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A method for release and multiple strand amplification of small quantities of DNA from endospores of the fastidious bacterium Pasteuria penetrans. / Mauchline, Tim H.; Mohan, Sharad; Schaff, J. E.; Opperman, Charles H.; Kerry, Brian R.; Davies, Keith; Hirsch, Penny R.

In: Letters in Applied Microbiology, Vol. 50, No. 5, 05.2010, p. 515-521.

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Mauchline, Tim H. ; Mohan, Sharad ; Schaff, J. E. ; Opperman, Charles H. ; Kerry, Brian R. ; Davies, Keith ; Hirsch, Penny R. / A method for release and multiple strand amplification of small quantities of DNA from endospores of the fastidious bacterium Pasteuria penetrans. In: Letters in Applied Microbiology. 2010 ; Vol. 50, No. 5. pp. 515-521.

Bibtex

@article{47bd298f6ad9444a9258bc91653baf8b,
title = "A method for release and multiple strand amplification of small quantities of DNA from endospores of the fastidious bacterium Pasteuria penetrans",
abstract = "Aims: To establish a reliable protocol to extract DNA from Pasteuria penetrans endospores for use as template in multiple strand amplification, thus providing sufficient material for genetic analyses. To develop a highly sensitive PCR-based diagnostic tool for P. penetrans. Methods and Results: An optimized method to decontaminate endospores, release and purify DNA enabled multiple strand amplification. DNA purity was assessed by cloning and sequencing gyrB and 16S rRNA gene fragments obtained from PCR using generic primers. Samples indicated to be 100{\%}P. penetrans by the gyrB assay were estimated at 46{\%} using the 16S rRNA gene. No bias was detected on cloning and sequencing 12 housekeeping and sporulation gene fragments from amplified DNA. The detection limit by PCR with Pasteuria-specific 16S rRNA gene primers following multiple strand amplification of DNA extracted using the method was a single endospore. Conclusions: Generation of large quantities DNA will facilitate genomic sequencing of P. penetrans. Apparent differences in sample purity are explained by variations in 16S rRNA gene copy number in Eubacteria leading to exaggerated estimations of sample contamination. Detection of single endospores will facilitate investigations of P. penetrans molecular ecology. Significance and Impact of the Study: These methods will advance studies on P. penetrans and facilitate research on other obligate and fastidious micro-organisms where it is currently impractical to obtain DNA in sufficient quantity and quality.",
keywords = "16S rRNA gene, bead-beating, endospores, gyrB, microLYSIS (R)-PLUS, multiple strand amplification, Pasteuria penetrans, phi29, ROOT-KNOT NEMATODES, PLANT-PARASITIC NEMATODES, HYPERPARASITIC BACTERIUM, OBLIGATE ENDOPARASITE, PHYLOGENETIC POSITION, MELOIDOGYNE-INCOGNITA, BIOLOGICAL-CONTROL, SP-NOV, PRATYLENCHUS, HETERODERA",
author = "Mauchline, {Tim H.} and Sharad Mohan and Schaff, {J. E.} and Opperman, {Charles H.} and Kerry, {Brian R.} and Keith Davies and Hirsch, {Penny R.}",
year = "2010",
month = "5",
doi = "10.1111/j.1472-765X.2010.02830.x",
language = "English",
volume = "50",
pages = "515--521",
journal = "Letters in Applied Microbiology",
issn = "0266-8254",
publisher = "Wiley-Blackwell",
number = "5",

}

RIS

TY - JOUR

T1 - A method for release and multiple strand amplification of small quantities of DNA from endospores of the fastidious bacterium Pasteuria penetrans

AU - Mauchline, Tim H.

AU - Mohan, Sharad

AU - Schaff, J. E.

AU - Opperman, Charles H.

AU - Kerry, Brian R.

AU - Davies, Keith

AU - Hirsch, Penny R.

PY - 2010/5

Y1 - 2010/5

N2 - Aims: To establish a reliable protocol to extract DNA from Pasteuria penetrans endospores for use as template in multiple strand amplification, thus providing sufficient material for genetic analyses. To develop a highly sensitive PCR-based diagnostic tool for P. penetrans. Methods and Results: An optimized method to decontaminate endospores, release and purify DNA enabled multiple strand amplification. DNA purity was assessed by cloning and sequencing gyrB and 16S rRNA gene fragments obtained from PCR using generic primers. Samples indicated to be 100%P. penetrans by the gyrB assay were estimated at 46% using the 16S rRNA gene. No bias was detected on cloning and sequencing 12 housekeeping and sporulation gene fragments from amplified DNA. The detection limit by PCR with Pasteuria-specific 16S rRNA gene primers following multiple strand amplification of DNA extracted using the method was a single endospore. Conclusions: Generation of large quantities DNA will facilitate genomic sequencing of P. penetrans. Apparent differences in sample purity are explained by variations in 16S rRNA gene copy number in Eubacteria leading to exaggerated estimations of sample contamination. Detection of single endospores will facilitate investigations of P. penetrans molecular ecology. Significance and Impact of the Study: These methods will advance studies on P. penetrans and facilitate research on other obligate and fastidious micro-organisms where it is currently impractical to obtain DNA in sufficient quantity and quality.

AB - Aims: To establish a reliable protocol to extract DNA from Pasteuria penetrans endospores for use as template in multiple strand amplification, thus providing sufficient material for genetic analyses. To develop a highly sensitive PCR-based diagnostic tool for P. penetrans. Methods and Results: An optimized method to decontaminate endospores, release and purify DNA enabled multiple strand amplification. DNA purity was assessed by cloning and sequencing gyrB and 16S rRNA gene fragments obtained from PCR using generic primers. Samples indicated to be 100%P. penetrans by the gyrB assay were estimated at 46% using the 16S rRNA gene. No bias was detected on cloning and sequencing 12 housekeeping and sporulation gene fragments from amplified DNA. The detection limit by PCR with Pasteuria-specific 16S rRNA gene primers following multiple strand amplification of DNA extracted using the method was a single endospore. Conclusions: Generation of large quantities DNA will facilitate genomic sequencing of P. penetrans. Apparent differences in sample purity are explained by variations in 16S rRNA gene copy number in Eubacteria leading to exaggerated estimations of sample contamination. Detection of single endospores will facilitate investigations of P. penetrans molecular ecology. Significance and Impact of the Study: These methods will advance studies on P. penetrans and facilitate research on other obligate and fastidious micro-organisms where it is currently impractical to obtain DNA in sufficient quantity and quality.

KW - 16S rRNA gene

KW - bead-beating

KW - endospores

KW - gyrB

KW - microLYSIS (R)-PLUS

KW - multiple strand amplification

KW - Pasteuria penetrans

KW - phi29

KW - ROOT-KNOT NEMATODES

KW - PLANT-PARASITIC NEMATODES

KW - HYPERPARASITIC BACTERIUM

KW - OBLIGATE ENDOPARASITE

KW - PHYLOGENETIC POSITION

KW - MELOIDOGYNE-INCOGNITA

KW - BIOLOGICAL-CONTROL

KW - SP-NOV

KW - PRATYLENCHUS

KW - HETERODERA

U2 - 10.1111/j.1472-765X.2010.02830.x

DO - 10.1111/j.1472-765X.2010.02830.x

M3 - Article

VL - 50

SP - 515

EP - 521

JO - Letters in Applied Microbiology

JF - Letters in Applied Microbiology

SN - 0266-8254

IS - 5

ER -