[Problems related to the use of real-time RT-PCR in environmental analysis].
Molecular biology techniques allow high sensitivity and specificity in the detection of enteric viruses in various environmental samples, and are considerably less costly and more rapid than traditional analytical methods. Real time RT-PCR technology allows accurate, efficient, and reproducible quantification of viral genes, by amplifying enteroviral RNA directly from an adequately treated environmental sample. It uses different chemical systems, including TaqMan and Syber Green probes, for detection of the amplificon. Both systems allow quantification of the initial number of copies in each cycle by comparing values with those of an external calibration curve (standard curve), generated by serial dilutions of a reference RNA sample with a known concentration. Difficulties in generating a standard curve for each enteric virus however, make standardization of the system time consuming. In an attempt to overcome this obstacle, we used an internal standard with a known concentration, to obtain a valid calibration curve for the quantification of environmental enteroviruses. A comparative analysis was performed with various commercially available extraction and amplification systems to evaluate the method's efficiency and reproducibility.
Donia
Domenica Tommasa
DT
Dipartimento di Sanità Pubblica e Biologia Cellulare Cattedra di Igiene, Università Tor Vergata. donia@med.uniroma2.it
Divizia
Maurizio
M
Panà
Augusto
A
D004770:Enterovirus / Q000302:isolation & purification*; D004783:Environmental Microbiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity