Specific and quantitative detection of PCR products from Clostridium piliforme, Helicobacter bilis, H. hepaticus, and mouse hepatitis virus infected mouse samples using a newly developed electrochemical DNA chip.
We developed a microfabricated electrochemical DNA chip for detection of polymerase chain reaction (PCR) products from 16S rRNA sequences of Clostridium piliforme (Cp), Helicobacter bilis (Hb) and Helicobacter hepaticus (Hh), and the nucleocapsid protein gene of mouse hepatitis virus (MHV). This chip does not require DNA labeling, and the hybridization signal can be detected as an anodic current. The average anodic currents of 9 (Cp), 5 (Hb), 8 (Hh) and 7 (MHV) PCR positive samples derived from feces of spontaneously infected mice (Cp, Hb and Hh) and MHV-contaminated tumor cells were 27.9+/-7.2, 31.9+/-8.1, 29.3+/-10.1, and 27.6+/-3.0 nA, respectively. On the other hand, the average anodic currents of 19 (Cp), 27 (Hb), 18 (Hh), and 13 (MHV) PCR negative samples were 0.3+/-2.9, 3.7+/-2.4, -1.0+/-1.7, and -2.3+/-2.7 nA, respectively. The anodic current increased with increasing concentrations of pathogens. For experimentally infected samples, the results of PCR/electrophoresis were in complete accord with those of this system when anodic currents of 6.1 (Cp), 8.5 (Hb), 2.4 (Hh), and 3.1 nA (MHV) were taken as the cut-off value. The results suggested that the electrochemical DNA chip system is useful for specific and quantitative detection of PCR products.
Goto
Kazuo
K
Central Institute for Experimental Animals, 1430 Nogawa, Miyamae, Kawasaki 216-0001, Japan. gotok@ciea.or.jp
Horiuchi
Hideki
H
Shinohara
Haruka
H
Motegi
Katsumi
K
Hashimoto
Koji
K
Hongo
Sadato
S
Gemma
Nobuhiro
N
Hayashimoto
Nobuhito
N
Itoh
Toshio
T
Takakura
Akira
A
D000818:Animals; D003013:Clostridium / Q000235:genetics / Q000302:isolation & purification*; D017931:DNA Primers; D004563:Electrochemistry; D016998:Helicobacter / Q000235:genetics / Q000302:isolation & purification*; D016481:Helicobacter Infections / Q000175:diagnosis / Q000662:veterinary; D044923:Helicobacter hepaticus / Q000235:genetics / Q000302:isolation & purification*; D051379:Mice; D008954:Models, Biological; D006517:Murine hepatitis virus / Q000235:genetics / Q000302:isolation & purification*; D019590:Nucleocapsid Proteins / Q000235:genetics / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation / Q000379:methods*; D016133:Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S / Q000378:metabolism; D012376:Rodent Diseases / Q000175:diagnosis; D012680:Sensitivity and Specificity
D017931:DNA Primers; D019590:Nucleocapsid Proteins; D012336:RNA, Ribosomal, 16S