[Construction of the method for quantitative detection of epidermal growth factor receptor variant III by real-time polymerase chain reaction with TaqMan probe and its application].
OBJECTIVE
To construct the recombinant plasmid and standard curve for detection of epidermal growth factor receptor variant III (EGFRv III) by real-time quantitive polymerase chain reaction (PCR) and establish the RT-PCR assay for accurate detection of EGFRv III.


METHODS
To amplify the DNA sequences of exon 1 and exon 8 to exon 9 of EGFR separately and fuse the above sequences by overhang extension PCR. Then constructing the recombinant plasmid of EGFRv III by highly specific primers with the fused sequence as its template. The recombinant plasmid of EGFRv III was then sent for sequence analysis. The gradient diluted recombinant plasmid were used as the template to amplify the EGFRv III by RT-PCR with TaqMan probe and then applied it in 32 laryngeal carcinoma tissues.


RESULTS
The recombinant plasmid of EGFRv III was successfully constructed. The gradient diluted recombinant plasmid could be used in RT-PCR for quantitative analysis of EGFRv III. In all, 5 tumor samples expressing EGFRv III were detected, with its positive rate of about 15.6%. The expression of EGFRv III was tumor specific with only one exception for extraordinarily low expression of EGFRv III in macroscopically normal laryngeal mucosas adjacent to the tumor.


CONCLUSIONS
RT-PCR with TaqMan probe was good at sensitivity, specificity, quantification and linear function. It can be a standard method for quantitative detection for EGFRv III. Expression of EGFRv III was detected in laryngeal carcinoma tissues with relatively low level. Whether EGFRv III was a suitable target for biological therapy in laryngeal carcinomas remained to be discussed.
Yang
Bei-Bei
BB
Department of Otorhinolaryngology, Second Affiliated Hospital of Medical College, Zhejiang University, Hangzhou 310009, China. shengbei@mail.hz.zj.cn
Zhang
Xing
X
Chen
Jia
J
Cao
Jiang
J
Chang
Hui-Yu
HY
D000328:Adult; D000368:Aged; D002294:Carcinoma, Squamous Cell / Q000235:genetics* / Q000473:pathology; D003199:Computer Systems; D004247:DNA / Q000235:genetics; D017931:DNA Primers; D066246:ErbB Receptors / Q000235:genetics*; D005091:Exons; D006801:Humans; D007822:Laryngeal Neoplasms / Q000235:genetics* / Q000473:pathology; D008297:Male; D008875:Middle Aged; D009367:Neoplasm Staging; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*
D017931:DNA Primers; C410723:epidermal growth factor receptor VIII; D004247:DNA; D066246:ErbB Receptors