Strain identification

Introduction

RAPD is a procedure for typing and fingerprinting isolates of a species. It can be used for epidemiological studies, such as investigations into hospital outbreaks and as a laboratory aid to keep track of cultures and to verify that mutants generated in the laboratory are genetically identical to the parental strain. In our hands, the use of one primer, R108, is sufficiently discriminatory to distinguish between the isolates of different strains.

Materials

  • Genomic DNA
  • Primers: R108: GTATTGCCCT (Aufauvre-Brown et al., 1992); 2: GCTGGTGG (Loudon et al., 1993); RC08: GGATGTCGAA (Crowhurst et al., 1991)
  • Taq polymerase 5U/µl
  • Reaction buffer 10 X: 200mM (NH4)2SO4, 750 mM Tris-Cl (pH 9.0 [at 25° C]), 0.1% (v/v) Tween® 20
  • dNTPs:10 X stock solution containing each nucleotide at 2mM
  • MgCl2: stock solution of 25 mM
  • Light mineral oil (d= 0.84 g/ml)
  • 18 megaohm water filter sterilised

Equipment

  • Digital pipettes: 0 - 10µl (2 X): one for PCR setup; one for DNA and 10 - 100µl
  • Sterile tips containing filters
  • 0.2 ml or 0.5 ml microcentrifuge tubes
  • UV transilluminator: 254 or 302/312 nm
  • Thermal cycler
  • Microcentrifuge
  • Vortex Mixer

Benches

  • DNA preparation area
  • PCR setup area
  • PCR gel analysis area

Preparation

  • UV treatment: treat the following components to 5 min UV irradiation at 254 nm: racks, pipettes, mineral oil, H2O, microcentrifuge tubes.
  • Genomic DNA: dilute samples to 20 ng/µl using H2O in 0.5 ml microcentrifuge tubes in DNA preparation area with dedicated tips and pipette.
  • Primers, dNTPs: prepare stock solutions of appropriate concentration in PCR setup area.

Procedure

NB Use aerosol resistant tips throughout

In PCR setup area:

1) Prepare master mix of components containing sufficient volume for all the sample tubes, the reagent control and one spare; add the components in the following order: 10 X buffer, MgCl2, 10 X dNTPs, primer, Taq (7.5 U per sample; final concentration in each sample tube 0.15 U/µl); mix gently by inversion and pulse contents to bottom of tube. We suggest that 50 µl reaction volumes are used; you might want to reduce this volume to 25 µl to save on Taqpolymerase.

2) Primer R108: require final concentration of 0.5 µM with 2.5 mM MgCl2

Primer 2: 0.5 µM primer with 2.5 mM MgCl2

Primer RC08: 1.0 µM primer with 4.0 mM MgCl2

3) Add appropriate volume of H2O to sample tubes; include a reagent control which contains all the components except for DNA; add master mix and 50 µl light mineral oil (if the thermal cycler has no heated lid); the samples tubes can be UV irradiated for 5 min at this stage if additional contamination precautions are required

In DNA preparation area:

4) Add 100 ng DNA to samples tubes, making sure DNA is placed beneath mineral oil; mix samples gently, pulse contents to bottom of tubes and place in thermal cycler

5) Set up thermal cycler with the following temperature and time profiles:

R108 and RC08: 4 min at 94° C, 1 min at 36° C, 1 min at 72° C for 1 cycle; 1 min at 94° C, 1 min at 36° C, 1 min at 72° C for 29 cycles and 5 cycles with 1 min at 94° C, 1 min at 36° C, 5 min at 72° C

Primer 2: 4 min at 94° C, 1 min at 30° C, 1 min at 72° C for 1 cycle; 1 min at 94° C, 1 min at 30° C, 1 min at 72° C for 29 cycles and 5 cycles with 1 min at 94° C, 1 min at 30° C, 5 min at 72° C

In PCR gel analysis area:

6) After completion of the reactions, remove 10 µl, add loading dye and load into the wells of a 1.8% agarose gel; run at 2.5 V/cm until the bromophenol blue is ¾ way down, stain in an ethidium bromide bath (50 µg/ml) and photograph under UV (302/312nm)

Timetable

RAPD setup Steps 1 - 4 1 hour
RAPD reaction Step 5 3 - 4 hours
RAPD analysis Step 6 4 - 5 hours

Tips and general comments

1) Athough it is not as critical as for standard PCR, care must be taken to avoid contamination, especially with post-amplification products - hence the need for dedicated bench areas and a reagent control

