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Detection of Pathogens and Ampicillin-resistance Genes Using Multiplex Padlock Probes
使用多重锁式探针检测病原体和氨苄西林耐药基因   

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Abstract

Diagnostic assays for pathogen identification and characterization are limited either by the number of simultaneously detectable targets, which rely on multiplexing methods, or by time constraints due to cultivation-based techniques. We recently presented a 100-plex method for human pathogen characterization to identify 75 bacterial and fungal species as well as 33 clinically relevant β-lactamases (Barišić et al., 2016). By using 16S rRNA gene sequences as barcode elements in the padlock probes, and two different fluorescence channels for species and antibiotic resistance identification, we managed to cut the number of microarray probes needed by half. Consequently, we present here the protocol of an assay with a runtime of approx. 8 h and a detection limit of 105 cfu ml-1. A total of 89% of β-lactamases and 93.7% of species were identified correctly.

Keywords: Multiplex detection(多重检测), Human pathogens(人体病原菌), Padlock probes(锁式探针), Species identification(物种鉴定), Antibiotic resistance identification(抗生素耐药鉴定)

Background

β-Lactamases are a class of antibiotic resistance genes which provide resistance to β-lactam antibiotics, which structurally mimic D-alanyl-D-alanine, a component of the bacterial cell wall and thereby inhibit bacterial cell wall synthesis. β-Lactamases are able to hydrolyze the central component of β-lactam antibiotics, the β-lactam ring, and render them useless (Kong et al., 2010). Today, over 1,000 β-lactamases are described and a huge potential environmental reservoir exists (Bush, 2010; Brandt et al., 2017). β-Lactamases are ancient enzymes and we classify them as class A, C, and D (serine β-lactamases) with a serine catalytic site, or as class B (metallo-β-lactamases) whose active center is zinc-dependent (Hall and Barlow, 2003 and 2004). Despite their high phylogenetic age, the serine β-lactamases probably share a common ancestor and acquired a high number of SNPs due to a permanent selection pressure. Additionally, to β-lactamases, more than 500 other antibiotic resistance genes exist (Zankari et al., 2012).

Current multiplexing methods reduce the number of simultaneously detectable targets while cultivation-dependent techniques are limited by time constraints. Given these facts and the high number of pathogens of clinical importance, new methods are needed for the fast characterization and identification of pathogens, virulence factors and antibiotic resistance genes.

The gold standard of infection diagnostics takes 2-3 days and is cultivation-dependent (Marik, 2014). Additionally, PCR methods provide results at a high sensitivity and low cost, but remain impractical due to the high number of clinically relevant targets (Mussap et al., 2007; Wellinghausen et al., 2009). The current multiplex-PCR protocols are not suitable for a high number of targets and the limitations can only be overcome by microfluidic-based assays, which run a high number of analyses in parallel.

Padlock probes are linear DNA probes, which upon annealing, circularize and are then used for rolling circle amplifications (Nilsson et al., 1994; Hardenbol et al., 2005). They allow for a higher number of multiplexing and can easily be integrated into established PCR-based assays. Recently, we presented a 100-plex method based on padlock probes for pathogen characterization (75 bacterial and fungal species) as well as 33 clinically important β-lactamases (Barišić et al., 2016). By adapting this method from our previous work (Barišić et al., 2013), we increased the sensitivity and specificity of the assay and we managed to cut down the number of microarray probes needed by half by using 16S rRNA sequences as barcode elements in the padlock probes and two different fluorescence channels for species identification and antibiotic resistance characterization. Here, we present an assay to overcome time limitations and to increase the number of detectable targets. Our assay allows for the detection of up to 105 cfu ml-1 in a total of 8 h. We were able to retrieve and correctly characterize 89% of β-lactamases and to identify 93.7% of all species.

Materials and Reagents

  1. Filter tips 10 µl, 20 µl, 200 µl and 1,250 µl (e.g., Biozym, catalog numbers: VT0200 , VT0220 , VT0240 and VT0270 )
  2. Safe-Lock tubes 1.5 ml (Eppendorf, catalog number: 022363204 )
  3. Falcon 15 ml conical centrifuge tubes (Corning, catalog number: 352196 )
  4. 2 ml screw-cap tube (e.g., Roche Molecular Systems, catalog number: 03358941001 )
  5. Ritter Riplate 384 well plate PP (Ritter, catalog number: 43001-0035 )
  6. Glass beads, acid-washed, 150-212 μm (Sigma-Aldrich, catalog number: G1145 )
  7. Glass beads, acid-washed, 425-600 μm (Sigma-Aldrich, catalog number: G8772 )
  8. Vantage Silylated Aldehyde Slides (CEL Associates, catalog number: VSS-25 )
  9. LifterSlip mSeries cover slips for microarray slides, 55 µl (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 25X60IM5439001LS )
  10. PCR tubes 0.2 ml (Eppendorf, catalog number: 0030124537 )
  11. Millex-GV 0.22 µm syringe filter units (Merck, catalog number: SLGV033RS )
  12. 50 ml tubes (e.g., Corning, catalog number: 430829 )
  13. Disposable syringes, e.g., Omnifix 50 ml LL (B. Braun Medical, catalog number: 8508577FN )
  14. The multiplex PCR primers (66 in total, Table S1) targeting the β-lactamase genes were ordered from Microsynth (Balgach, Switzerland) (Note 3)
  15. The padlock probes (66 in total, Figure 1, Table S2) targeting the β-lactamases were ordered from Integrated DNA Technologies (Coralville, IA, USA)


    Figure 1. Schematic illustration of a padlock probe. The 3’ and the 5’ target recognition arms bind to the multiplex PCR products of the β-lactamase genes. During the binding process, the padlock probes are circularized and subsequently ligated. The maximum distance of the padlock probe binding region to the 3’ or 5’-ends of the PCR product should not exceed 200 base pairs. Since the padlock probes gets concatenated with the PCR product upon the ligation reaction, longer distances cause an inhibition of subsequent RCAs. The C2CA sequence is needed for the circle-to-circle amplification and comprises an AluI restriction site for monomerization of the amplification products. The barcode sequence is derived from the 16S rRNA gene. This allowed us to halve the number of microarray probes because the C2CA products and 16S rRNA gene PCR products are detected on the same microarray probe but in different fluorescence channels.

