Detection of Bacillus anthracis from spores and cells by loop-mediated isothermal amplification without sample preparation.
Loop-mediated isothermal amplification (LAMP) is a technique capable of rapidly amplifying specific nucleic acid sequences without specialized thermal cycling equipment. In addition, several detection methods that include dye fluorescence, gel electrophoresis, turbidity and colorimetric change, can be used to measure or otherwise detect target amplification. To date, publications have described the requirement for some form of sample nucleic acid extraction (boiling, lysis, DNA purification, etc.) prior to initiating a LAMP reaction. We demonstrate here, the first LAMP positive results obtained from vegetative cells and spores of Bacillus anthracis without nucleic acid extraction. Our data show that the simple addition of cells or spores to the reaction mixture, followed by heating at 63°C is all that is required to reproducibly amplify and detect target plasmid and chromosomal DNA via colorimetric change. The use of three primer sets targeting both plasmids and the chromosome of B. anthracis allows for the rapid discrimination of non-pathogenic bacteria from pathogenic bacteria within 30 min of sampling. Our results indicate that direct testing of B. anthracis spores and cells via LAMP assay will greatly simplify and shorten the detection process by eliminating nucleic acid purification. These results may allow more rapid detection of DNA from pathogenic organisms present in field and environmental samples.
Biosciences and Biotechnology Division, Physics and Life Sciences Directorate, Lawrence Livermore National Laboratory, Livermore, CA 94550, United States. dugan3@llnl.gov
Lawrence
L
Dugan
Jane
J
Bearinger
Aubree
A
Hinckley
Cheryl
C
Strout
Brian
B
Souza
D001408:Bacillus anthracis / Q000166:cytology / Q000235:genetics* / Q000502:physiology; D001426:Bacterial Proteins / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D057230:Limit of Detection; D050296:Microbial Viability; D058889:Molecular Typing; D021141:Nucleic Acid Amplification Techniques; D013171:Spores, Bacterial / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial