Intercalation activating fluorescence DNA probe and its application to homogeneous quantification of a target sequence by isothermal sequence amplification in a closed vessel.
We developed a completely homogeneous and isothermal method of detecting RNA sequences and demonstrated ultrarapid and direct quantification of pathogenic gene expression with high sensitivity. The assay is based on performing isothermal RNA sequence amplification in the presence of our novel DNA probe, an intercalation activating fluorescence DNA probe, and measuring the fluorescence intensity of the reaction mixture. When detecting mecA gene expression of methicillin-resistant Staphylococcus aureus, we quantified starting copies ranging from 10 to 10(7) copies within 10min. The primer sequences were designed to bind to secondary structure-free sites of the target RNA, which enabled a totally isothermal protocol to quantify mRNA specifically in a sample of existing genomic DNA. When we applied this to quantifying the expression of marker genes of Vibrio parahaemolyticus and Mycobacterium bovis BCG strain, the results correlated well with the viability of each bacterium. We also demonstrated monitoring Pab gene expression of M. bovis BCG during cultivation with antibiotics. The present method can potentially realize rapid antimicrobial susceptibility testing of slowly growing organisms, such as tuberculosis.
Ishiguro
Takahiko
T
Tokyo Research Laboratories, Tosoh Corporation, 2743-1 Hayakawa, Ayase-shi, Kanagawa 252-1123, Japan. isiguro@tosoh.co.jp
Saitoh
Juichi
J
Horie
Ryuichi
R
Hayashi
Toshinori
T
Ishizuka
Tetsuya
T
Tsuchiya
Shigeo
S
Yasukawa
Kiyoshi
K
Kido
Takahiro
T
Nakaguchi
Yoshitsugu
Y
Nishibuchi
Mitsuaki
M
Ueda
Kunihiro
K
D001483:Base Sequence; D017931:DNA Primers / Q000378:metabolism; D015342:DNA Probes / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D007364:Intercalating Agents / Q000378:metabolism*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009163:Mycobacterium bovis / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012329:RNA, Bacterial / Q000032:analysis / Q000235:genetics; D012680:Sensitivity and Specificity; D013211:Staphylococcus aureus / Q000235:genetics; D013696:Temperature; D014736:Vibrio parahaemolyticus / Q000235:genetics
D017931:DNA Primers; D015342:DNA Probes; D004269:DNA, Bacterial; D005456:Fluorescent Dyes; D007364:Intercalating Agents; D012329:RNA, Bacterial