We describe a novel HPV 9G DNA chip test for the accurate and reliable genotyping of human papillomavirus (HPV). The HPV 9G DNA chip test established its efficiency in terms of a signal-to-background ratio (SBR) of 200, which is 50 times superior to commercial HPV DNA chips, and 100% target-specific hybridization at 25°C. We compared the genotyping results for the 439 clinical samples by the HPV 9G DNA chip test with the sequencing results for the MY11/GP6؉ (M2) primer set-mediated PCR products. The discrimination of HPV genotypes in the 151 HPV-positive clinical samples by the HPV 9G DNA chip test were 100% identical with the sequencing analysis. The clinical sensitivities of HPV genotyping by the HPV 9G DNA chip test and a commercial HPV DNA chip test were 100% and 88%, respectively. However, the clinical specificities of HPV genotyping by the HPV 9G DNA chip test and the commercial HPV DNA chip test were 100% and 94%, respectively. The 100% clinical sensitivity and specificity of the HPV 9G DNA chip test make it a promising diagnostic tool for HPV genotyping.
With
the increasing rise of antibiotic-resistant pathogens, a simple
and rapid detection of antibiotic resistance gene (ARG) is crucial
to mitigate the spreading of antibiotic resistance. DNA-binding zinc
finger proteins (ZFPs) can be engineered to recognize specific double-stranded
(ds) DNA sequences in ARG. Here, we designed a simple and rapid method
to detect ARG in bacteria utilizing engineered ZFPs and 2D nanosheet
graphene oxide (GO) as a sensing platform. Our approach relies on
the on and off effect of fluorescence signal in the presence and absence
of target ARG, respectively. By taking advantage of the unique quenching
capability of GO due to its electronic property, quantum dot (QD)-labeled
ZFPs are adsorbed onto the GO sheets, and their fluorescence signal
is quenched by proximal GO sheets through fluorescence resonance energy
transfer (FRET). In the presence of target DNA, ZFP binding to the
target DNA induces dissociation from GO, thereby restoring the fluorescence
signal. Our system detects target DNA through restoration of QD emission
as the restored signal increases directly with target DNA concentrations.
Engineered ZFPs were able to detect specific dsDNA of the tetracycline
resistance gene tetM with high specificity after
only 10 min incubation on our GO-based sensing system. Our sensing
system employed one-step FRET-based ZFP and GO combined technology
to enable rapid and quantitative detection of ARG, providing a limit
of detection as low as 1 nM. This study demonstrated the application
of GO in conjunction with engineered DNA-binding domains for the direct
detection of dsDNA with great potential as a rapid and reliable screening
and detecton method against the growing threat of antibiotic resistant
bacteria.
We introduce the phenomenon of molecular recognition to immobilize oligonucleotides on AMCA slides for the production of 9G DNAChips. Facile and efficient method for the immobilization of the oligonucleotides appended with consecutive nine guanine bases is described. The 9G DNAChips shows more than 90% hybridization efficiency at 25 °C in 30 min.
The flaws in the present probe selection methods restrained the development of the DNA chip technology and its applications. The presented generalized probe selection method for the DNA chips elaborates the length of the probe, the melting temperatures, the specificity of the probe, and the position where the probe may bind to the targets.
According to the proposed DAGON method, the CRP and PSA antigens with the concentrations of 1 pg ml(-1) to 10 pg ml(-1) range can be easily differentiated in the buffer matrix. Moreover, it is for the first time that the multiple antigens with the concentrations of 1 pg ml(-1) and 0.1 pg ml(-1) can be detected in the mixture of the proteins without an amplification technique.
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