Evaluation of a rapid PCR-based epidemiological typing method for routine studies of Mycobacterium tuberculosis.
Restriction fragment length polymorphism (RFLP) based on the insertion sequence IS6110 is used to investigate episodes of suspected transmission of infection of tuberculosis but usually takes a number of weeks from receipt of request to obtain a result. Often investigations would benefit from a more rapid method, possibly one containing an amplification step. The method employed uses a simple DNA extraction followed by a PCR step involving a single primer. Restriction enzyme analysis was performed when the patterns obtained from the PCR products were indistinguishable, especially when only single similar-size bands were obtained. The isolates used were strains of Mycobacterium tuberculosis submitted for epidemiological investigations as part of (i) possible contact-outbreak (22 episodes involving between 2 and 20 patients), (ii) possible incidents of laboratory cross-contamination (21 episodes), and (iii) possible change in drug resistance pattern or a case of reinfection (1 patient). The PCR products giving similar patterns were then subjected to restriction enzyme analysis. In conclusion it has been shown that this method is rapid, with results within 1 to 2 days of the request being received; is reproducible; and gives the same results as does RFLP. The restriction enzyme analysis stage has improved the efficiency of the technique.
Yates
Malcolm D
MD
PHLS Mycobacterium Reference Unit, Public Health Laboratory, Dulwich Hospital, East Dulwich Grove, London SE22 8QF, United Kingdom. m_d_yates@hotmail.com
Drobniewski
Francis A
FA
Wilson
Stuart M
SM
D015373:Bacterial Typing Techniques; D016358:Contact Tracing; D004251:DNA Transposable Elements / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification; D004196:Disease Outbreaks; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000145:classification* / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D015203:Reproducibility of Results; D013997:Time Factors; D014397:Tuberculosis, Pulmonary / Q000453:epidemiology* / Q000382:microbiology
D004251:DNA Transposable Elements; D004269:DNA, Bacterial