RESUMO
Fluorogenic quantitative polymerase chain reaction(FQ-PCR) is an emerging technique with high specificity,susceptibility,automatism and has been widely used for the detection and quantification of microorganism,some genetic diseases and cancer.The design and establishment of standard FQ-PCR laboratory are required not only by gene expansion and diagnosis but also by biosafty and veracity.The aim of the paper is to introduce the structural characteristics,instruments requirement and comfort parameters of FQ-PCR laboratory.
RESUMO
AIM: To establish a fluorogenic quantitative polymerase chain reaction (FQ-PCR) method for the routine examination of c-erbB-2 gene expression in breast cancer. METHODS: The c-erbB-2 standard gene was obtained by in vitro amplification of cloned c-erbB-2 fragment in plasmid PGEM-T easy vector. FQ-PCR product was detected by using a 7700 ABI PRISM sequence detector system and c-erbB-2 standard curve was obtained to quantity c-erbB-2 in unknown samples. RESULTS: “S” kinetics curve of FQ-PCR amplification was generated by relating the fluorescence signal intensity (△Rn) to the cycle number. The standard curve of c-erbB-2 was constructed by the linear relationship between the cycle threshold (ct) and the log of starting copy number. The high correlation (0.999) revealed the reliability of FQ-PCR. CONCLUSION: The FQ-PCR is a rapid, sensitive, reliable method for quantity of c-erbB-2 gene expression.