established. The applicant of the present invention also discovered a multi-drug resistant methicillin-resistant Staphylococcus aureus (MRSA) strain resistant to 18 kinds of antibiotics in the early stage. Through the analysis of the whole genome sequencing results, it was shown that the strain carries both the cfr gene and the lsa (E ) drug resistance gene, this strain is reported for the first time at home and abroad, and whether these two drug resistance genes coexist in other bacteria or bacterial plasmids for a long time needs to be explored for a long time.

SUMMARY OF THE INVENTION

The first purpose of the present invention is to solve the deficiencies of the prior art, and to provide a methicillin-resistant Staphylococcus aureus that simultaneously carries multiple drug resistance genes cfr and lsa(E). This bacterium is reported for the first time at home and abroad. Important materials for seeking bacterial resistance mechanisms can be used to screen novel functional microorganisms/drugs/antibacterial materials.

The methicillin-resistant Staphylococcus aureus carrying the multi-drug resistance genes cfr and lsa(E) at the same time of the present invention belongs to Staphylococcus aureus and is named as Staphylococcus aureus Sta2868B2. On May 30, 2020, it was deposited in the Guangdong Provincial Microbial Culture Collection Center (GDMCC), address: 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou City, preservation number: GDMCC No: 61037.

The Staphylococcus aureus Sta2868B2 of the present invention is a typical beta-hemolytic, reduced potassium tellurite, plasma coagulase-positive strain, identified as Staphylococcus aureus by API STAPH, with a coincidence rate of 77.7%. Its biochemical characteristics are as follows: D-dextrose, D-fructose, D-mannose, D-maltose, D-lactose, D-trehalose, D-mannitol, potassium nitrate, β-naphthyl phosphate, pyruvate Sodium, D-cane two ponds, N-acetylglucosamine, L-arginine and urea were positive, with typical physiological and biochemical characteristics of Staphylococcus aureus. With reference to the antibiotic micro-dilution method specified by the American Clinical and Laboratory Standards Institute (CLSI) 2015 edition, the minimum inhibitory concentration (MIC) of cefoxitin against Staphylococcus aureus Sta2868B2 was 32 μg According to the regulations of CLSI (2015), when the MIC value of Staphylococcus aureus to cefoxitin is ≥8 μg/mL, the bacteria can be determined to be methicillin-resistant Staphylococcus aureus. At the same time, Sta2868B2 of Staphylococcus aureus was confirmed by PCR to contain the mecA gene, which further confirmed that the strain was methicillin-resistant Staphylococcus aureus.

Further, Staphylococcus aureus Sta2868B2 of the present invention is resistant to penicillin antibiotics (penicillin, amoxicillin/clavulanic acid, ampicillin, cefepime, ceftazidime), aminoglycoside antibiotics (gentamicin, kanamycin) respectively. , streptomycin), chloramphenicol antibiotics (chloramphenicol, florfenicol), lincamide antibiotics (clindamycin), macrocyclic lipid antibiotics (erythromycin, telithromycin), Pleuromutilin antibiotics (tiamulin), quinolone antibiotics (ciprofloxacin, norfloxacin), tetracycline antibiotics (tetracycline), oxazolidinone antibiotics (linezolid), sulfonamide antibiotics (co-trimoxazole) and streptavidin antibiotics (quinupristin/dalfopristin) were resistant.

Staphylococcus aureus Sta2868B2 can be cultured in TSA, BHI and NA media.

Staphylococcus aureus Sta2868B2 of the present invention simultaneously carries two multidrug resistance genes of cfr and lsa(E), the nucleotide sequence of cfr is as shown in SEQ ID NO1, and the nucleotide sequence of lsa(E) is as shown in SEQ ID NO: 2 shown.

Therefore, the second object of the present invention is to provide a double PCR primer capable of detecting Staphylococcus aureus Sta2868B2, including the upstream primer cfr-F, the downstream primer cfr-R and the lsa(E) gene for amplifying the cfr gene fragment The upstream primers lsa(E)-F and the downstream primers lsa(E)-R of the fragment, the nucleotide sequences of each primer are as follows:

Upstream primer cfr-F: 5'-CCCAAGCTTATGAATTTTAATAATAAAACAAGT-3';

Downstream primer cfr-R: 5'-CCGGAATTCCTATTGGCTATTTTGATAAT-3';

Upstream primer lsa(E)-F: 5'-TTGTACGGAATGTATGG-3';

Downstream primer lsa(E)-R: 5'-TTGCCTCTATTAAGCACTCTT-3'.

