Identification and characterization of Bacillus anthracis by multiplex PCR on DNA chip.
Bacillus anthracis can be identified by detecting virulence factor genes located on two plasmids, pXO1 and pXO2. Combining multiplex PCR with arrayed anchored primer PCR and biotin-avidin alkaline phosphatase indicator system, we developed a qualitative DNA chip method for characterization of B. anthracis, and simultaneous confirmation of the species identity independent of plasmid contents. The assay amplifies pag gene (in pXO1), cap gene (in pXO2) and Ba813 gene (a B. anthracis specific chromosomal marker), and the results were indicated by an easy-to-read profile based on the color reaction of alkaline phosphatase. About 1 pg of specific DNA fragments on the chip wells could be detected after PCR. With the proposed method, the avirulent (pXO1+/2-, pXO1-/2+ and pXO1-/2-) strains of B. anthracis and distinguished 'anthrax-like' strains from other B. cereus group bacteria were unambiguously identified, while the genera other than Bacillus gave no positive signal.
Wang
Shi-Hua
SH
Department of Analytical Biochemistry, Biotech Wuchang, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China.
Wen
Ji-Kai
JK
Zhou
Ya-Feng
YF
Zhang
Zhi-Ping
ZP
Yang
Rui-Fu
RF
Zhang
Ji-Bin
JB
Chen
Jia
J
Zhang
Xian-En
XE
D000469:Alkaline Phosphatase; D001408:Bacillus anthracis / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D003124:Colorimetry / Q000295:instrumentation* / Q000379:methods; D004867:Equipment Design; D019544:Equipment Failure Analysis; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation* / Q000379:methods; D016133:Polymerase Chain Reaction / Q000295:instrumentation* / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013045:Species Specificity; D018511:Systems Integration; D037521:Virulence Factors / Q000032:analysis / Q000235:genetics*
D037521:Virulence Factors; D000469:Alkaline Phosphatase