Restriction site insertion-PCR (RSI-PCR) for rapid discrimination and typing of closely related microbial strains

Daniele Daffonchio; Sara Borin; Arianna Consolandi; Claudia Sorlini

doi:10.1016/S0378-1097(99)00467-X

Restriction site insertion-PCR (RSI-PCR) for rapid discrimination and typing of closely related microbial strains

Daniele Daffonchio^*, Sara Borin, Arianna Consolandi, Claudia Sorlini

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

13 Scopus citations

Abstract

Taking advantage of point mutations between DNA sequences of closely related microbial strains, PCR primers modified with respect to the target sequence at positions 2-5 near the 3' end were designed to obtain a fragment harbouring an artificial restriction site specific for a given strain. The modified forward primer coupled with a specific reverse primer allows for the amplification of DNA fragments which can be digested with the specific endonuclease only in those strains where the restriction site is inserted by the DNA polymerase. The effectiveness of the method, named restriction site insertion-PCR (RSI-PCR), was tested on isolates of the 'Bacillus cereus group' for the rapid typing and discrimination of these closely related strains. Copyright (C) 1999 Federation of European Microbiological Societies.

Original language	English (US)
Pages (from-to)	77-83
Number of pages	7
Journal	FEMS Microbiology Letters
Volume	180
Issue number	1
DOIs	https://doi.org/10.1016/S0378-1097(99)00467-X
State	Published - Nov 1 1999
Externally published	Yes

Keywords

3' End primer modification
Bacillus cereus group
Point mutation
Restriction analysis
Restriction site insertion-PCR
Strain identification

ASJC Scopus subject areas

Microbiology
Molecular Biology
Genetics

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10.1016/S0378-1097(99)00467-X

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title = "Restriction site insertion-PCR (RSI-PCR) for rapid discrimination and typing of closely related microbial strains",

abstract = "Taking advantage of point mutations between DNA sequences of closely related microbial strains, PCR primers modified with respect to the target sequence at positions 2-5 near the 3' end were designed to obtain a fragment harbouring an artificial restriction site specific for a given strain. The modified forward primer coupled with a specific reverse primer allows for the amplification of DNA fragments which can be digested with the specific endonuclease only in those strains where the restriction site is inserted by the DNA polymerase. The effectiveness of the method, named restriction site insertion-PCR (RSI-PCR), was tested on isolates of the 'Bacillus cereus group' for the rapid typing and discrimination of these closely related strains. Copyright (C) 1999 Federation of European Microbiological Societies.",

keywords = "3' End primer modification, Bacillus cereus group, Point mutation, Restriction analysis, Restriction site insertion-PCR, Strain identification",

author = "Daniele Daffonchio and Sara Borin and Arianna Consolandi and Claudia Sorlini",

note = "Funding Information: The study was performed in the ambit of the ELECTRO Project {\textquoteleft}Electrochemical treatment of fresh animal manure for reducing environment and health risk{\textquoteright}, financed by the European Community (E.C. contract no. FAIR5-PL97-3506). We thank Prof. M. Mock and Dr. G. Patra, who kindly gave us the total DNA of B. anthracis, Prof. S. Scherer and Dr. R. Mayr for providing us the strains of B. weihenstephanensis and Prof. L.K. Nakamura for the B. pseudomycoides strains.",

year = "1999",

month = nov,

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doi = "10.1016/S0378-1097(99)00467-X",

language = "English (US)",

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pages = "77--83",

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AU - Daffonchio, Daniele

AU - Borin, Sara

AU - Consolandi, Arianna

AU - Sorlini, Claudia

N1 - Funding Information: The study was performed in the ambit of the ELECTRO Project ‘Electrochemical treatment of fresh animal manure for reducing environment and health risk’, financed by the European Community (E.C. contract no. FAIR5-PL97-3506). We thank Prof. M. Mock and Dr. G. Patra, who kindly gave us the total DNA of B. anthracis, Prof. S. Scherer and Dr. R. Mayr for providing us the strains of B. weihenstephanensis and Prof. L.K. Nakamura for the B. pseudomycoides strains.

PY - 1999/11/1

Y1 - 1999/11/1

N2 - Taking advantage of point mutations between DNA sequences of closely related microbial strains, PCR primers modified with respect to the target sequence at positions 2-5 near the 3' end were designed to obtain a fragment harbouring an artificial restriction site specific for a given strain. The modified forward primer coupled with a specific reverse primer allows for the amplification of DNA fragments which can be digested with the specific endonuclease only in those strains where the restriction site is inserted by the DNA polymerase. The effectiveness of the method, named restriction site insertion-PCR (RSI-PCR), was tested on isolates of the 'Bacillus cereus group' for the rapid typing and discrimination of these closely related strains. Copyright (C) 1999 Federation of European Microbiological Societies.

AB - Taking advantage of point mutations between DNA sequences of closely related microbial strains, PCR primers modified with respect to the target sequence at positions 2-5 near the 3' end were designed to obtain a fragment harbouring an artificial restriction site specific for a given strain. The modified forward primer coupled with a specific reverse primer allows for the amplification of DNA fragments which can be digested with the specific endonuclease only in those strains where the restriction site is inserted by the DNA polymerase. The effectiveness of the method, named restriction site insertion-PCR (RSI-PCR), was tested on isolates of the 'Bacillus cereus group' for the rapid typing and discrimination of these closely related strains. Copyright (C) 1999 Federation of European Microbiological Societies.

KW - 3' End primer modification

KW - Bacillus cereus group

KW - Point mutation

KW - Restriction analysis

KW - Restriction site insertion-PCR

KW - Strain identification

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JF - FEMS Microbiology Letters

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Restriction site insertion-PCR (RSI-PCR) for rapid discrimination and typing of closely related microbial strains

Abstract

Keywords

ASJC Scopus subject areas

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