Long-term storage and safe retrieval of DNA from microorganisms for molecular analysis using FTA matrix cards.
We assessed the potential use of Whatman FTA paper as a device for archiving and long-term storage of bacterial cell suspensions of over 400 bacterial strains representing 61 genera, the molecular applications of immobilised DNA on FTA paper, and tested its microbial inactivation properties. The FTA paper extracted bacterial DNA is of sufficiently high quality to successfully carryout the molecular detection of several key genes including 16S rRNA, esp (Enterococcus surface protein), Bft (Bacteroides fragilis enterotoxin) and por (porin protein) by PCR and for DNA fingerprinting by random amplified polymorphic DNA-PCR (RAPD-PCR). To test the long-term stability of the FTA immobilised DNA, 100 of the 400 archived bacterial samples were randomly selected following 3 years of storage at ambient temperature and PCR amplification was used to monitor its success. All of the 100 samples were successfully amplified using the 16S rDNA gene as a target and confirmed by DNA sequencing. Furthermore, the DNA was eluted into solution from the FTA cards using a new alkaline elution procedure for evaluation by real-time PCR-based assays. The viability of cells retained on the FTA cards varied among broad groups of bacteria. For the more fragile gram-negative species, no viable cells were retained even at high cell densities of between 10(7) and 10(8) colony forming units (cfu) ml(-1), and for the most robust species such as spore-formers and acid-fast bacteria, complete inactivation was achieved at cell densities ranging between 10(1) and 10(4) cfu ml(-1). The inactivation of bacterial cells on FTA cards suggest that this is a safe medium for the storage and transport of bacterial nucleic acids.
Rajendram
D
D
Molecular Identification Services Unit, Health Protection Agency Centre For Infections, 61 Colindale Avenue, London, UK.
Ayenza
R
R
Holder
F M
FM
Moran
B
B
Long
T
T
Shah
H N
HN
D001419:Bacteria / Q000235:genetics* / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000235:genetics; D001427:Bacterial Toxins / Q000235:genetics; D016172:DNA Fingerprinting; D004269:DNA, Bacterial / Q000235:genetics* / Q000302:isolation & purification*; D004275:DNA, Ribosomal / Q000032:analysis / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics; D008666:Metalloendopeptidases / Q000235:genetics; D050296:Microbial Viability; D016133:Polymerase Chain Reaction; D018272:Porins / Q000235:genetics; D011309:Preservation, Biological / Q000009:adverse effects* / Q000379:methods*; D019105:Random Amplified Polymorphic DNA Technique; D013997:Time Factors
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D008565:Membrane Proteins; D018272:Porins; C116707:enterococcal surface protein, esp; C405031:Bacteroides fragilis toxin; D008666:Metalloendopeptidases