An improved, real-time PCR assay for the detection of GC-rich and low abundance templates of Mycobacterium tuberculosis.
The detection of low abundance mRNA and/or GC-rich targets is very difficult using real-time PCR, often requiring laborious optimization procedures. This work shows that formamide is a useful PCR additive, increasing the sensitivity and specificity of SYBR Green real-time PCR to detect low abundance mycobacterial RNA from infected samples.
Grassi
Manuela
M
Institute Neurobiology and Molecular Medicine, Molec. Med. Section, National Research Council, Via del Fosso del Cavaliere, 100, 00133, Rome, Italy.
Volpe
Elisabetta
E
Colizzi
Vittorio
V
Mariani
Francesca
F
D001482:Base Composition; D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes / Q000737:chemistry; D005559:Formamides / Q000737:chemistry; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000302:isolation & purification; D009930:Organic Chemicals / Q000737:chemistry; D011804:Quinolines; D012333:RNA, Messenger / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013698:Templates, Genetic; D014376:Tuberculosis / Q000382:microbiology
D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D005559:Formamides; D009930:Organic Chemicals; D011804:Quinolines; D012333:RNA, Messenger; C098022:SYBR Green I; C031066:formamide