  16. The 5’-amino-modified microarray probes (274 in total, Table S3) were ordered from Microsynth (Balgach, Switzerland) (Note 3)
  17. Nuclease-free water for PCR application comes with the Mastermix 16S Basic kit or can be purchased separately (e.g., Fresenius Kabi, Aqua bidest. ‘Fresenius’, no catalog number)
  18. Ultrapure water, hereafter simply referred to as water or H2O (Note 1)
  19. The universal bacterial 16S rRNA primers 45f++ (5’-GCYTAAYACATGCAAGTCGARCG-3’) and 783R (5’-TGGACTACCAGGGTATCTAATCCT-3’) were ordered from Integrated DNA Technologies (Coralville, IA, USA)
  20. The fungal 18S rRNA (ITS region) primers ITS3 (5’-GCATCGATGAAGAACGCAGC-3’) and ITS4+ (5’-TCCT-CCGCTTATTGATATGCTTAAGT-3’) were ordered from Integrated DNA Technologies (Coralville, IA, USA)
  21. The circle-to-circle amplification (C2CA) oligonucleotides C2CA- (5’-TACTCGAGGAGCTGCATACAC-3’) and C2CA+ (5’-GTGTATGCAGCTCCTCGAGTA-3’) were ordered from Integrated DNA Technologies (Coralville, IA, USA)
  22. The Cy5-labelled hybridization control (complimentary to ‘Bsrev’) (5’-Cy5-AAGCTCACTGGCCGTCGTTTTAAA-3’) was ordered from Microsynth (Balgach, Switzerland)
  23. T4 DNA ligase (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EL0011 )
  24. T4 polynucleotide kinase (10 U/µl) supplied with 10x reaction buffer A (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EK0031 )
  25. ATP solution (100 mM) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0441 )
  26. Mastermix 16S Basic, DNA-free (Molzym, catalog number: S-040-0250 ) containing a 2.5x complete master mix, Moltaq 16S DNA polymerase and PCR-grade water (Note 2)
  27. dNTP mix (10 mM each) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0192 )
  28. Cy5-dCTP (1 mM solution) (GE Healthcare, catalog number: PA55021 )
  29. VentR (exo-) DNA polymerase (New England Biolabs, catalog number: M0257S ) supplied with 10x ThermoPol reaction buffer and 100 mM MgSO4
  30. ExpressHyb hybridization solution (Takara Bio, Clontech, catalog number: 636831 )
  31. Ampligase thermostable DNA ligase (5 U/µl) and Ampligase 10x reaction buffer (Epicentre, catalog number: A32750 )
  32. Bovine serum albumin (BSA), molecular biology grade (New England Biolabs, catalog number: B9000S )
  33. phi29 DNA polymerase supplied with 10x phi29 DNA polymerase reaction buffer (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EP0091 )
  34. AluI restriction enzyme (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: ER0011 )
  35. Atto532-dCTP (MoBiTec, Göttingen, Germany)
  36. SDS solution 10% for molecular biology (AppliChem, catalog number: A0676 )
  37. Tryptic soy broth, also referred to as CASO medium (Casein-peptone soymeal-peptone broth) (Merck, catalog number: 105459 )
  38. PBS (10x), pH 7.2 (Thermo Fisher Scientific, GibcoTM, catalog number: 70013 )
  39. Betaine monohydrate (Sigma-Aldrich, catalog number: B2754 )
  40. UltraPure SSC, 20x (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15557036 )
  41. CASO medium (see Recipes)
  42. 1x phosphate-buffered saline (PBS) (see Recipes)
  43. 2x spotting buffer (6x SSC, 3 M betaine) (see Recipes)

Equipment

  1. Pipettes (e.g., Sartorius, catalog numbers: 728020 , 728050 , 728060 and 728070 )
  2. Microbiological incubator shaker (e.g., IKA, model: KS 4000 i control )
  3. Tabletop centrifuge for 1.5 ml tubes (e.g., Eppendorf, model: 5424 )
  4. Roche MagNA Lyser Instrument (Basel, Switzerland)
  5. Thermomixer comfort (Eppendorf, Hamburg, Germany)
  6. Epoch Microplate spectrophotometer (Biotek, Winooski, VT, USA)
  7. Biosan DNA/RNA UV-cleaner box (Warren, MI, USA) (recommended, see Note 4)
  8. Thermal cycler (e.g., Thermo Fisher Scientific, Applied Biosystems, model: GeneAmpTM PCR System 2700 ) (Paisley, UK)
  9. Heraeus Megafuge 40R with a TX-750 rotor (Thermo Fisher Scientific, Thermo ScientificTM, model: HeraeusTM MegafugeTM 40R , catalog number: 75004518; TX-750 rotor: Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 75003180 ) and inserts for plates (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 75003617 ) and Falcon tubes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 75003608 )
  10. Slide humidity incubation box (e.g., LabScientific, catalog number: HIC-3 )
  11. GeneMachines Omnigrid 100 contact arrayer (Madison, WI, USA)
  12. International Microarray Pin Stealth 3 SMP3 (Telechem, catalog number: SMP3 )
  13. Agilent SureScan DNA Microarray Scanner (Santa Clara, CA, USA)
  14. Autoclave
  15. Sartorius arium pro UV ultrapure water system (Sartorius, model: arium® pro )

Software

  1. GenePix Pro 6.0 Software (Molecular Devices LLC, Sunnyvale, CA, USA)
  2. Microsoft Excel or any other data analysis software
  3. ARB software package for microarray probe design (Note 3)
  4. Primer3 for primer design (Note 3)

Procedure

  1. Preparation of oligonucleotides
    1. Dissolve the 5’ amino modified microarray probes to 100 µM in nuclease-free water.
    2. Dissolve the 33-plex PCR primers to 100 µM in nuclease-free water, then create a 100 µM primer mix by pipetting equal volume of all 66 primers (e.g., 6 µl each) into one 1.5 ml Eppendorf tube.
    3. Dissolving and phosphorylation of the padlock probes (Note 8).
      1. Dissolve the padlock probes to 200 µM in nuclease-free water, then create a 2 µM per probe Master mix by pipetting 1 volume of padlock probe (e.g., 66 x 10.00 µl) and 0.34 volumes of nuclease-free water (e.g., 66 x 10.00 x 0.515 = 340.00 µl in total) into a 1.5 ml Eppendorf tube.
      2. Phosphorylation of the padlock probe Master mix is done by combining 10 µl of T4 polynucleotide kinase (0.2 U/µl final conc.), 50 µl of 10x reaction buffer A (1x final conc.), 50 µl of 10 mM ATP (1 mM final conc.), 250 µl of the padlock probe mix (1 µM per probe final conc.) and 140 µl PCR-grade water (final volume 500 µl).
      3. Incubate for 30 min at 37 °C, then for 10 min at 65 °C.
    4. Store microarray probes, padlock probes, primers and the respective master mixes at -20 °C.

  2. Growth of bacterial cells and preparation of genomic DNA extracts (Note 5)
    1. Inoculate a pure culture (e.g., from a glycerol stock) or a clinical isolate (e.g., from an agar culture) in approx. 5 ml CASO medium (see Recipes) in a 15 ml Falcon tube (Note 6). Do not completely screw the cap to allow for proper aeration of the bacteria.
    2. Incubate samples for 14-18 h at 37 °C in an orbital shaking incubator at 300 rpm (Note 7).
    3. Vortex the Falcon tube and transfer 2 ml of the culture to a 2 ml Eppendorf tube.
    4. Pellet the bacteria by centrifugation for 5 min at 2,400 x g at room temperature.
    5. Resuspend the pellet in 1 ml 1x PBS (see Recipes).
    6. Centrifuge the suspension for 5 min at 2,400 x g at room temperature.
    7. Resuspend the pellet in 1 ml sterile ddH2O and transfer to a 2 ml screw-cap tube suitable for the MagNA Lyser.
    8. Add acid-washed beads of 150-212 µm and acid-washed beads of 425-600 µm diameter at equal volumes to the tube, so that the beads make up about one third of the tube.
    9. Disrupt the bacterial and fungal cells using a MagNA Lyser for 30 sec at 6,500 rpm at room temperature.
    10. Leave sample 5 min at room temperature.
    11. Repeat step A9 once.
    12. For thermal lysis, incubate the sample for 20 min at 95 °C in a thermomixer.
    13. Centrifuge for 10 min at maximum speed.
    14. Transfer the supernatant to a new tube.