The third object of the present invention is to provide a kind of detection method of Staphylococcus aureus Sta2868B2, comprises the following steps:

Extracting the genomic DNA of the sample to be tested, performing PCR amplification with the above-mentioned double PCR primers, and then performing electrophoresis or sequencing on the PCR product to determine the result;

When the double PCR amplification product contains a band with a size of 1050 bp, the sample to be tested contains a cfr drug resistance gene;

When the double PCR amplification product contains a band with a size of 675 bp, the sample to be tested contains the lsa(E) drug resistance gene;

When the double PCR amplification product contains both a 1050bp band and a 675bp band, the sample to be tested contains both cfr and lsa(E) multidrug resistance genes, which is Staphylococcus aureus Sta2868B2;

When the double PCR amplification product does not contain a band with a size of 1050 bp and does not contain a band with a size of 675 bp, the sample to be tested does not contain cfr and lsa(E) multidrug resistance genes.

The PCR amplification, the reaction system configuration: 2 × PCR Master Mix 12.5 μL, 10 μmol/L upstream and downstream primers cfr-F and cfr-R 0.5-1 μL each, 10 μmol/L upstream and downstream primers lsa(E)-F and 0.5-1 μL of lsa(E)-R, 1 μL of DNA of the sample to be tested, and then supplemented with deionized water to make the total reaction volume 25.0 μL.

For the PCR amplification, the reaction procedure was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30s, annealing at 47.9°C-54.7°C for 30s, and denaturation at 72°C for 90s, a total of 35 cycles; extension at 72°C for 5 min, and incubation at 4°C.

The present invention also provides a kit for detecting Staphylococcus aureus Sta2868B2, which contains the above double PCR primers and PCR reaction reagents.

The applicant of the present invention has discovered a multi-drug resistant methicillin-resistant Staphylococcus aureus (MRSA)-Sta2868B2 strain resistant to 18 antibiotics. The strain contains a plasmid carrying the cfr gene, and the chromosome carries a The multi-drug resistance region contains the lsa(E) drug resistance gene. This is the first time that cfr and lsa(E) coexist in an MRSA isolate, which has not been reported at home and abroad.

Compared with the prior art, the present invention has the following beneficial effects: the present invention provides a methicillin-resistant Staphylococcus aureus-Sta2868B2 carrying multi-drug resistance genes cfr and lsa(E) at the same time. The strain is resistant to 11 types of antibiotics, especially linezolid, and can be used as a model material for screening new functional microorganisms/drugs/antibacterial materials, and has good application prospects.

The invention provides a primer set and a double PCR detection method for simultaneously detecting multiple drug resistance genes cfr and lsa(E). This method has good stability and specificity. Two pairs of optimal primers are selected after comparing multiple sets of primers; the specificity of the reaction can be further improved by adjusting the annealing temperature. The detection has high specificity, no cross-reaction with each other and with drug resistance genes, and also has good repeatability; in addition, the invention can detect two drug resistance genes at one time, and the whole reaction is completed within 2 hours, save time and energy. At the same time, the use of the present invention does not require an expensive fluorescent PCR instrument, nor does it need to synthesize expensive probes and corresponding reagents, the detection cost is low, the operation is simple, and it is beneficial to the application and popularization of laboratories in hospitals and grass-roots departments.

Staphylococcus aureus Sta2868B2 was deposited on May 30, 2020 in Guangdong Microbial Culture Collection Center (GDMCC), address: 5th Floor, Building 59, Yard, No. 100 Middle Xianlie Road, Guangzhou City, preservation number: GDMCC No: 61037.

