title: Quantitative real-time PCR assay for rapid detection of plant and human pathogenic Macrophomina phaseolina from field and environmental samples.
abstract: A real-time qPCR assay was developed to detect and quantify Macrophomina phaseolina abundance in rhizosphere soil and plant tissue. Both TaqMan and SYBR green techniques were targeted on ~ 1 kb sequence characterized amplified region (SCAR) of M. phaseolina and two sets of specific primers were designed for SYBR green (MpSyK) and TaqMan (MpTqK) assays. No cross-hybridization and no fluorescent signal exceeding the baseline threshold was observed in TaqMan and SYBR green assays, respectively. The minimum detection limit or sensitivity of TaqMan assay was 30 fg/μL of M. phaseolina DNA and limit of quantification of M. phaseolina viable population was estimated as 0.66 × 10(5) CFU/g soil(-1) equivalent to 10 pg/μL of target DNA. This is the first report which demonstrated real-time qPCR assays with greater specificity and sensitivity to detect M. phaseolina population in soil and plant materials.
authors.affiliation: National Bureau of Agriculturally Important Microorganisms (NBAIM), Post Box 06, Maunath Bhanjan, Uttar Pradesh-275 101, India. kishore_bandam@yahoo.co.in
authors.forename: Bandamaravuri Kishore
authors.initials: BK
authors.lastname: Babu
authors.forename: Sukumar
authors.initials: S
authors.lastname: Mesapogu
authors.forename: Anu
authors.initials: A
authors.lastname: Sharma
authors.forename: Saida Reddy
authors.initials: SR
authors.lastname: Somasani
authors.forename: Dilip K
authors.initials: DK
authors.lastname: Arora
mesh_terms: D001203:Ascomycota / Q000737:chemistry / Q000302:isolation & purification* / Q000472:pathogenicity; D001483:Base Sequence; D052160:Benzothiazoles; D016172:DNA Fingerprinting; D015342:DNA Probes; D004271:DNA, Fungal / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification*; D003959:Diamines; D006801:Humans; D057230:Limit of Detection; D009930:Organic Chemicals; D010944:Plants / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D058441:Rhizosphere; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D012988:Soil Microbiology; D013045:Species Specificity
chemical_list: D052160:Benzothiazoles; D015342:DNA Probes; D004271:DNA, Fungal; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I