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Titolo:
Use of the bead beater for preparation of Mycobacterium paratuberculosis template DNA in milk
Autore:
Odumeru, J; Gao, A; Chen, S; Raymond, M; Mutharia, L;
Indirizzi:
Univ Guelph, Lab Serv Div, Guelph, ON N1H 8J7, Canada Univ Guelph Guelph ON Canada N1H 8J7 Serv Div, Guelph, ON N1H 8J7, Canada Univ Guelph, Dept Microbiol, Guelph, ON N1G 2W1, Canada Univ Guelph Guelph ON Canada N1G 2W1 icrobiol, Guelph, ON N1G 2W1, Canada
Titolo Testata:
CANADIAN JOURNAL OF VETERINARY RESEARCH-REVUE CANADIENNE DE RECHERCHE VETERINAIRE
fascicolo: 4, volume: 65, anno: 2001,
pagine: 201 - 205
SICI:
0830-9000(200110)65:4<201:UOTBBF>2.0.ZU;2-E
Fonte:
ISI
Lingua:
ENG
Soggetto:
CONDITIONS SIMULATING PASTEURIZATION; POLYMERASE CHAIN-REACTION; CROHNS-DISEASE; MICROBIAL DNA; COWS MILK; RAW-MILK; PCR; EXTRACTION; IS900; INACTIVATION;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Agriculture,Biology & Environmental Sciences
Citazioni:
28
Recensione:
Indirizzi per estratti:
Indirizzo: Odumeru, J Univ Guelph, Lab Serv Div, 95 Stone Rd W, Guelph, ON N1H 8J7, Canada Univ Guelph 95 Stone Rd W Guelph ON Canada N1H 8J7 8J7, Canada
Citazione:
J. Odumeru et al., "Use of the bead beater for preparation of Mycobacterium paratuberculosis template DNA in milk", CAN J VET R, 65(4), 2001, pp. 201-205

Abstract

Mycobacterium paratuberculosis is a recognized chronic enteric pathogen that can affect many different species of animals, including primates. It hasbeen suggested that this organism is associated with Crohn's disease in humans, and that milk is a potential source of human exposure to this organism. The limit of the detection of M. paratuberculosis in milk samples by direct PCR was 10(5) cfu/mL if the traditional boiling method was used for template DNA preparation. In this study, an improved method for template DNA preparation was examined. The method involves the use of a bead beater, which breaks up bacterial cell wall mechanically by vibrating bacteria with microbeads at high speed. The effectiveness of this method for lysing M. paratuberculosis cells was compared to that of the freeze-thaw method, and use of commercial kits such as the InstaGene Matrix and the QIAamp Tissue Kit. The bead beater procedure was tested in combination with various cell lysis and template DNA preparation procedures to determine which of these steps improved the limit of detection of PCR assay that amplifies a 413 by fragment of the IS900 gene. Results showed that the use of the bead beater, in combination with the use of lysis buffer, boiling, and isopropanol precipitation, decreased the limit of detection of M. paratuberculosis in milk by the PCR to 10(2) cfu/mL. The limit of detection was further decreased to 10 cfu/mL when 0.0037% bovine serum albumin was included in the PCR reaction mixtures. The improved assay was 10- to 10(4)-fold more sensitive than the PCR assays using template DNA prepared by other lysis procedures including boiling alone, freeze-thaw plus boiling, or use of commercial kits for lysis.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 20/09/20 alle ore 07:45:33