2) Primers may need to be re-titrated for each new synthesis. The concentrations suggested above are starting points and the lowest concentration that gives the highest number of bands should be used. It is probably better to use primers that have only been desalted rather than purified in any way e.g. using a reverse-phase cartridge

3) RAPD is not a very reliable procedure and great care must be taken to ensure reproducible results: maintain a set of conditions once they have been established - this applies particularly to the thermal cycler and reaction buffer. If a typing study is being carried out, analyse all the isolates together and carry out each reaction at least three times to check for the reproducibility of scored bands. Some reactions will peter out towards the top of the gel and will need to be repeated

4) It is preferable to dilute the DNA sample in H2O and not to add too much DNA. Inhibition of the reaction can occur, especially if the original DNA was resuspended in a buffer containing EDTA. DNA in the range 25 to 100 ng should give satisfactory results

References

A. fumigatus

Aufauvre-Brown A, Cohen J, Holden DW. Use of randomly amplified polymorphic DNA markers to distinguish isolates of Aspergillus fumigatus. Journal of Clinical Microbiology, 1992, 30:2991-2993

Loudon KW, Burnie JP, Coke AP, Matthews RC. Application of polymerase chain reaction to fingerprinting Aspergillus fumigatus by random amplification of polymorphic DNA. Journal of Clinical Microbiology, 1993, 31:1117-1121

Loudon KW, Coke AP, Burnie JP, Lucas GS, Yin JAL. Invasive aspergillosis - clusters and sources. Journal of Medical and Veterinary Mycology, 1994, 32:217-224

Paugam A, Bougnoux ME, Robert F, Dupouycamet J, Fierobe L, Dhainaut JF, Girardin H. Use of randomly amplified polymorphic DNA markers (RAPD) to demonstrate nosocomial contamination in a case of lethal invasive aspergillosis. Journal of Hospital Infection, 1995, 29:158-161

Lin DM, Lehmann PF, Hamory BH, Padhye AA, Durry E, Pinner RW, Lasker BA. Comparison of 3 typing methods for clinical and environmental isolates ofAspergillus fumigatus

Journal of Clinical Microbiology, 1995, 33:1596-1601

Anderson MJ, Gull K, Denning DW. Molecular typing by random amplification of polymorphic DNA and M13 Southern hybridization of related paired isolates ofAspergillus fumigatus Journal of Clinical Microbiology, 1996, 34:87-93

Leenders A, Vanbelkum A, Janssen S, Demarie S, Kluytmans J, Wielenga J, Lowenberg B, Verbrugh H. Molecular epidemiology of apparent outbreak of invasive aspergillosis in a hematology ward Journal of Clinical Microbiology, 1996, 34:345-351

Rath PM, Marggraf G, Dermoumi H, Ansorg R. Use of phenotypic and genotypic fingerprinting methods in the strain identification of Aspergillus fumigatusMycoses, 1995, 38:429-434

Soriaroyer C, Bougnoux ME, Sulahian A, Ribaud P, Traore F, Garin YIF, Gluckman E, Derouin F. RAPD typing of Aspergillus fumigatus strains - epidemiologic applications on a hospital site Journal de Mycologie Medicale, 1996, 6:49-55

Soriaroyer C. Molecular typing of Aspergillus fumigatus by random amplified polymorphic DNA (RAPD) - Response. Journal de Mycologie Medicale, 1996, 6:197-198

Belkacemi L, Hopwood V, Barton RC, Evans EGY. Molecular typing of Aspergillus fumigatus by random amplified polymorphic DNA (RAPD) Journal de Mycologie Medicale, 1996, 6:197

Rath PM, Ratjen F, Ansorg R. Genetic diversity among isolates of Aspergillus fumigatus in patients with cystic fibrosis. Zentralblatt fur Bakteriologie 1997, 285:450-455

Rath PM, Ansorg R. Value of environmental sampling and molecular typing of Aspergilli to assess nosocomial sources of aspergillosis. Journal of Hospital Infection, 1997, 37:47-53

Chaib F, Carlotti A, Couble A, Villard J, Piens MA, Chapuis F, Mondon P, Lebeau B, Grillot R, Symoens F, Nolard N, Tortorano AM, Viviani MA, Sanchezsousa A, Rodriguez E, Mallie M Bastide JM. Differentiation of colonizing and environmental Aspergillus fumigatus isolates by molecular typing methods. Journal de Mycologie Medicale, 1997, 7:179-186

Year prepared: 

1998