  3. Microarray processing
    1. Prepare the Ritter 384-well spotting plate by adding 18 µl of the 2x spotting buffer (see Recipes) to every well to be used, and then add 18 µl of microarray oligonucleotide to every well (final concentration of every oligonucleotide is now 50 µM in 1x spotting buffer). Take note of which oligonucleotides are in which wells and how your spotter transfers the oligonucleotides to the glass slides (Note 4).
    2. Set the Omnigrid contact arrayer to an air humidity of 60% and set up the machine to print the oligonucleotides in 4 replicates per slide (Notes 9 and 10).
    3. Spot the oligonucleotides onto glass slides with aldehyde surface using SMP3 pins which produce spots with 100 µm in diameter.
    4. After spotting, allow the oligonucleotides to attach for approx. 5 h in the humid atmosphere.
    5. The glass slides are not washed after spotting.
    6. Store the slides at room temperature.

  4. Species identification
    1. 16S rRNA gene PCR
      1. Prepare the PCR master mix in 0.2 ml PCR tubes for n + 1 reactions, using the Molzym Mastermix 16S Basic kit, according to Table 1. Include at least one negative control (NTC) where you replace the genomic DNA extract with water. A primer mix consisting of equal quantities of all four primers at 15 µM can be prepared in advance to avoid repetitive pipetting. In this case, 1.20 µl of the primer mix is used. Keep samples chilled on ice until they are placed into the thermal cycler (Note 11).

        Table 1. Multiplex PCR Master mix for 16S rRNA gene identification


      2. Run this reaction in parallel with ‘33-plex pre-amplification PCR for β-lactamase-encoding gene identification’ using the PCR program outlined in Table 2 in a thermal cycler with heated lid (Figure 2).
      3. Keep the samples at 4 °C after the PCR run is finished.

        Table 2. Multiplex PCR program for 16S rRNA gene identification and for pre-amplification of β-lactamase-encoding gene information



        Figure 2. Schematic illustration of the detection principle of the assay. For the species identification, the 16S rRNA gene is amplified using universal 16S primers (A1). Subsequently, the PCR products are labeled using a linear PCR and Cy5-modified cytosines (A2). In parallel, β-lactamase genes are pre-amplified in a 33-plex PCR (B1). Then, the padlock probes are hybridized (B2) and ligated (B3) using the β-lactamase PCR products as template and amplified in a first rolling circle amplification (RCA) (B4). The RCA products comprise a C2CA sequence and corresponding 16S rRNA barcode sequences. In a next reaction, the RCA products are monomerized (B5) using a restriction enzyme and circularized (B6) to serve as a new template for a second RCA (B7). These C2CA products are labelled in a linear PCR reaction using Atto532-modified cytosines (B8). Finally, the labelled amplification products from the 16S rRNA gene PCR and from the multiplex padlock assay are pooled together and hybridized to a microarray detecting the products at two different fluorescence wavelengths (C).

    2. Linear PCR for labelling of the PCR products
      1. Prepare the linear PCR master mix in 0.2 ml PCR tubes for n + 1 reactions according to Table 3 with the PCR products from the previous PCR. Keep samples chilled until placed in the thermal cycler.

        Table 3. Linear PCR Master mix for labelling of the PCR products


      2. Run this reaction in parallel with ‘Labelling of C2CA products in a linear PCR’ using the PCR program outlined in Table 4 in a thermal cycler with heated lid.

        Table 4. Linear PCR program for labelling of the PCR and C2CA products


  5. Multiplex β-lactamase-encoding gene identification
    1. 33-plex pre-amplification PCR for β-lactamase-encoding gene identification
      1. Prepare the PCR master mix in 0.2 ml PCR tubes for n + 1 reactions, using the Molzym Mastermix 16S Basic kit, according to Table 5. Include at least one NTC. The primer concentration corresponds to 76 nM each. Keep samples chilled until placed in the thermal cycler (Note 11).

        Table 5. Multiplex pre-amplification PCR for β-lactamase-encoding gene identification


      2. Run the PCR program as described in Table 2.
    2. Hybridization and circularization by ligation of padlock probes
      1. Prepare the ligation mixture (10 µl per sample) according to Table 6 for n + 1 samples.

        Table 6. Enzymatic reaction mix for circularization by enzymatic ligation of padlock probes. A padlock master mix concentration of 66 nM equals to 1 nM per probe.


      2. Add 10 µl of the ligation mixture to the PCR products from the 33-plex PCR. The final concentration of every padlock probe is 100 pM each.
      3. Incubate at 95 °C for 5 min, then at 60 °C for 1 h in a thermal cycler with heated lid.
    3. First rolling circle amplification
      1. Prepare the reaction Master mix for the first rolling circle amplification (RCA) as described in Table 7, using the ligation products from the previous reaction, for n + 1 reactions.

        Table 7. Reaction mix for the first RCA


      2. Incubate for 20 min at 37 °C.
      3. Inactivate the polymerase by a 2 min incubation at 65 °C.
    4. Hybridization of C2CA oligonucleotides and monomerization by enzymatic restriction
      1. Prepare the restriction mixture as described in Table 8 for n + 1 reactions.

        Table 8. Reaction mix for the enzymatic restriction reaction


      2. Add 5 µl of the restriction mixture to the first RCA mixture (total volume is now 25 µl).
      3. Incubate at 37 °C for 5 min.
      4. Incubate at 65 °C for 5 min to inactivate the restriction enzyme.
    5. Circularization, ligation and second RCA
      1. Prepare the ligation and second RCA Master mix as described in Table 9 for n + 1 reactions using the products from the previous reaction.

        Table 9. Circularization, ligation and second RCA


      2. Add 10 µl of the master mix to the previous reaction.
      3. Incubate for 20 min at 37 °C.
      4. Inactivate the ligation and RCA reaction by incubation for 2 min at 65 °C.
    6. Labelling of C2CA products in a linear PCR
      1. Prepare the linear PCR master mix as described in Table 10 for n + 1 reactions and finally add 6 µl C2CA products.

        Table 10. Labelling of C2CA products in a linear PCR


      2. Run the PCR program described in Table 4.

  6. Microarray analysis
    1. Prepare the detection mix for each sample by pooling 15 µl labelled PCR products, 15 µl labelled C2CA products, 1 µl Cy5-labelled ‘hybridization control’ oligonucleotide at a concentration of 50 µM and 30 µl ExpressHyb hybridization solution.
    2. Cover the microarray slide with a lifter slip (slides are not washed before use).
    3. Pipette the whole detection mix under the lifter slip.
    4. Put the slide in a humid incubation chamber (Note 12).
    5. Incubate at 65 °C for 45 min.
    6. Remove lifter slips.
    7. Wash slides in 2x SSC + 0.1% SDS for 5 min in a slide rack or staining jar while agitating.
    8. Wash slides in 0.2x SSC for 2 min in a slide rack or staining jar while agitating.
    9. Wash slides in H2O for 1 min in a slide rack or staining jar while agitating.
    10. Dry slides by centrifugation (2 min, 175 x g [900 rpm]) (Note 13).
    11. Scan the slides in a microarray scanner using the correct parameters for detection of Cy5 and Atto532 fluorophores.

Data analysis

  1. The images for both fluorescence channels are imported into GenePix Pro 6.0.
  2. Create a .gal file to overlay an array containing the positions and names of your spotted microarray probes.
  3. Using the ‘Analyze’ function, mean and median intensities as well as standard deviations are calculated for the respective spots (and many more values).
  4. Export the results as text file and import them into Microsoft Excel or any other software suitable for data analysis (e.g., GraphPad Prism, R, IBM SPSS or other spreadsheet software).

