[Development of a quantitative real-time polymerase chain reaction for detecting Bartonella henselae].
OBJECTIVE
To develop a quantitative real-time polymerase chain reaction (PCR) for detecting Bartonella henselae.


METHODS
According to the 16S-23S rRNA intervening sequences (IVS) specific for B. henselae, one pair of primers and one TaqMan-MGB probe were designed. A quantitative real-time PCR was developed with the primers, the probe, and the IVS, a standard template, in DNA sequence detection system (ABI 7900HT).


RESULTS
The standard curve was established with the standard template and the relationship between the value of threshold cycle (Ct) and the DNA copy number was linear (r = 0.997). The sensitivity of this quantitative real-time PCR was about 1000 times higher than that of a common PCR used to detect homologous DNA. By this quantitative real-time PCR, the DNA sample of B. henselae was positively detected but not from other rickettsial or bacterial DNA samples. The variation coefficients of intra- and inter-assay reproducibility were 0.2%-1.9%. Using the real-time quantitative PCR to detect samples from mice that were experimentally infected with B. henselae, the small amount of B. henselae DNA was detected in blood samples on days 2, 3, and 5 and large amount of B. henselae DNA was detected in spleen samples on days 1 and 2 after infection.


CONCLUSIONS
Results from our study suggested that this quantitative real-time PCR was highly specific, sensitive and with good repeatability for detection of B. henselae. It seemed quite useful for rapid detection of tiny DNA of B. henselae in various samples and laboratory diagnosis of bartonellosis caused by B. henselae.
Zhang
Jing-bo
JB
Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing 100071, China.
Wen
Bo-hai
BH
Chen
Mei-ling
ML
Li
Li-li
LL
Qiu
Ling
L
Niu
Dong-sheng
DS
D000818:Animals; D001474:Bartonella Infections / Q000175:diagnosis*; D018416:Bartonella henselae / Q000235:genetics*; D004269:DNA, Bacterial / Q000032:analysis*; D051379:Mice; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D004269:DNA, Bacterial