Optical detection of human papillomavirus type 16 and type 18 by sequence sandwich hybridization with oligonucleotide-functionalized Au nanoparticles.
The importance of detecting and subtyping human papillomaviruses (HPVs) in clinical and epidemiological studies has been well addressed. In detecting the most common types of HPV, type 16 (HPV-16) and type 18 (HPV-18), in the cervical mucous of patients in a simple and rapid manner, the assay of a label-free colorimetric DNA sensing method based on sequence sandwich hybridization with oligonucleotide-functionalized Au nanoparticles (AuNPs) was fabricated in this study. Specific oligonucleotide probes were designed for the sequence detection within the L1 gene of HPV-16 and HPV-18, and the probes were capped onto AuNPs, as AuNP probes. The target HPV sequences in clinical specimens were obtained by an asymmetric polymerase chain reaction (PCR) with universal primers, which can amplify the target sequences from several HPV serotypes, including HPV-16 and HPV-18. The DNA sandwich hybridization between the target sequences and the specific AuNP probes was performed at a temperature closer to the theoretical melting temperature of the DNA hybridization. Next, the procedure of increasing salt concentration and cooling the hybridizing solution was immediately utilized to discriminate the target sequences of HPV-16 or HPV-18. If the target sequences were not complementary to sequences of AuNP probes, the AuNPs would aggregate because no duplex DNA formation occurred such that the color of the reaction solution changed from red to purple. If the AuNP probes were a perfect match to the target sequences and a full DNA sandwich hybridization occurred, the reaction solution maintained its red color. A total of 70 mucous specimens from patients with cervical intraepithelial neoplasia were tested by the AuNP probes sandwich hybridization. The results show that there were 33, 16, 5, and 16 cases detected with HPV-16, HPV-18, both HPV-16 and HPV-18 (HPV-16/HPV-18), and neither HPV-16 nor HPV-18, respectively.
Chen
Sz-Hau
SH
Department of Biological Science and Technology, National Chiao Tung University, Hsinchu 30068, Taiwan.
Lin
Kun-I
KI
Tang
Chuan-Yi
CY
Peng
Sheng-Lung
SL
Chuang
Yao-Chen
YC
Lin
Yi-Rou
YR
Wang
Jui-Ping
JP
Lin
Chih-Sheng
CS
D003124:Colorimetry / Q000379:methods*; D004279:DNA, Viral / Q000235:genetics; D006046:Gold / Q000737:chemistry; D052162:Human papillomavirus 16 / Q000235:genetics* / Q000302:isolation & purification*; D052161:Human papillomavirus 18 / Q000235:genetics* / Q000302:isolation & purification*; D017403:In Situ Hybridization / Q000379:methods*; D053758:Nanoparticles / Q000737:chemistry; D017422:Sequence Analysis, DNA / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods
D004279:DNA, Viral; D006046:Gold