Useofpolymerase chain reaction forearly identification ofMycobacterium tuberculosis in positive cultures

Aims:To developa readily applicable polymerase chainreaction (PCR)based technique whichwouldpermittheidentification ofMycobacterium tuberculosis complexisolates fromBactecphials atan earlier stagethancurrently available methods. Methods:Mycobacterial cells cultured in Bactec12Bmediumwereharvested by centrifugation. The cells werelysedby heating indistilled water. Oligonucleotide primers basedonthesequence ofthegene codingfortheimmunogenicprotein MPB64 werethenusedtoamplify a 240 basepairfragment ofDNA directly from thecrudecelllysate. ThePCR product wasvisualised underultraviolet light following electrophoresis ofanaliquot inan agarose gelcontaining ethidium bromide. Thesensitivity ofthePCRwasadjusted so thatabout600cfuofM tuberculosis gavea positive result. Thelowest growthindexat whichthismethodofidentification might beapplied toBactecphials was determinedandanumberofroutine cultures giving apositive growthindexexamined. Results: M tuberculosis was positively identified atthelowest growthindex, as determined bytheBactecsystem.Of45 routine cultures examined, withgrowth indexesranging from6 to999,the15 confirmedby conventional means to contain M tuberculosis were correctly identified from1mlofculture medium. Conclusions: Themethoddescribed can beusedtoidentify M tuberculosis isolates cultured intheBactecsystematthe earliest detectable riseingrowth index. It may therefore allowculturedmycobacteria tobeidentified atanearlier stage thanconventional methodsorthecommercially available DNA probesadapted forusewiththeBactecsystem.