Description of drawings:

Fig. 1 is the colony morphology diagram of methicillin-resistant Staphylococcus aureus Sta2868B2 strain;

Fig. 2 is the microscopic observation morphological diagram of methicillin-resistant Staphylococcus aureus Sta2868B2 strain;

Fig. 3 is the API Staph biochemical identification schematic diagram of methicillin-resistant Staphylococcus aureus Sta2868B2 strain;

Figure 4 shows the drug susceptibility results of the KB method for the methicillin-resistant Staphylococcus aureus Sta2868B2 strain, A: AMP (ampicillin), AMC (amoxicillin/clavulanic acid), FEP (cefepime), FOX (cefocillin) D); B: AK (amikacin), P (penicillin), CAZ (ceftazidime), CN (gentamicin); C: K (kanamycin), C (chloramphenicol), DA ( clindamycin), S (streptomycin); D: CIP (ciprofloxacin), NOR (norfloxacin), E (erythromycin), TEL (telithromycin); E: RD (limex Fuping), TE (tetracycline), SXT (co-trimoxazole), LZD (linezolid); F: TEC (teicoplanin), FD (fusidic acid), QD (quinupristine/dalton) fopristin), F (nitrofurantoin);

Fig. 5 is the PCR amplification diagram of mecA gene of methicillin-resistant Staphylococcus aureus Sta2868B2 strain;

Figure 6 shows the electrophoresis results after PCR amplification of cfr and lsa (E) primers with gradient annealing temperature. The numbers marked on the channels represent the annealing temperature.

Figure 7 shows the optimization results of the annealing temperature of the double PCR detection method of cfr and lsa (E). The numbers marked on the channels represent the annealing temperature.

Figure 8 shows the detection results of the double PCR detection method of cfr and lsa(E) in actual samples. M: DL2000DNA Marker; Lane 1-2: cfr+lsa(E)-strain; Lane 3-7: cfr-lsa(E)+ positive strain; Lane 8: cfr+lsa(E)+ strain; Lane 9: cfr-lsa(E)-strain; C: blank control.

Detailed ways:

Those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. If no specific technology or condition is indicated in the examples, the technology or condition described in the literature in the field or the product specification is used. The reagents or instruments used without the manufacturer's indication are conventional products that can be obtained through purchase.

Example 1:

1. Isolation, identification and culture of strains

Staphylococcus aureus Sta2868B2 was isolated from quick-frozen dumplings of a supermarket brand in Hangzhou, China. The collected samples were tested qualitatively and quantitatively at the same time. The detection method was slightly adjusted on the basis of the national standard "Food Microbiological Inspection" GB 4789.10-2010. Sampling 25g (mL) and adding 225mL of normal saline into a sterile homogenizing bag and shake well to make a 1:10 sample solution, and take 1mL from it and add it to 9mL of 10% sodium chloride tryptone soybean broth In the test tube of 1:100, the sample solution of 1:100 was prepared, and the sample solution of 1:1000 was prepared according to the above method; each gradient was 3 parallel. The sample solutions of gradient concentrations were streaked on the Staphylococcus aureus color plate medium, and cultured at 37°C for 24-48h. Its typical Staphylococcus aureus colonies are pink globules with smooth edges on the chromogenic plate (Figure 1). The target colonies were transferred from the NA plates to brain heart infusion nutrient broth (BHI) and recovered at 37°C overnight. Under sterile conditions, the bacterial solution was added to a tube with a final concentration of 40% glycerol, stored in a -40°C refrigerator, and stored in a freeze-dried tube, thereby obtaining the strain Sta2868B2.

The purified strain Sta2868B2 was identified in terms of morphological characteristics, physiology and biochemistry, serotype and molecular biology.

Staining microscopy: smear suspicious colonies, carry out Gram staining, and observe the morphology by microscopy. Staphylococcus aureus was Gram-positive and formed a prototype grape cluster under the microscope (Figure 2).

Plasma coagulase test: inoculate a single colony into 5 mL of BHI medium and culture at 37°C for 18-24h. Aspirate 1 mL of the culture medium, add it to plasma coagulase, and incubate at 37°C. After 2.5h, observe whether the coagulation occurs once every hour. If it does not coagulate after 6h, incubate overnight to observe and verify.

API Staph identification: Scrape pink single colonies from the chromogenic plate, prepare a cell suspension with appropriate turbidity with normal saline, and use API Staph biochemical identification reagent strips for identification (Figure 3).

