Detection Of Rickettsiae By Real-time Quantitative PCR

Many rickettsial infections are important emergent or reemergent diseases. There is an urgent need to develop methods to rapidly detect their pathogens in various samples in order to immediately treat the rickettisal infections.Real-time quantitative PCR (RtPCR) is regarded as the most accurate and sensitive methods for rapidly detecting pathogens in various samples. According to the specific gene sequences of Coxiella burnetii, Bartonelae henselae, Bartonela quintana, Ehrlichiae chaffeensis, and Anaplasma phigocytophila, we designed the primers and probes to develop five real-time quantitative PCR methods, respectively. The results showed that the sensitivity of these RtPCRs were about 100 times higher than that of the nested PCR used to detect homologous DNA, accompanied by high species-specificity and good repeatability. The time required for RtPCR assay was approximately 2h, which was only 1/3 of time of the common PCR.Using RtPCR specific for C. burnetii and B. henselae to detect the blood samples from BALB/c mince experimentally infected with C. burnetii and B. henselae, respectively, the positive results were obtained on day 1 postinfection. The results suggest that the two RtPCRs may be used to in early diagnosis of the homologous rickettsial disease. The spleen samples from the mice infected with C. burnetii or B. henselae were examined by RtPCR specific for C. burnetii and B. henselae, respectively and the load level of the infectious agent was correlated with the progression of the infection. In addition, the coxiella loads in spleens of mice immunized with whole cells antigen of C. burnetii were significantly lower than that of the mice unimmunized and the coxiella loads in spleens of mice immunized with two-boosters were markedly lower than that of mice immunized with one-booster in RtPCR assay specific for C. burnetii. The results suggest that the RtPCR of detecting C. burnetii is very useful for evaluation of the immunoprotective efficiency of thevaccine candidate against Q fever in animal models.The RtPCR specific for A. phigocytophila was used to detect ticks samples and spleen samples of wild mice in forest. The positive and negative results were corresponded with that detected by nested PCR specific for the pathogen. However, because of its quantitative assay, the sensitivity and reliability of the RtPCR assay was much better than that of the PCR detection.hi conclusion, the RtPCRs developed in this study are highly specific and sensitive methods to rapidly detect C. burnetii, B. henselae, B. quintana, E. chaffeensis, and A. phigocytophila, respectively and they may be very useful in detecting these rickettsial agents in various samples for early diagnoses and epidemiological study of these rickettsial infections, as well as in evaluating of protective efficiency of vaccine candidates against these rickettsial infection.