Notes

  1. We use a Sartorius arium pro UV ultrapure water system. The lack of a hollow-fiber ultrafilter leads to incomplete removal of endotoxins, microorganisms, DNases and RNases, resulting in water which is not PCR-grade.
  2. Please consider that most enzymatic kits are not DNA-free. Enzymes such as polymerases are expressed and purified from cell cultures. In general, antibiotic resistance genes are used as selection markers and contamination of these and other genes may remain in the purified enzyme. Specialized kits have optimized purification procedures to remove such DNA contaminations.
  3. Microarray probe and primer design: The 5’-amino-modified microarray probes were designed with the ‘Probe Design’ function of the ARB software package. In summary, rRNA sequences were downloaded from GenBank and imported into ARB. Generated probes were optimized regarding maximum non-group hits, melting temperature, G+C content and minimum hairpin loops. Melting temperature and secondary structure were assessed using the ‘Oligo’ function of ARB. Finally, probes were manually optimized by adding or removing nucleotides regarding duplex formation and melting temperature. Using the ‘Probe Match’ function of ARB, the oligonucleotides were checked for their specificity. The multiplex PCR primers targeting the 33 beta-lactamase genes were designed using Primer3. When designing primers for multiplex PCR, it is important to avoid primer dimer formation and to have low differences in the primer melting temperature (here: 55 °C).
  4. Although not a requisite, we highly recommend to preparing oligonucleotide dilutions and Master mixes for PCR in a DNA/RNA-free environment to avoid cross-contamination. Genomic material (including PCR products) is added outside of the PCR hood to the enzymatic reactions. Sterilize the UV hood between different steps by UV irradiation.
  5. The preparation of genomic DNA extracts using bead-based cell disruption protocols has the advantage that genomic material of both Gram-positive and Gram-negative bacteria can be extracted simultaneously, while most commercially available kits require different protocols for these two types.
  6. Addition of antibiotics to the medium might be necessary because bacteria tend to lose their antibiotic resistance genes and plasmids due to reduced fitness and competition in the absence of antibiotics in liquid cell cultures.
  7. Bacterial or fungal growth is indicated by a cloudy haze. Bacteria should optimally be harvested at the end of the exponential phase (OD600 around 2).
  8. The phosphorylation of the 5’-end of the padlock probes is required in order to connect the 3’- with the 5’-end in the ligation reaction. We recommend ordering the padlock probes directly phosphorylated at the 5’-end. However, the cost of the oligonucleotides increases significantly if they are ordered with modifications.
  9. Spotting at low humidity causes the spots to dry too fast, resulting in torus-shaped spots. If the humidity is too high, spots become bigger and they might bleed into each other and create uneven, heterogeneous spots.
  10. The silylated aldehyde slides are delivered clean and there is no need for cleaning the slides before spotting. However, after clamping the glass slides into the mount, we recommend to blow off small splinters of glass with a high-pressure air pistol.
  11. In our case, 1 µl of genomic DNA extract corresponded to a concentration ranging between 11-48 ng µl-1.
  12. Be careful when handling the incubation chamber having water at the bottom. To avoid spilling water, put towels at the bottom, which still release humidity.
  13. Using our centrifuge (Heraeus 40R) and swinging bucket rotor, 900 rpm equal to 175 x g. Do not spin your slides at higher velocities to prevent damage to your slides or centrifuge.

Recipes

  1. CASO medium
    Weight 30 g tryptic soy broth and dissolve in 1 L water
    Autoclave for 15 min at 121 °C. Store at 4 °C
  2. 1x phosphate-buffered saline (PBS)
    Dilute 100 ml 10x PBS in 850 ml water
    Adjust the pH using HCl or NaOH to 7.2, and adjust the final volume to 1 L
    Store at room temperature
  3. 2x spotting buffer (6x SSC, 3 M betaine)
    Dissolve 20.27 g betaine monohydrate in 15 ml 20x SSC buffer
    Adjust the volume to 50 ml with water
    Sterile-filter the solution with a 0.20 µm syringe filter and transfer to a new 50 ml tube
    Store at room temperature

Acknowledgments

This protocol has been adapted from Barišić et al. (2016), Journal of medical microbiology, 65(1), pp. 48-55.

References

  1. Barišić, I., Petzka, J., Schoenthaler, S., Vierlinger, K., Noehammer, C. and Wiesinger-Mayr, H. (2016). Multiplex characterization of human pathogens including species and antibiotic-resistance gene identification. J Med Microbiol 65(1): 48-55.
  2. Barišić, I., Schoenthaler, S., Ke, R., Nilsson, M., Noehammer, C. and Wiesinger-Mayr, H. (2013). Multiplex detection of antibiotic resistance genes using padlock probes. Diagn Microbiol Infect Dis 77(2): 118-125.
  3. Brandt, C., Braun, S.D., Stein, C., Slickers, P., Ehricht, R., Pletz, M.W. and Makarewicz, O., (2017). In silico serine β-lactamases analysis reveals a huge potential resistome in environmental and pathogenic species. Sci Rep 7.
  4. Bush, K., (2010). Alarming β-lactamase-mediated resistance in multidrug-resistant Enterobacteriaceae. Curr Opin Microbiol 13(5): 558-564.
  5. Hall, B. G. and Barlow, M. (2003). Structure-based phylogenies of the serine β-lactamases. J Mol Evol 57(3): 255-260.
  6. Hall, B. G. and Barlow, M. (2004). Evolution of the serine β-lactamases: past, present and future. Drug Resist Updat 7(2): 111-123.
  7. Hardenbol, P., Yu, F., Belmont, J., Mackenzie, J., Bruckner, C., Brundage, T., Boudreau, A., Chow, S., Eberle, J., Erbilgin, A., Falkowski, M., Fitzgerald, R., Ghose, S., Iartchouk, O., Jain, M., Karlin-Neumann, G., Lu, X., Miao, X., Moore, B., Moorhead, M., Namsaraev, E., Pasternak, S., Prakash, E., Tran, K., Wang, Z., Jones, H. B., Davis, R. W., Willis, T. D. and Gibbs, R. A. (2005). Highly multiplexed molecular inversion probe genotyping: over 10,000 targeted SNPs genotyped in a single tube assay. Genome Res 15(2): 269-275.
  8. Kong, K. F., Schneper, L. and Mathee, K. (2010). Beta‐lactam antibiotics: from antibiosis to resistance and bacteriology. APMIS 118(1) 1-36.
  9. Marik, P. E. (2014). Don’t miss the diagnosis of sepsis! Crit Care 18(5): 529.
  10. Mussap, M., Molinari, M. P., Senno, E., Gritti, P., Soro, B., Mannelli, S. and Fabris, C. (2007). New diagnostic tools for neonatal sepsis: the role of a real-time polymerase chain reaction for the early detection and identification of bacterial and fungal species in blood samples. J Chemother 19 Suppl 2: 31-34.
  11. Nilsson, M., Malmgren, H., Samiotaki, M., Kwiatkowski, M., Chowdhary, B. P. and Landegren, U. (1994). Padlock probes: circularizing oligonucleotides for localized DNA detection. Science 265(5181): 2085-2088.
  12. Wellinghausen, N., Kochem, A. J., Disque, C., Muhl, H., Gebert, S., Winter, J., Matten, J. and Sakka, S. G. (2009). Diagnosis of bacteremia in whole-blood samples by use of a commercial universal 16S rRNA gene-based PCR and sequence analysis. J Clin Microbiol 47(9): 2759-2765.
  13. Zankari, E., Hasman, H., Cosentino, S., Vestergaard, M., Rasmussen, S., Lund, O., Aarestrup, F. M. and Larsen, M. V. (2012). Identification of acquired antimicrobial resistance genes. J Antimicrob Chemother 67(11): 2640-2644.