Strain Sta2868B2 is a typical β-hemolytic, reduced potassium tellurite, positive plasma coagulase strain, identified as Staphylococcus aureus by API STAPH, with a coincidence rate of 77.7%. Its biochemical characteristics are as follows: D-dextrose, D-fructose, D-mannose, D-maltose, D-lactose, D-trehalose, D-mannitol, potassium nitrate, β-naphthyl phosphate, pyruvate Sodium, D-cane two ponds, N-acetylglucosamine, L-arginine and urea were positive, with typical physiological and biochemical characteristics of Staphylococcus aureus. With reference to the antibiotic microdilution method specified by the American Clinical and Laboratory Standards Institute (CLSI) 2015 edition for drug resistance identification, the minimum inhibitory concentration (MIC) of cefoxitin for the bacteria was 32 μg/mL, According to the regulations of CLSI (2015), when the MIC value of Staphylococcus aureus to cefoxitin is ≥8 μg/mL, the bacteria can be determined to be methicillin-resistant Staphylococcus aureus. At the same time, the strain was confirmed by PCR to contain the mecA gene, which further confirmed that the strain was methicillin-resistant Staphylococcus aureus.

By comprehensively judging the appearance, morphology, Gram staining and biochemical reaction of the strain Sta2868B2, the strain Sta2868B2 can be identified as Staphylococcus aureus and named as Staphylococcus aureus Sta2868B2, which was preserved in Guangdong on May 30, 2020 Provincial Microorganism Culture Collection Center (GDMCC), Address: 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou City, preservation number: GDMCC No: 61037.

2. Analysis of drug susceptibility characteristics

The drug susceptibility of Staphylococcus aureus strain Sta2868B2 was confirmed by gradient dilution method and KB method. The gradient dilution method used Staphylococcus aureus ATCC29213 as the quality control strain, and the KB method used Staphylococcus aureus ATCC25923 as the quality control strain. Both methods refer to the determination according to the methods and standards of the American Committee for Clinical Testing Standards (CLSI).

Gradient dilution method: each group was operated in parallel for 3 times, and the average concentration was obtained. Select a sterile 96-well flat-bottom microplate, add 200 μL MH broth medium to the first well of each row as a negative control; add 100 μL Staphylococcus aureus Sta2868B2 bacterial solution and 100 μL MH broth to the second well of each row for culture Base as a positive control; add 100 μL of MH broth medium to the 3rd to 12th wells of each row, then add 100 μL of the drug to be tested in the 3rd well of each row, mix evenly, and remove 100 μL and transfer it to the 4th well. And so on until the 12th well was mixed, 100 μL was discarded, and finally 100 μL of the mixed bacterial suspension of Sta2868B2 of the present invention (5×10 ⁵ cfu/mL) was added to each well. The culture plate was placed in a 37°C incubator for 18h to observe the results and determine its MIC value. Select the black background for observation, and take the drug concentration in the lowest concentration well with clear and translucent solution as the MIC.

KB method: The strain (Sta2868B2 of Staphylococcus aureus) was activated by NA plate, then diluted with physiological saline to a final concentration of 1×10 ⁷ cfu/mL and spread on MH plate. The surface of the medium was incubated at 37°C for 24h. The size of the inhibition zone was measured with a vernier caliper, accurate to 0.01 mm.

Select antibiotics amoxicillin-clavulanic acid, ampicillin, cefepime, penicillin, ceftazidime, amikacin, gentamicin, kanamycin, streptomycin, chloramphenicol, clindamycin, erythromycin tetracycline, telithromycin, ciprofloxacin, norfloxacin, tetracycline, linezolid, rifampicin, co-trimoxazole, quinupristin/darfupristin, tiraconine, nitrofurantoin, brown mold Drug susceptibility confirmation was carried out with ketamine, florfenicol, peptidomycin, vancomycin and daptomycin, among which amoxicillin-clavulanate, ampicillin, cefepime, penicillin, ceftazidime, amikacin, acetaminophen Damycin, kanamycin, streptomycin, telithromycin, ciprofloxacin, norfloxacin, tetracycline, co-trimoxazole, quinupristin/dafupristin, tiraconine, nitrofurantoin The KB method was used for confirmation of and fucoidin (Fig. 4), and the other antibiotics were multiplexed by the serial dilution method. Experiments show that the MIC value of Staphylococcus aureus Sta2868B2 cefoxitin is 32 μg/mL, which is methicillin-resistant Staphylococcus aureus, and is resistant to 11 types of antibiotics, especially linezolid. The specific drug susceptibility results of Staphylococcus aureus Sta2868B2 are shown in Table 1.

