A primer and probe set for detecting multiple types of EGFR exon 19 deletions.
EGFR exon 19 deletion is an important indicator for tyrosine kinase inhibitor treatment in non-small cell lung cancer. However, detection of exon 19 deletions faces a challenge: there are more than 30 types of mutations reported at the hotspot. Moreover, considering the application in body fluid samples, assays with high sensitivity and specificity are necessary for the detection of rare mutant alleles. Here, we describe a single tube reaction which could detect at least 29 types of exon 19 deletions with only an unlabeled peptide nucleic acid (PNA) clamp and a pair of DNA probes. The PNA clamp was used to inhibit amplification of wild-type templates; and the DNA probes were used to generate melting peaks for multiple types of mutations. Under optimal condition, the assay was able to detect as low as 0.01% mutant DNA in wild-type background, and had a limit of detection of 10 pg genomic DNA. Feasibility of the assay was tested in body fluid samples from lung cancer patients. The assay detected 100% and 60% of deletions in pleural effusions and plasma, respectively. We believe the present assay can be used in the clinical laboratories and has potential to be adapted for a microfluidic device.
Graduate Institute of Biomedical Sciences, Chang Gung University, Taoyuan, Taiwan.
Tai-Long
TL
Chen
Division of Hematology-Oncology, Department of Internal Medicine, Chang Gung Memorial Hospital, Taoyuan, Taiwan; School of Medicine, College of Medicine, Chang Gung University, Taoyuan, Taiwan.
John Wen-Cheng
JW
Chang
Department of Thoracic Medicine, Chang Gung Memorial Hospital, Taoyuan, Taiwan.
Chih-Liang
CL
Wang
Department of Thoracic Medicine, Chang Gung Memorial Hospital, Taoyuan, Taiwan; Department of Respiratory Therapy, Chang Gung University, Taoyuan, Taiwan.
Cheng-Ta
CT
Yang
Graduate Institute of Biomedical Sciences, Chang Gung University, Taoyuan, Taiwan; Department of Laboratory Medicine, Chang Gung Memorial Hospital, Taoyuan, Taiwan.
Mei-Chia
MC
Wang
Graduate Institute of Biomedical Sciences, Chang Gung University, Taoyuan, Taiwan; Department of Thoracic Medicine, Chang Gung Memorial Hospital, Taoyuan, Taiwan; Department of Medical Biotechnology and Laboratory Science, Chang Gung University, Taoyuan, Taiwan. Electronic address: ccchiou@mail.cgu.edu.tw.
Chiuan-Chian
CC
Chiou
D001483:Base Sequence; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics*; D015342:DNA Probes / Q000235:genetics*; D066246:ErbB Receptors / Q000235:genetics*; D005260:Female; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics*; D008297:Male; D020135:Peptide Nucleic Acids / Q000235:genetics*; D016066:Pleural Effusion, Malignant / Q000235:genetics*; D017384:Sequence Deletion
D015342:DNA Probes; D020135:Peptide Nucleic Acids; C512478:EGFR protein, human; D066246:ErbB Receptors
EGFR mutation; Fluorescent probe; Forster resonance energy transfer; Melting analysis; Peptide nucleic acid clamp