简介

用于病原体鉴定和表征的诊断测定法由依赖于多重方法的同时可检测目标的数量或由于基于培养的技术的时间限制来限制。 我们最近提出了一种用于人类病原体鉴定的100plex方法,以鉴定75种细菌和真菌物种以及33种临床相关β-内酰胺酶(Barišić等,2016)。 通过使用16S rRNA基因序列作为挂锁探针中的条形码元件,以及用于物种和抗生素抗性鉴定的两种不同的荧光通道,我们设法将需要的微阵列探针的数量减少一半。 因此,我们在这里介绍一个运行时间约为的测定方案。 8 h,检测限为105 cfu ml-1。 正确鉴定了89%的β-内酰胺酶和93.7%的物种。
【背景】β-内酰胺酶是一类提供抗β-内酰胺抗生素的抗生素抗性基因,其结构模拟D-丙氨酰-D-丙氨酸,细菌细胞壁的一个组分,从而抑制细菌细胞壁合成。 β-内酰胺酶能够水解β内酰胺抗生素β-内酰胺环的中心成分,并使其无效(Kong et al。,2010)。今天,描述了超过1000种β-内酰胺酶,并且存在巨大的潜在环境储层(Bush,2010; Brandt等,2017)。 β-内酰胺酶是古代酶,我们将它们分类为具有丝氨酸催化位点的A,C和D(丝氨酸β-内酰胺酶),或者其活性中心是锌依赖性的B(金属-β-内酰胺酶)(Hall和Barlow,2003和2004)。尽管它们的系统发育年龄高,丝氨酸β-内酰胺酶可能共享共同的祖先,并且由于永久的选择压力而获得了大量的SNP。另外,对于β-内酰胺酶,存在500多种抗生素抗性基因(Zankari等,2012)。
   当前的多路复用方法减少同时可检测的目标数量,而依赖于培养的技术受时间限制。鉴于这些事实和大量具有临床重要性的病原体,需要新的方法来快速表征和鉴定病原体,毒力因子和抗生素抗性基因。
   感染诊断的黄金标准需要2-3天的时间,而且依赖于培养(Marik,2014)。此外,PCR方法以高灵敏度和低成本提供结果,但是由于临床相关靶标数量高(Mussap et al。,2007; Wellinghausen et al。,2009),仍然不切实际。目前的多重PCR方案不适用于大量的靶标,并且限制只能通过基于微流控的测定来克服,其中并行进行大量分析。
   挂锁探针是线性DNA探针,其在退火后圆化,然后用于滚环扩增(Nilsson等人,1994; Hardenbol等人,2005)。它们允许更多数量的复用,并且可以容易地整合到基于PCR的测定中。最近,我们提出了一种基于用于病原体鉴定的挂锁探针(75种细菌和真菌物种)以及33种临床上重要的β-内酰胺酶的100-plex方法(Barišić等,2016)。通过从以前的工作(Barišić等人,2013)适应这种方法,我们增加了测定的灵敏度和特异性,我们设法通过使用16S rRNA序列作为条形码元素,将所需的微阵列探针数量减少了一半挂锁探针和两个不同的荧光通道,用于物种鉴定和抗生素抗性表征。在这里,我们提出了一种克服时间限制和增加可检测目标数量的测定法。我们的测定允许在总共8小时内检测高达105 cfu ml-1。我们能够检索和正确表征89%的β-内酰胺酶,并确定了所有物种的93.7%。

关键字:多重检测, 人体病原菌, 锁式探针, 物种鉴定, 抗生素耐药鉴定

材料和试剂

  1. 过滤嘴10μl,20μl,200μl和1,250μl(例如,Biozym,目录号:VT0200,VT0220,VT0240和VT0270)
  2. 安全锁管1.5 ml(Eppendorf,目录号:022363204)
  3. Falcon 15 ml锥形离心管(Corning,目录号:352196)
  4. 2ml螺旋盖管(例如,Roche Molecular Systems,目录号:03358941001)
  5. Ritter Riplate 384孔板PP(Ritter,目录号:43001-0035)
  6. 150-212μm酸洗的玻璃珠(Sigma-Aldrich,目录号:G1145)
  7. 玻璃珠,酸洗,425-600微米(Sigma-Aldrich,目录号:G8772)
  8. 有机硅烷化醛幻灯片(CEL Associates,目录号:VSS-25)
  9. LifterSlip m系列用于微阵列载玻片的盖子,55μl(Thermo Fisher Scientific,Thermo Scientific TM,目录号:25X60IM5439001LS)
  10. PCR管0.2ml(Eppendorf,目录号:0030124537)
  11. Millex-GV 0.22μm注射器过滤器(默克,目录号:SLGV033RS)
  12. (例如,Corning,目录号:430829)
  13. 一次性注射器,例如,Omnifix 50ml LL(B.Braun Medical,目录号:8508577FN)
  14. 多重PCR引物(总共66个,表S1 )来自Microsynth(Balgach,Switzerland)(注3)订购了β-内酰胺酶基因。
  15. 挂锁探针(总共66个,图1,的靶向β-内酰胺酶的表S2

    图1.挂锁探针的示意图。 3'和5'靶标识别臂结合β-内酰胺酶基因的多重PCR产物。在结合过程中,挂锁探针被环化并随后连接。挂锁探针结合区域与PCR产物的3'或5'末端的最大距离不应超过200个碱基对。由于挂钩探针在连接反应时与PCR产物连接,因此较长的距离会导致后续RCA的抑制。 C2CA序列是环到圆扩增所必需的,并且包含用于扩增产物单体化的AluI限制性位点。条形码序列来源于16S rRNA基因。这允许我们将微阵列探针的数量减半,因为在相同的微阵列探针上检测到C2CA产物和16S rRNA基因PCR产物,但是在不同的荧光通道中。

