Non-isotopic microtitre plate-based assay for detecting products of polymerase chain reaction amplification: application to detection of the tdh gene of Vibrio parahaemolyticus.
A non-isotopic microtitre plate-based assay method was devised for detection of products of the polymerase chain reaction. This assay involves affinity immobilization of the biotinylated amplification products in microtitre plate wells and their fluorescence detection by their hybridization with an oligonucleotide probe linked to alkaline phosphatase. An advantage of this procedure is that the immobilization and hybridization are carried out simultaneously in the wells, thus shortening the assay time. The assay method was applied to the detection of the tdh gene of Vibrio parahaemolyticus. Seven copies of the target chromosome could be detected in about 45 min after 35 cycles of amplification.
Tada
J
J
Central Research Laboratory, Shimadzu Corporation, Kyoto, Japan.
Ohashi
T
T
Nishimura
N
N
Ozaki
H
H
Fukushima
S
S
Takano
J
J
Nishibuchi
M
M
Takeda
Y
Y
D000469:Alkaline Phosphatase; D015415:Biomarkers; D001710:Biotin; D004269:DNA, Bacterial / Q000032:analysis*; D005453:Fluorescence; D005798:Genes, Bacterial; D006460:Hemolysin Proteins / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000295:instrumentation; D014736:Vibrio parahaemolyticus / Q000235:genetics* / Q000472:pathogenicity; D014774:Virulence
D015415:Biomarkers; D004269:DNA, Bacterial; D006460:Hemolysin Proteins; D015345:Oligonucleotide Probes; D001710:Biotin; D000469:Alkaline Phosphatase