A Highly Sensitive Quantitative PCR for the Detection of Bartonella bacilliformis by Targeting a Multiple-Copy DNA Segment

Abstract

Carrion’s disease is a human disease caused by an infection with Bartonella bacilliformis. Sand fly is believed to be the transmitting vector. Acute infection without treatment is life-threatening with fatality rates as high as 88%. PCR based diagnostic assays have been developed for detecting B. bacilliformis DNA in clinical samples. Genome sequence analysis of B. bacilliformis had identified a segment of 1,162 bp which is present at three different locations. In this study we have developed a qPCR assay targeting this Multiplecopy DNA Segment (MTSeq) to reach a higher sensitivity. The assay sensitivity was evaluated by three different sets of primers. The best set of primers yielded the detection limit of 3.3 bacteria per reaction. DNA extracted from sand flies fed on blood containing B. bacilliformis was also tested. Flies fed on Day 1 and 3 were determined as positive for B. bacilliformis; the results were consistent with the earlier study targeting pap31 gene. The consistency of the qPCR targeting the MTSeq was evaluated using samples containing 8.3 or 3.3 copies of genomic DNA. We demonstrated that 18 out of 36 reactions (50%) were positive for samples containing 8.3 copies of genome; similarly 12 out of 36 reactions (33%) were positive for samples containing 3.3 copies of genome. At the same time, only 8 (25%) and 2 (6%) reactions out of 36 reactions showed positive using primers targeting pap31, respectively. These results have demonstrated that qPCR targeting MTSeq is more sensitive for detecting B. bacilliformis than previous nucleic acid based method targeting pap31.