  16. 5'-氨基修饰的微阵列探针(总共274个,表S3 )由Microsynth(瑞士Balgach)订购(注3)
  17. 用于PCR应用的无核酸酶水应用于Mastermix 16S Basic试剂盒,也可以单独购买(例如,,Fresenius Kabi,Aqua bidest。“Fresenius',无目录号)
  18. 超纯水,以下简称为水或H 2 O(注1)
  19. 从Integrated DNA Technologies(Coralville,IA,USA)订购通用细菌16S rRNA引物45f ++(5'-GCYTAAYACATGCAAGTCGARCG-3')和783R(5'-TGGACTACCAGGGTATCTAATCCT-3')
  20. 从Integrated DNA Technologies(Coralville,IA,USA)订购真菌18S rRNA(ITS区域)引物ITS3(5'-GCATCGATGAAGAACGCAGC-3')和ITS4 +(5'-TCCT-CCGCTTATTGATATGCTTAAGT-3')
  21. 订购圆圈扩增(C2CA)寡核苷酸C2CA (5'-TACTCGAGGAGCTGCATACAC-3')和C2CA + (5'-GTGTATGCAGCTCCTCGAGTA-3')来自Integrated DNA Technologies(Coralville,IA,USA)
  22. 从Microsynth(Balgach,Switzerland)订购了Cy5标记的杂交控制(免疫'Bsrev')(5'-Cy5-AAGCTCACTGGCCGTCGTTTAAA-3')
  23. T4 DNA连接酶(Thermo Fisher Scientific,Thermo Scientific TM,目录号:EL0011)
  24. 提供10x反应缓冲液A(Thermo Fisher Scientific,Thermo Scientific& TM,目录号:EK0031)的T4多核苷酸激酶(10U /μl)
  25. ATP溶液(100mM)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:R0441)
  26. 含有2.5x完全母体混合物,Moltaq 16S DNA聚合酶和PCR级水(注2)的Mastermix 16S Basic,无DNA(Molzym,目录号:S-040-0250)
  27. dNTP混合物(每种10mM)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:R0192)
  28. Cy5-dCTP(1mM溶液)(GE Healthcare,目录号:PA55021)
  29. 通过10x ThermoPol反应缓冲液和100mM MgSO 4递送的Ventin R(exo-)DNA聚合酶(New England Biolabs,目录号:M0257S)
  30. ExpressHyb杂交溶液(Takara Bio,Clontech,目录号:636831)
  31. Ampligase热稳定DNA连接酶(5U /μl)和Ampligase 10x反应缓冲液(Epicentre,目录号:A32750)
  32. 牛血清白蛋白(BSA),分子生物学级(New England Biolabs,目录号:B9000S)
  33. 用10x phi29 DNA聚合酶反应缓冲液(Thermo Fisher Scientific,Thermo Scientific TM,目录号:EP0091)提供的phi29 DNA聚合酶
  34. AluⅠ限制酶(Thermo Fisher Scientific,Thermo Scientific TM,目录号:ER0011)
  35. Atto532-dCTP(MoBiTec,Göttingen,Germany)
  36. SDS溶液10%用于分子生物学(AppliChem,目录号:A0676)
  37. 胰蛋白胨大豆肉汤,也称为CASO培养基(酪蛋白胨大豆蛋白胨肉汤)(默克,目录号:105459)
  38. PBS(10x),pH7.2(Thermo Fisher Scientific,Gibco TM,目录号:70013)
  39. 甜菜碱一水合物(Sigma-Aldrich,目录号:B2754)
  40. UltraPure SSC,20x(Thermo Fisher Scientific,Invitrogen TM,目录号:15557036)
  41. CASO培养基(见食谱)
  42. 1x磷酸盐缓冲盐水(PBS)(见食谱)
  43. 2x点样缓冲液(6x SSC,3M甜菜碱)(参见食谱)

设备

  1. 移液器(例如,,Sartorius,目录号:728020,728050,728060和728070)
  2. 微生物孵化器振荡器(例如,IKA,型号:KS 4000 i对照)
  3. 用于1.5ml管的台式离心机(例如,Eppendorf,型号:5424)
  4. 罗氏MagNA Lyser仪器(瑞士巴塞尔)
  5. Thermomixer舒适(Eppendorf,汉堡,德国)
  6. Epoch微孔板分光光度计(Biotek,Winooski,VT,USA)
  7. Biosan DNA / RNA UV清洁盒(Warren,MI,USA)(推荐见附注4)
  8. 热循环仪(例如Thermo Fisher Scientific,Applied Biosystems,型号:GeneAmp PCR System 2700)(Paisley,UK)
  9. 具有TX-750转子的Heraeus Megafuge 40R(Thermo Fisher Scientific,Thermo Scientific TM,型号:Heraeus TM Megafuge TM 40R,目录号: 75004518; TX-750转子:Thermo Fisher Scientific,Thermo Scientific TM,目录号:75003180)和板用插入件(Thermo Fisher Scientific,Thermo Scientific TM,目录号: 75003617)和Falcon管(Thermo Fisher Scientific,Thermo Scientific TM,目录号:75003608)
  10. 幻灯片湿度保存盒(例如, LabScientific,目录号:HIC-3)
  11. GeneMachines Omnigrid 100接触阵列器(美国威斯康星州麦迪逊)
  12. 国际微阵列针隐形3 SMP3(Telechem,目录号:SMP3)
  13. Agilent SureScan DNA微阵列扫描仪(Santa Clara,CA,USA)
  14. 高压灭菌器
  15. Sartorius arium pro UV超纯水系统(Sartorius,型号:arium ® pro)

软件

  1. GenePix Pro 6.0软件(Molecular Devices LLC,Sunnyvale,CA,USA)
  2. Microsoft Excel或任何其他数据分析软件
  3. 用于微阵列探针设计的ARB软件包(注3)
  4. Primer3用于底漆设计(注3)

程序

  1. 寡核苷酸的制备
    1. 将5'氨基修饰的微阵列探针在不含核酸酶的水中溶解至100μM
    2. 将33-plex PCR引物溶解于不含核酸酶的水中的100μM,然后通过将等体积的所有66个引物(例如,每个6μl)移液到一个1.5ml Eppendorf中来产生100μM引物混合物管。
    3. 挂锁探针的溶解和磷酸化(注8)。
      1. 在无核酸酶的水中将挂锁探针溶解至200μM,然后通过吸取1体积的挂锁探针(例如,66×10.00μl)和0.34体积的核酸酶 - 游离水(例如,总共66×10.00×0.515 =340.00μl)放入1.5ml Eppendorf管中。
      2. 挂锁探针的磷酸化主混合物通过将10μlT4多核苷酸激酶(0.2U /μl最终浓度),50μl10x反应缓冲液A(1x最终浓度),50μl10mM ATP(1mM最终浓度),250μl挂锁探针混合物(每个探针最终浓度1μM)和140μlPCR级水(终体积500μl)。
      3. 在37℃下孵育30分钟,然后在65℃孵育10分钟。
    4. 存储微阵列探针,挂锁探针,底漆和相应的主混合物在-20°C
  2. 细菌细胞的生长和基因组DNA提取物的制备(注5)
    1. 接种来自甘油储备液的纯培养物(例如,/或从琼脂培养物)或临床分离物(例如,从琼脂培养物)将5 ml CASO培养基(见食谱)放入15 ml Falcon管中(注6)。不要完全拧上盖子,以适当通风细菌。
    2. 在37℃下,以300rpm在轨道振荡培养箱中孵育14-18小时的样品(注7)。
    3. 旋转Falcon管,并将2ml培养物转移到2ml Eppendorf管中
    4. 通过在室温下以2,400×g离心5分钟使细菌颗粒化
    5. 将沉淀重悬于1ml 1x PBS中(参见食谱)
    6. 在室温下以2,400×g离心悬浮液5分钟。
    7. 将沉淀重悬于1ml无菌ddH 2 O中,并转移到适合MagNA Lyser的2ml螺旋盖管中。
    8. 将150-212μm的酸洗珠子和等体积的直径为425-600μm的酸洗珠子与管接合,使得珠粒占管的约三分之一。
    9. 使用MagNA Lyser在室温下以6,500 rpm破坏细菌和真菌细胞30秒。
    10. 在室温下放置样品5分钟。
    11. 重复步骤A9一次。
    12. 对于热裂解,在95℃在温热混合器中孵育样品20分钟。
    13. 以最大速度离心10分钟。
    14. 将上清液转移到新管中
  3. 微阵列处理
    1. 通过向每个孔中加入18μl的2x点样缓冲液(参见食谱)来制备Ritter 384孔斑点板,然后向每个孔中加入18μl微阵列寡核苷酸(每个寡核苷酸的终浓度现在为50μM) 1x点样缓冲液)。注意哪些寡核苷酸在哪个孔中以及您的识别剂如何将寡核苷酸转移到载玻片上(注4)
    2. 将Omnigrid接触排列器设置为60%的空气湿度,并设置机器以每张幻灯片4次重复打印寡核苷酸(注9和10)。
    3. 使用产生直径为100μm的斑点的SMP3引脚将寡核苷酸定位在具有醛表面的载玻片上
    4. 点样后,让寡核苷酸附着大约潮湿气氛中5小时。
    5. 发现后,玻璃片不会洗涤。
    6. 在室温下存放载玻片。