Table 1 The drug-resistant phenotype of methicillin-resistant Staphylococcus aureus Sta2868B2 strain

Note: FFC, florfenicol; CHL, chloramphenicol; CLI, clindamycin; TIA, peptidomycin; LZD, linezolid; ERY, erythromycin; FOX, cefoxitin; VAN, vancomycin RIP, rifampicin; DAP, daptomycin; AMP, ampicillin; FEP, cefepime; CAZ, ceftazidime; GEN, gentamicin; KAN, kanamycin; STR, streptomycin ; TEL, telithromycin; CIP, ciprofloxacin; NOR, norfloxacin; TET, tetracycline; SXT, co-trimoxazole; QD, quinupristin/dalfopristin

3. Detection of drug resistance gene of methicillin-resistant Staphylococcus aureus Sta2868B2 strain

Since Staphylococcus aureus Sta2868B2 is resistant to the selected β-amide antibiotics, in order to further confirm whether it is methicillin-resistant Staphylococcus aureus, PCR was used to confirm whether the Staphylococcus aureus strain Sta2868B2 contained mecA/ mecC gene. The mecA/mecC gene encodes penicillin-binding protein 2a (PBP2a), which makes the related strains have a very low affinity for β-lactam antibiotics. When other PBPs are inhibited by β-lactam antibiotics, PBP2a is not inhibited and replaces other PBPs, which play a role in catalyzing cell wall synthesis, allowing bacteria to survive. The primers and amplification methods of mecA and mecC genes were reported in the previous literature. The single-plex PCR method was used, and the primer sequences were shown in Table 2. PCR amplification conditions were as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 45s, annealing at 56°C for 45s, and extension at 72°C for 50s for 35 cycles; and a final extension at 72°C for 8 min. The reaction system (25 μL) contained: 12.5 μL 2×DreamTaq mastermix, 9.5 μL ultrapure water, 40 ng template DNA, 0.5 μmol/L upstream and downstream primers. 5 μL of the PCR product was loaded on a 2.0% agarose gel for electrophoresis separation (120V, 25min), using a 2000bp DNAMarker, and its electrophoresis chart is shown in 5.

Table 2 Staphylococcus aureus mecA/mecC primers and amplified fragments

Example 2

According to the present invention, the specific primers for detecting the cfr gene and the multidrug resistance gene lsa(E) primers are designed, and the double PCR primers which can simultaneously detect the multidrug resistance genes cfr and lsa(E) are determined, including the primers for amplifying the cfr gene The upstream primer cfr-F of the fragment, the downstream primer cfr-R and the upstream primer lsa(E)-F and the downstream primer lsa(E)-R of the lsa(E) gene fragment, the nucleotide sequences of each primer are as follows:

Upstream primer cfr-F: 5'-CCCAAGCTTATGAATTTTAATAATAAAACAAGT-3';

Downstream primer cfr-R: 5'-CCGGAATTCCTATTGGCTATTTTGATAAT-3';

Upstream primer lsa(E)-F: 5'-TTGTACGGAATGTATGG-3';

Downstream primer lsa(E)-R: 5'-TTGCCTCTATTAAGCACTCTT-3'.

(1) Configuration of the cfr gene reaction system: 12.5 μL of 2×PCR Master Mix, 0.5 μL of 10 μmol/L upstream and downstream primers cfr-F and cfr-R, 1 μL of Staphylococcus aureus Sta2868B2 DNA, and then added deionized water to make up. Bring the total volume of the reaction to 25.0 μL.

PCR reaction program: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30s, annealing at 47.9°C to 65.1°C for 30s, extension at 72°C for 90s, for 35 cycles; the final extension at 72°C for 10 min, and incubation at 4°C.

(2) The configuration of the reaction system of the lsa(E) gene: 2×PCR Master Mix 12.5 μL, 10 μmol/L upstream and downstream primers lsa(E)-F and lsa(E)-R 0.5 μL each, Staphylococcus aureus Sta2868B2 DNA 1 μL, and then add deionized water to make up, making the total reaction volume 25.0 μL.

PCR reaction program: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30s, annealing at 47.9°C to 65.1°C for 30s, extension at 72°C for 90s, for 35 cycles; the final extension at 72°C for 10 min, and incubation at 4°C.

As shown in Figure 6, within the annealing temperature of 47.5 ℃ ~ 58.3 ℃, both have good amplification effect, and sequencing, methicillin-resistant Staphylococcus aureus Sta2868B2 strain carries these two multi-drug resistance genes at the same time, The nucleotide sequence of cfr is shown in SEQ ID NO:1, and the nucleotide sequence of lsa(E) is shown in SEQ ID NO:2.

Example 3

Annealing temperature optimization results for the duplex PCR assay for cfr and lsa(E):

(1) Double PCR reaction system configuration: 2×PCR Master Mix 12.5 μL, 10 μmol/L upstream and downstream primers cfr-F and cfr-R 0.5-1 μL each, 10 μmol/L upstream and downstream primers lsa(E)-F and lsa( E)-R each 0.5-1 μL, Staphylococcus aureus Sta2868B2 DNA 1 μL, and then supplemented with deionized water to make the total reaction volume 25.0 μL.

(2) Double PCR reaction procedure: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30s, annealing at 47.9°C-63.5°C for 30s, denaturation at 72°C for 90s, a total of 35 cycles; extension at 72°C for 5 min, and incubation at 4°C.

(3) Optimization results: The optimization results of the annealing temperature are shown in Figure 7. When the annealing temperature is 49.5 °C, the amplification effects of the two are consistent, and the electrophoresis bands are also brighter, so the cfr and lsa(E) in the reaction system The optimum annealing temperature is 50°C.

Example 4

Utilize the double PCR primer set and detection method of cfr and lsa(E) provided by the present invention, comprising the following steps:

(1) Double PCR reaction system configuration: 2×PCR Master Mix 12.5 μL, 10 μmol/L upstream and downstream primers cfr-F and cfr-R 0.5-1 μL each, 10 μmol/L upstream and downstream primers lsa(E)-F and lsa( E)-R each 0.5-1 μL, the sample DNA to be tested is 1 μL, and then supplemented with deionized water to make the total reaction volume 25.0 μL.

(2) Double PCR reaction procedure: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30s, annealing at 47.9°C～54.7°C for 30s, denaturation at 72°C for 90s, a total of 35 cycles; extension at 72°C for 5 min, incubation at 4°C.

(3) Detection, analysis and determination of PCR amplification products: Take 8 μL PCR amplification products, electrophoresis on a 1.5% agarose gel with 0.01% GelRed nucleic acid dye, and detect PCR amplification products on a gel imaging system. Result judgment:

If the double PCR amplification product contains a band with a size of 1050 bp, the sample to be tested contains a cfr drug resistance gene;

If the double PCR amplification product contains a band with a size of 675 bp, the sample to be tested contains the lsa(E) drug resistance gene;

If the double PCR amplification product contains both a 1050bp band and a 675bp band, the sample to be tested contains both cfr and lsa(E) multidrug resistance genes;

If the double PCR amplification product does not contain a band with a size of 1050 bp and does not contain a band with a size of 675 bp, the sample to be tested does not contain cfr and lsa(E) multidrug resistance genes.

Example 5

Specificity determination of the double PCR detection method for the multidrug resistance genes cfr and lsa(E):

A total of 9 strains of cfr-positive strains, lsa(E)-positive strains, cfr-lsa(E)-positive strains and cfr-lsa(E)-negative strains were used, respectively. After DNA was extracted by conventional methods, PCR reaction was carried out with the method of Example 4. and result judgment. The strains of the bacteria used and the test results are shown in Table 3 below. In Table 3, in the column of test results, "+" indicates positive, "-" indicates negative; "√" indicates that the results are consistent with known strains. The electrophoresis results of PCR products are shown in Figure 8; among them, Lane 1-2 is cfr+lsa(E)- strain; Lane 3-7 is cfr-lsa(E)+ strain; Lane 8 is cfr+lsa(E)+ strain; Lane 9 is cfr-lsa(E)-strain; M is 2000Maker; C is blank control.

