A simple and sensitive method for detection of Bacillus anthracis by loop-mediated isothermal amplification.
OBJECTIVE
To develop a rapid and simple system for detection of Bacillus anthracis using a loop-mediated isothermal amplification (LAMP) method and determine the suitability of LAMP for rapid identification of B. anthracis infection.


RESULTS
A specific LAMP assay targeting unique gene sequences in the bacterial chromosome and two virulence plasmids, pXO1 and pXO2, was designed. With this assay, it was possible to detect more than 10 fg of bacterial DNA per reaction and obtain results within 30-40 min under isothermal conditions at 63 degrees C. No cross-reactivity was observed among Bacillus cereus group and other Bacillus species. Furthermore, in tests using blood specimens from mice inoculated intranasally with B. anthracis spores, the sensitivity of the LAMP assay following DNA extraction methods using a Qiagen DNeasy kit or boiling protocol was examined. Samples prepared by both methods showed almost equivalent sensitivities in LAMP assay. The detection limit was 3.6 CFU per test.


CONCLUSIONS
The LAMP assay is a simple, rapid and sensitive method for detecting B. anthracis.


CONCLUSIONS
The LAMP assay combined with boiling extraction could be used as a simple diagnostic method for identification of B. anthracis infection.
Kurosaki
Y
Y
First Department of Forensic Science, National Research Institute of Police Science, Kashiwa, Japan.
Sakuma
T
T
Fukuma
A
A
Fujinami
Y
Y
Kawamoto
K
K
Kamo
N
N
Makino
S-I
SI
Yasuda
J
J
D000818:Animals; D000881:Anthrax / Q000382:microbiology*; D001408:Bacillus anthracis / Q000235:genetics* / Q000302:isolation & purification* / Q000472:pathogenicity; D004269:DNA, Bacterial / Q000235:genetics; D005260:Female; D057230:Limit of Detection; D051379:Mice; D008807:Mice, Inbred BALB C; D021141:Nucleic Acid Amplification Techniques; D010957:Plasmids; D012680:Sensitivity and Specificity; D013047:Specific Pathogen-Free Organisms; D014774:Virulence
D004269:DNA, Bacterial