  4. 物种识别
    1. 16S rRNA基因PCR
      1. 根据表1,使用Molzym Mastermix 16S Basic试剂盒,在0.2ml PCR管中准备PCR主混合物进行n + 1反应,包括至少一个阴性对照(NTC),您可以用水替代基因组DNA提取物。可以预先制备由等量的所有四种引物在15μM组成的引物混合物,以避免重复移液。在这种情况下,使用1.20μl的底漆混合物。保持样品在冰上冷冻,直到放入热循环仪(注11)
        表1.用于16S rRNA基因鉴定的多重PCR主混合物


      2. 在具有加热盖的热循环仪中使用表2中列出的PCR程序(图2),使用“33-复合体预扩增PCR进行β-内酰胺酶编码基因鉴定”进行该反应(图2)。
      3. PCR运行结束后,将样品保持在4°C
        表2.用于16S rRNA基因鉴定和β-内酰胺酶编码基因信息的预扩增的多重PCR程序



        图2.测定检测原理的示意图对于物种鉴定,使用通用16S引物(A1)扩增16S rRNA基因。随后,使用线性PCR和Cy5修饰的胞嘧啶(A2)标记PCR产物。同时,β-内酰胺酶基因在33plex PCR(B1)中预扩增。然后,使用β-内酰胺酶PCR产物作为模板,将挂锁探针杂交(B2)并连接(B3),并在第一滚环扩增(RCA)(B4)中扩增。 RCA产品包含C2CA序列和相应的16S rRNA条形码序列。在下一个反应中,使用限制酶和环化(B6)将RCA产物单体化(B5)作为第二个RCA(B7)的新模板。这些C2CA产物在使用Atto532修饰的胞嘧啶(B8)的线性PCR反应中进行标记。最后,将来自16S rRNA基因PCR和多重挂锁测定的标记的扩增产物汇集在一起并与两个不同荧光波长(C)检测产物的微阵列杂交。

    2. 用于PCR产物标记的线性PCR
      1. 根据表3,用0.2ml PCR管制备线性PCR主混合物,用n + 1反应制备来自先前PCR的PCR产物。保持样品冷却至放置在热循环仪中。

        表3.用于PCR产物标记的线性PCR主混合物


      2. 使用表4中概述的PCR程序在具有加热盖的热循环仪中,使用“线性PCR”中的C2CA产物标记进行该反应。

        表4. PCR和C2CA产品标记的线性PCR程序


  5. 多重β-内酰胺酶编码基因鉴定
    1. 用于β-内酰胺酶编码基因鉴定的33倍预扩增PCR
      1. 根据表5,使用Molzym Mastermix 16S Basic试剂盒,在0.2ml PCR管中准备PCR主混合物进行n + 1反应,包括至少一个NTC。引物浓度各自为76nM。将样品冷却至放置在热循环仪中(注11)
        表5:β-内酰胺酶编码基因鉴定的多重预扩增PCR


      2. 按照表2所述运行PCR程序。
    2. 通过挂锁探针进行杂交和环化
      1. 根据表6,为n + 1个样品准备连接混合物(每个样品10μl)
        表6.通过酶连接挂锁探针进行环化的酶反应混合物。 66 nM的挂锁主混合浓度等于每个探针1 nM。


      2. 从33plex PCR中将10μl连接混合物加入到PCR产物中。每个挂锁探头的最终浓度分别为100 pM
      3. 在95℃下孵育5分钟,然后在加热盖子的热循环仪中在60℃孵育1小时。
    3. 第一滚圈放大
      1. 使用上述反应的连接产物,对于n + 1反应,制备如表7所述的第一次滚环扩增(RCA)的反应主混合物。

        表7.第一个RCA的反应混合


      2. 在37°C孵育20分钟。
      3. 在65℃下孵育2分钟使聚合酶失活。
    4. C2CA寡核苷酸的杂交和酶促限制的单体化
      1. 如表8所述,为n + 1反应制备限制性混合物。

        表8.酶限制反应的反应混合物


      2. 将5μl限制性混合物加入到第一个RCA混合物中(总体积现在为25μl)
      3. 在37℃孵育5分钟。
      4. 在65℃孵育5分钟以灭活限制酶。
    5. 循环,结扎和第二次RCA
      1. 使用上述反应的产物,准备连接和第二个RCA主混合物,如表9所述,用于n + 1反应。

        表9.循环,结扎和第二RCA


      2. 将10μl的主混合物加入上一反应。
      3. 在37°C孵育20分钟。
      4. 在65°C温育2分钟,灭活连接和RCA反应
    6. 在线性PCR中标记C2CA产物
      1. 按照表10所述准备线性PCR主混合物,进行n + 1反应,最后加入6μlC2CA产物
        表10.线性PCR中C2CA产物的标记


      2. 运行表4中描述的PCR程序。

  6. 微阵列分析
    1. 通过将15μl标记的PCR产物,15μl标记的C2CA产物,1μlCy5标记的“杂交对照”寡核苷酸以50μM的浓度和30μlExpressHyb杂交溶液合并,为每个样品准备检测组合。
    2. 用提升器滑盖覆盖微阵列滑块(幻灯片在使用前未洗涤)。
    3. 移动整个检测组合在升降机下。
    4. 将幻灯片置于潮湿的孵育室中(注12)
    5. 在65℃孵育45分钟。
    6. 取下升降单。
    7. 在2×SSC + 0.1%SDS中将载玻片在载玻片上或染色瓶中搅拌5分钟
    8. 在载玻片上或0.2×SSC中洗涤玻片2分钟,同时搅拌
    9. 在玻璃架或染色瓶中,在H 2 O 2中洗涤玻片1分钟,同时搅拌。
    10. 通过离心(2分钟,175×g / 900rpm)干燥载玻片(注13)。
    11. 使用正确的参数检测Cy5和Atto532荧光团,在微阵列扫描仪中扫描载玻片。

数据分析

  1. 将两个荧光通道的图像导入GenePix Pro 6.0。
  2. 创建一个.gal文件,覆盖一个数组,其中包含您所发现的微阵列探针的位置和名称。
  3. 使用“分析”功能,计算各个斑点的平均值和中值强度以及标准偏差(以及更多值)。
  4. 将结果导出为文本文件,并将其导入Microsoft Excel或适用于数据分析的任何其他软件(例如,,GraphPad Prism,R,IBM SPSS或其他电子表格软件)。