The double PCR detection test results of the multi-drug resistance genes cfr and lsa (E) of table 3 of the present invention

It can be seen from FIG. 7 that the results of the double PCR are consistent with the actual situation, indicating that the primer set and the detection method of the present invention have strong specificity.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, but not to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that , the technical solutions of the present invention may be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention.

sequence listing

<110> Guangdong Institute of Microbiology (Guangdong Microbiological Analysis and Testing Center)

<120> A methicillin-resistant Staphylococcus aureus carrying drug-resistant genes cfr and lsa(E) at the same time and its detection method

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1050

<212> DNA

<213> Staphylococcus aureus Sta2868B2 (Staphylococcus aureus)

<400> 1

atgaatttta ataataaaac aaagtatggt aaaatacagg aatttttaag aagtaataat 60

gagcctgatt atagaataaa acaaataacc aatgcgattt ttaaacaaag aattagtcga 120

tttgaggata tgaaggttct tccaaaatta cttagggagg atttaataaa taattttgga 180

gaaacagttt tgaatatcaa gctcttagca gagcaaaatt cagagcaagt tacgaaagtg 240

ctttttgaag tatcaaagaa tgagagagta gaaacggtaa acatgaagta taaagcaggt 300

tgggagtcat tttgtatatc atcacaatgc ggatgtaatt ttgggtgtaa attttgtgct 360

acaggcgaca ttggattgaa aaaaaaccta actgtagatg agataacaga tcaagtttta 420

tacttccatt tattaggtca tcaaattgat agcatttctt ttatgggaat gggtgaagct 480

ctagccaacc gtcaagtatt tgatgctctt gattcgttta cggatcctaa tttatttgca 540

ttaagtcctc gtagactttc tatatcaacg attggtatta tacctagtat caaaaaaata 600

acccaggaat atcctcaagt aaatcttaca ttttcattac actcacctta tagtgaggaa 660

cgcagcaaat tgatgccaat aaatgataga tacccaatag atgaggtaat gaatatactc 720

gatgaacata taagattaac ttcaaggaaa gtatatatag cttatatcat gttgcctggt 780

gtaaatgatt ctcttgagca tgcaaacgaa gttgttagcc ttcttaaaag tcgctataaa 840

tcagggaagt tatatcatgt aaatttgata cgatacaatc ctacaataag tgcacctgag 900

atgtatggag aagcaaacga agggcaggta gaagcctttt acaaagtttt gaagtctgct 960

ggtatccatg tcacaattag aagtcaattt gggattgata ttgacgctgc ttgtggtcaa 1020

ttatatggta attatcaaaa tagccaatag 1050

<210> 2

<211> 804

<212> DNA

<213> Staphylococcus aureus Sta2868B2 (Staphylococcus aureus)

<400> 2

atgttaaaac aaaaagaatt aattgcaaac gttaagaatc ttactgagtc agatgaacga 60

attacagctt gtatgatgta tggatcgttt accaaaggag aaggtgacca atactctgat 120

atagagttct atatattttt gaaagatagt ataacctcga actttgattc atccaactgg 180

ttgtttgacg tagctccgta cttgatgctt tataaaaatg agtacggaac agaggtagtt 240

atttttgata atcttatacg tggggaattt catttccttt ctgaaaaaga tatgaacata 300

atcccctcgt ttaaagattc aggttatatt cctgatacga aggctatgct tatttacgat 360

gaaacagggc aattagaaaa ttatttatca gagataagtg gtgcaagacc aaatagactt 420

actgaagaaa atgctaattt tttgttgtgt aatttctcta atctatggtt gatgggaatc 480

aacgttctaa aaagaggaga atatgctcgt tcattagaac tcttatcaca acttcaaaaa 540

aatacactac aacttatacg tatggcagaa aaaaatgctg ataattggct aaacatgagt 600

aaaaaccttg aaaaagaaat tagccttgaa aattataaaa aatttgcaaa gaccactgct 660

cgattagata aggtagaatt atttgaagcc tataaaaatt ctttgctatt agttatggat 720

ttgcaaagtc accttattga acaatacaac ttaaaagtta cacatgacat tttagaaaga 780

ttgttgaatt acattagtga atag 804

Claims (1)

1. Staphylococcus aureus Sta2868B2, deposit number: GDMCC No: 61037.

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