Comparison of Four Rapid DNA Extraction Techniques for Conventional Polymerase Chain Reaction Testing of Three Mycobacterium spp. that Affect Birds

Lisa A Tell, Janet E Foley, Martha L. Needham, Richard L. Walker

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

Mycobacteriosis is an avian disease that is most commonly caused by Mycobacterium avium or Mycobacterium genavense. In order to optimize molecular laboratory tests for diagnosing mycobacteriosis in birds, we compared four methods of rapid DNA extraction with isolates of M. avium, M. genavense, and Mycobacterium fortuitum. DNA extraction methods included enzymatic lysis, boiling for 30 min followed by enzymatic lysis, four cycles of freezing and thawing followed by enzymatic lysis, and bead beating followed by enzymatic lysis. The DNA yield and purity for the four methods were evaluated by spectrophotometry and compared. The bead beating with enzymatic lysis technique yielded significantly purer and higher concentrations of extracted DNA compared with other DNA extraction methods. All four methods yielded extraction products for all three organisms that were successfully amplified by polymerase chain reaction (PCR) for a fragment in the 65-kD heat shock protein gene. Subjectively, the PCR amplification products were most abundant for samples extracted by bead beating with enzymatic lysis.

Original languageEnglish (US)
Pages (from-to)1486-1490
Number of pages5
JournalAvian Diseases
Volume47
Issue number4
StatePublished - Oct 2003

Fingerprint

Mycobacterium
Birds
polymerase chain reaction
Mycobacterium genavense
Polymerase Chain Reaction
birds
DNA
mycobacterial diseases
Mycobacterium avium
testing
Bird Diseases
Mycobacterium fortuitum
bird diseases
methodology
Spectrophotometry
Heat-Shock Proteins
boiling
heat shock proteins
rapid methods
thawing

Keywords

  • Avian
  • DNA extraction
  • Mycobacterium avium
  • Mycobacterium fortuitum
  • Mycobacterium genavense
  • Polymerase chain reaction

ASJC Scopus subject areas

  • Immunology
  • Cancer Research
  • veterinary(all)

Cite this

Comparison of Four Rapid DNA Extraction Techniques for Conventional Polymerase Chain Reaction Testing of Three Mycobacterium spp. that Affect Birds. / Tell, Lisa A; Foley, Janet E; Needham, Martha L.; Walker, Richard L.

In: Avian Diseases, Vol. 47, No. 4, 10.2003, p. 1486-1490.

Research output: Contribution to journalArticle

@article{b78818e1750f4f66b7fc99dc43542c61,
title = "Comparison of Four Rapid DNA Extraction Techniques for Conventional Polymerase Chain Reaction Testing of Three Mycobacterium spp. that Affect Birds",
abstract = "Mycobacteriosis is an avian disease that is most commonly caused by Mycobacterium avium or Mycobacterium genavense. In order to optimize molecular laboratory tests for diagnosing mycobacteriosis in birds, we compared four methods of rapid DNA extraction with isolates of M. avium, M. genavense, and Mycobacterium fortuitum. DNA extraction methods included enzymatic lysis, boiling for 30 min followed by enzymatic lysis, four cycles of freezing and thawing followed by enzymatic lysis, and bead beating followed by enzymatic lysis. The DNA yield and purity for the four methods were evaluated by spectrophotometry and compared. The bead beating with enzymatic lysis technique yielded significantly purer and higher concentrations of extracted DNA compared with other DNA extraction methods. All four methods yielded extraction products for all three organisms that were successfully amplified by polymerase chain reaction (PCR) for a fragment in the 65-kD heat shock protein gene. Subjectively, the PCR amplification products were most abundant for samples extracted by bead beating with enzymatic lysis.",
keywords = "Avian, DNA extraction, Mycobacterium avium, Mycobacterium fortuitum, Mycobacterium genavense, Polymerase chain reaction",
author = "Tell, {Lisa A} and Foley, {Janet E} and Needham, {Martha L.} and Walker, {Richard L.}",
year = "2003",
month = "10",
language = "English (US)",
volume = "47",
pages = "1486--1490",
journal = "Avian Diseases",
issn = "0005-2086",
publisher = "American Association of Avian Pathologists",
number = "4",

}

TY - JOUR

T1 - Comparison of Four Rapid DNA Extraction Techniques for Conventional Polymerase Chain Reaction Testing of Three Mycobacterium spp. that Affect Birds

AU - Tell, Lisa A

AU - Foley, Janet E

AU - Needham, Martha L.

AU - Walker, Richard L.

PY - 2003/10

Y1 - 2003/10

N2 - Mycobacteriosis is an avian disease that is most commonly caused by Mycobacterium avium or Mycobacterium genavense. In order to optimize molecular laboratory tests for diagnosing mycobacteriosis in birds, we compared four methods of rapid DNA extraction with isolates of M. avium, M. genavense, and Mycobacterium fortuitum. DNA extraction methods included enzymatic lysis, boiling for 30 min followed by enzymatic lysis, four cycles of freezing and thawing followed by enzymatic lysis, and bead beating followed by enzymatic lysis. The DNA yield and purity for the four methods were evaluated by spectrophotometry and compared. The bead beating with enzymatic lysis technique yielded significantly purer and higher concentrations of extracted DNA compared with other DNA extraction methods. All four methods yielded extraction products for all three organisms that were successfully amplified by polymerase chain reaction (PCR) for a fragment in the 65-kD heat shock protein gene. Subjectively, the PCR amplification products were most abundant for samples extracted by bead beating with enzymatic lysis.

AB - Mycobacteriosis is an avian disease that is most commonly caused by Mycobacterium avium or Mycobacterium genavense. In order to optimize molecular laboratory tests for diagnosing mycobacteriosis in birds, we compared four methods of rapid DNA extraction with isolates of M. avium, M. genavense, and Mycobacterium fortuitum. DNA extraction methods included enzymatic lysis, boiling for 30 min followed by enzymatic lysis, four cycles of freezing and thawing followed by enzymatic lysis, and bead beating followed by enzymatic lysis. The DNA yield and purity for the four methods were evaluated by spectrophotometry and compared. The bead beating with enzymatic lysis technique yielded significantly purer and higher concentrations of extracted DNA compared with other DNA extraction methods. All four methods yielded extraction products for all three organisms that were successfully amplified by polymerase chain reaction (PCR) for a fragment in the 65-kD heat shock protein gene. Subjectively, the PCR amplification products were most abundant for samples extracted by bead beating with enzymatic lysis.

KW - Avian

KW - DNA extraction

KW - Mycobacterium avium

KW - Mycobacterium fortuitum

KW - Mycobacterium genavense

KW - Polymerase chain reaction

UR - http://www.scopus.com/inward/record.url?scp=0346752412&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0346752412&partnerID=8YFLogxK

M3 - Article

C2 - 14709001

AN - SCOPUS:0346752412

VL - 47

SP - 1486

EP - 1490

JO - Avian Diseases

JF - Avian Diseases

SN - 0005-2086

IS - 4

ER -