Rendering of mycobacteria safe for molecular diagnostic studies and development of a lysis method for strand displacement amplification and PCR.
Two criteria must be met before mycobacterial specimens can be tested by DNA amplification methods: (i) the sample must be rendered noninfectious, and (ii) the organisms must be lysed to free the DNA. Previous publications reporting DNA amplification of mycobacteria have concentrated on lysis and amplification procedures and have not addressed the issue of sample safety. We have shown that heating of samples below 100 degrees C may not consistently kill mycobacteria; however, heating at 100 degrees C in a boiling-water bath or a forced-air oven for a minimum of 5 min kills mycobacteria, including Mycobacterium thermoresistibile. Furthermore, heating at 100 degrees C for 30 min consistently lyses mycobacteria to produce short fragments of DNA that are suitable for amplification by PCR and strand displacement amplification. This procedure works with clinical samples digested by the n-acetyl cysteine-NaOH method as well as with suspensions of organisms in phosphate buffer. This paper also demonstrates the feasibility of using strand displacement amplification with clinical specimens.
Zwadyk
P
P
Veterans Affairs Medical Center, Durham, North Carolina 27705.
Down
J A
JA
Myers
N
N
Dey
M S
MS
D000111:Acetylcysteine; D001433:Bacteriolysis; D001483:Base Sequence; D002021:Buffers; D003264:Containment of Biohazards / Q000379:methods*; D004269:DNA, Bacterial / Q000302:isolation & purification*; D004202:Disinfectants / Q000494:pharmacology; D005240:Feasibility Studies; D006358:Hot Temperature; D006801:Humans; D008969:Molecular Sequence Data; D009161:Mycobacterium / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D009164:Mycobacterium Infections / Q000382:microbiology*; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction; D012449:Safety; D012680:Sensitivity and Specificity; D012972:Sodium Hydroxide; D013997:Time Factors
D002021:Buffers; D004269:DNA, Bacterial; D004202:Disinfectants; D012972:Sodium Hydroxide; D000111:Acetylcysteine