笔记

  1. 我们使用Sartorius arium pro UV超纯水系统。缺乏中空纤维超滤器导致内毒素,微生物,DNA酶和核糖核酸酶的不完全去除,导致不是PCR级的水。
  2. 请考虑大多数酶试剂盒不含DNA。从细胞培养物中表达和纯化酶如聚合酶。通常使用抗生素抗性基因作为选择标记,并且这些和其他基因的污染可能残留在纯化的酶中。专业工具包优化了纯化程序,以消除这些DNA污染
  3. 微阵列探针和引物设计:使用ARB软件包的“探针设计”功能设计5'-氨基修饰的微阵列探针。总之,从GenBank下载rRNA序列并导入ARB。生成的探针针对最大非组命中,熔解温度,G + C含量和最小发夹环进行了优化。使用ARB的“Oligo”功能评估熔融温度和二级结构。最后,通过添加或去除关于双链体形成和熔融温度的核苷酸手动优化探针。使用ARB的“探针匹配”功能检测寡核苷酸的特异性。使用Primer3设计靶向33个β-内酰胺酶基因的多重PCR引物。当设计多重PCR引物时,重要的是避免引物二聚体形成,并且引物熔解温度差异较小(这里:55°C)。
  4. 虽然不是必需的,但我们强烈建议在无DNA / RNA的环境中制备寡核苷酸稀释液和主混合物用于PCR,以避免交叉污染。将基因组材料(包括PCR产物)加入PCR罩外进行酶促反应。通过紫外线照射在不同的步骤之间灭菌紫外线罩
  5. 使用基于珠的细胞破坏方案的基因组DNA提取物的制备具有可以同时提取革兰氏阳性和革兰氏阴性菌的基因组材料的优点,而大多数市售试剂盒对于这两种类型需要不同的方案。 >
  6. 可能需要向培养基中加入抗生素,因为在液体细胞培养物中不存在抗生素时,由于细菌的适应性和竞争性降低,细菌往往会失去其抗生素抗性基因和质粒。
  7. 细菌或真菌生长由多云雾霾指示。细菌应在指数期结束时最佳收获(OD 600)约2)。
  8. 需要使用挂锁探针5'-末端的磷酸化,以便在连接反应中连接3'-与5'末端。我们建议订购挂锁探针直接在5'端磷酸化。然而,如果寡核苷酸的修改被命令,则寡核苷酸的成本显着增加
  9. 在低湿度下发现,斑点干燥得太快,导致圆环形斑点。如果湿度过高,斑点变大,可能会渗透到相互之间,造成不均匀的异质斑点
  10. 甲硅烷基化的醛载玻片被清洁,并且在发现之前不需要清洁载玻片。然而,在将玻璃滑入夹具之后,我们建议用高压空气手枪吹掉玻璃碎片。
  11. 在我们的例子中,1μl的基因组DNA提取物对应于11-48ngμl -1 的浓度。
  12. 处理底部有水的孵化室时要小心。为了避免溢出水,将毛巾放在底部,这仍然释放湿度。
  13. 使用我们的离心机(Heraeus 40R)和摆动叶轮转子,900 rpm等于175 x g。不要以更高的速度旋转幻灯片,以防止损坏幻灯片或离心机。

食谱

  1. CASO媒体
    重量30克胰蛋白胨大豆肉汤,溶于1升水中 在121℃高压灭菌15分钟。储存于4°C
  2. 1x磷酸盐缓冲盐水(PBS)
    稀释100毫升10倍的PBS在850毫升的水中 使用HCl或NaOH将pH调节至7.2,并将最终体积调整至1L
    在室温下存放
  3. 2x斑点缓冲液(6x SSC,3M甜菜碱)
    将20.27g甜菜碱一水合物溶于15ml 20x SSC缓冲液中 用水调节体积至50ml 用0.20微米注射器过滤器对溶液进行无菌过滤,并转移到新的50ml管中 在室温下存放

致谢

该方案已经从Barišić等人(2016),Journal of medical microbiology,65(1),pp。48-55中改编。

参考

  1. Barišić,I.,Petzka,J.,Schoenthaler,S.,Vierlinger,K.,Noehammer,C.and Wiesinger-Mayr,H。(2016)。  人类病原体的多重表征,包括物种和抗生素抗性基因鉴定。 J Med Microbiol 65(1):48-55。
  2. Barišić,I.,Schoenthaler,S.,Ke,R.,Nilsson,M.,Noehammer,C.and Wiesinger-Mayr,H。(2013)。  使用挂锁探针多重检测抗生素抗性基因。诊断微生物感染D >是 77(2):118-125。
  3. (a)=“ke-insertfile”href = “http://www.nature.com/articles/srep43232”target =“_ blank”> 丝印蛋白β-内酰胺酶分析揭示了环境和致病物种中巨大的潜在抵抗力。



  4. 布什,K.,(2010)。报警β-内酰胺酶介导的多药耐药肠杆菌科耐药性。 Curr Opin Microbiol 13(5):558-564。
  5. Hall,BG和Barlow,M。(2003)。  丝氨酸β-内酰胺酶的基于结构的系统发育 J.Mol Evol 57(3):255-260。
  6. Hall,BG和Barlow,M。(2004)。  丝氨酸β-内酰胺酶的进化:过去,现在和将来。药物抗性更新 7(2):111-123。
  7. Hardenbol,P.,Yu,F.,Belmont,J.,Mackenzie,J.,Bruckner,C.,Brundage,T.,Boudreau,A.,Chow,S.,Eberle,J.,Erbilgin, Falkowski,M.,Fitzgerald,R.,Ghose,S.,Iartchouk,O.,Jain,M.,Karlin-Neumann,G.,Lu,X.,Miao,X.,Moore,B.,Moorhead,M ,Namsaraev,E.,Pasternak,S.,Prakash,E.,Tran,K.,Wang,Z.,Jones,HB,Davis,RW,Willis,TD和Gibbs,RA(2005)。< a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/15687290”target =“_ blank”>高度多重分子反转探针基因分型:在单个管中基因分型的超过10,000个靶向SNPs测定。 Genome Res 15(2):269-275。
  8. Kong,K. F.,Schneper,L.和Mathee,K。(2010)。 β-内酰胺类抗生素:从抗生素到抗性和细菌学。 APMIS 118(1)1-36。
  9. Marik,PE(2014)。不要错过诊断脓毒症! Crit Care 18(5):529.
  10. Mussap,M.,Molinari,MP,Senno,E.,Gritti,P.,Soro,B.,Mannelli,S.and Fabris,C。(2007)。  新生儿败血症的新诊断工具:实时聚合酶链反应对早期检测和鉴定细菌的作用和血液样品中的真菌物种。 J Chemother 19 Suppl 2:31-34。
  11. Nilsson,M.,Malmgren,H.,Samiotaki,M.,Kwiatkowski,M.,Chowdhary,BPand Landegren,U。(1994)。  挂锁探针:用于局部DNA检测的环化寡核苷酸 科学 265(5181):2085-2088 。
  12. Wellinghausen,N.,Kochem,AJ,Disque,C.,Muhl,H.,Gebert,S.,Winter,J.,Matten,J.and Sakka,SG(2009)。< a class = insertfile“href =”http://www.ncbi.nlm.nih.gov/pubmed/19571030“target =”_ blank“>通过使用基于商业通用的16S rRNA基因的PCR和全血样品诊断菌血症序列分析。 J Clin Microbiol 47(9):2759-2765。
  13. Zankari,E.,Hasman,H.,Cosentino,S.,Vestergaard,M.,Rasmussen,S.,Lund,O.,Aarestrup,FM and Larsen,MV(2012)。< a class =插入文件“href =”http://www.ncbi.nlm.nih.gov/pubmed/22782487“target =”_ blank“>获得性抗微生物抗性基因的鉴定抗微生物化学品 67(11):2640-2644。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Conzemius, R. and Barišić, I. (2017). Detection of Pathogens and Ampicillin-resistance Genes Using Multiplex Padlock Probes. Bio-protocol 7(16): e2504. DOI: 10.21769/BioProtoc.2504.
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