In situ PCR法による腸管出血性大腸菌の検出

@article{1997InSP,
  title={In situ PCR法による腸管出血性大腸菌の検出},
  author={黒川 顕 and 谷 佳津治 and 那須 正夫},
  journal={Japanese journal of bacteriology},
  year={1997},
  volume={52},
  pages={513-518}
}
Direct in situ PCR法は特定のDNA配列を標的として菌体内でPCRを行い, 増幅されたDNAを蛍光基質などでラベルすることにより, 蛍光顕微鏡下で特定遺伝子を持つ細菌を単個細胞レベルで特異的に検出する方法である。PCRには腸管出血性大腸菌の Vero 毒素をコードする遺伝子 slt-I, slt-II に特異的なEVT, EVSプライマーを用いた。増幅されたDNAはジゴキシゲニンにより標識し, アルカリホスファターゼ標識した抗ジゴキシゲニン抗体および蛍光基質としてHNPP/Fast Red TRを用いた。その結果, E. coli O157を蛍光顕微鏡下で特異的にまた高感度に単個細胞レベルで検出することができた。 
8 Citations
In Situ Polymerase Chain Reaction Visualization of Vibrio halioticoli Using Alginate Lyase Gene AlyVG2
TLDR
Two-stage PI-PCR adding the extension and digoxigenin-labeling step of the amplified fragment into the first amplification stage allowed us to differentiate V. halioticoli cells from V. pelagius cells. Expand
rRNA-targeted fluorescent in situ hybridization analysis of bacterial community structure in river water.
TLDR
An improved in situ hybridization technique, HNPP-FISH, using 2-hydroxy-3-naphthoic acid 2'-phenylanilide phosphate (HNPP) and Fast Red TR was applied to analyse the community structure of planktonic bacteria in river water and should be a useful tool for the analysis of microbial community composition. Expand
Detection of Bacteria Carrying the stx2 Gene by In Situ Loop-Mediated Isothermal Amplification
TLDR
Loop-mediated isothermal amplification (LAMP) was used to detect stxA2 in Escherichia coli O157:H7 cells, causing less cell damage than in situ PCR and allowing use of fluorescent antibody labeling in the bacterial mixture after the DNA amplification. Expand
Monitoring of Ralstonia eutropha KT1 in Groundwater in an Experimental Bioaugmentation Field by In Situ PCR
ABSTRACT Ralstonia eutropha KT1, which degrades trichloroethylene, was injected into the aquifer after activation with toluene, and then the number of bacteria was monitored by in situ PCR targetingExpand
Abundance and distribution of bacteria carrying sltII gene in natural river water
TLDR
Direct in situ PCR with HNPP/Fast Red TR was used to enumerate bacteria carrying the sltII gene in river water, indicating that such bacteria are commonly distributed in natural river water. Expand
Isolation of Bacillus thuringiensis strains that contain Dipteran‑specific cry genes from Ilha Bela (São Paulo, Brazil) soil samples
TLDR
Seven bacterial isolates from 76 colonies of B. thuringiensis were potentially able to control A. aegypti larvae, and these are promising isolates for the biological control of A. Expand
Isolation of Bacillus thuringiensis strains that contain Dipteran-specific cry genes from Ilha Bela (São Paulo, Brazil) soil samples.
TLDR
This research isolated 76 colonies of B. thuringiensis from 30 soil samples that were taken from Ilha Bela, Brazil to find bacterial isolates that were capable of controlling A. aegypti larvae, and found seven isolates potentially able to control the mosquito's larvae. Expand
Occurrence of Escherichia coil O157
TLDR
Results indicate that viable E. coli O157: H7 occurs in river water in Osaka Prefecture, Japan. Expand

References

SHOWING 1-9 OF 9 REFERENCES
An improved technique for the in situ detection of DNA after polymerase chain reaction amplification.
TLDR
A nonisotopic PCR in situ technique, employing a single primer pair and target sequences as short as 115 base pairs, that can detect one target molecule per cell, and may permit target detection by direct incorporation of labeled nucleotides. Expand
Fluorescent Antibodies Applied to Direct Epifluorescent Filter Technique for Microscopic Enumeration of Escherichia coli 0157:H7 in Milk and Juice.
TLDR
This is the first known demonstration of the combination of DEFT and antibody probe technology for the specific enumeration of a microbe directly in food without a growth or enrichment step. Expand
Nucleotide sequence analysis and comparison of the structural genes for Shiga-like toxin I and Shiga-like toxin II encoded by bacteriophages from Escherichia coli 933
TLDR
The nucleotide sequence of the Shiga-like toxin type II (SLT-II) structural genes cloned from bacteriophage 933W of the enterohemorrhagic Escherichia coli O157:H7 strain 933 was determined and the regulation proposed for the SLT -II operon is similar to that previously proposed for SLT-I. Expand
Bacterial gene transfer by natural genetic transformation in the environment.
TLDR
The current understanding of the biology of transformation is summarized to provide the platform on which aspects of bacterial transformation in water, soil, and sediments and the habitat of pathogens are discussed. Expand
Immunomagnetic separation as a sensitive method for isolating Escherichia coli O157 from food samples.
Minced beef samples inoculated with Escherichia coli O157 were cultured in buffered peptone water supplemented with vancomycin, cefsulodin and cefixime (BPW-VCC) and subcultured to cefixime telluriteExpand
Detection of specific bacterial cells with 2-hydroxy-3-naphthoic acid-2'-phenylanilide phosphate and fast red TR in situ hybridization
TLDR
An in situ hybridization technique with HNPP (2-hydroxy-3-naphthoic acid-2'-phenylanilide phosphate) and Fast Red TR was used to detect specific bacterial cells at the single-cell level to identify small or low-rRNA-content bacterial cells in the natural environment. Expand
Purification and characterization of a Shigella dysenteriae 1-like toxin produced by Escherichia coli.
A toxin from an enteropathogenic strain of Escherichia coli (E. coli H30) was purified to apparent homogeneity from cell lysates. The steps used to isolate the E. coli H30 toxin included FrenchExpand
An improved selective medium for the isolation of Escherichia coli O157.
TLDR
Inclusion of rhamnose and cefixime in sorbitol-MacConkey agar improves its selectivity for the isolation of VT+ E. coli O157. Expand