PCR approach for the detection of Trypanosoma brucei and T. equiperdum and their differentiation from T. evansi based on maxicircle kinetoplast DNA.
The goal of this study was to develop a PCR approach based on the sequence of maxicircle kinetoplast DNA (kDNA) of Trypanosoma brucei to distinguish T. brucei/T. equiperdum from T. evansi and to evaluate its diagnostic use for their detection in blood samples. Primers derived from the sequence of the maxicircle kDNA of T. brucei, encoding the NADH dehydrogenase subunit 5 (nad5) gene, were used to test the PCR-amplification from T. brucei (including T. b. brucei and T. b. rhodesiense), T. equiperdum, T. evansi, T. vivax and T. congolense. A primer pair to a nuclear DNA region incorporated into a separate PCR was employed to control for the presence of amplifiable genomic DNA (representing the subgenus Trypanozoon) in each sample subjected to the PCR. Products of approximately 395bp were amplified from all T. brucei and T. equiperdum samples tested using the nad5-PCR, but not from T. evansi DNA samples or any of the control samples representing T. vivax, T. congolense, or host. The current PCR approach allows the rapid differentiation of T. brucei/T.equiperdum from T. evansi and can detect the equivalent of 20-25 cells of T. brucei or T. equiperdum in purified genomic DNA or infected blood samples.
Li
Feng-Jun
FJ
Center for Parasitic Organisms and State Key Laboratory of Biocontrol, School of Life Sciences, Zhongshan (Sun Yat-sen) University, Guangzhou 510275, PR China.
Gasser
Robin B
RB
Lai
De-Hua
DH
Claes
Filip
F
Zhu
Xing-Quan
XQ
Lun
Zhao-Rong
ZR
D000818:Animals; D001483:Base Sequence; D018105:DNA, Kinetoplast / Q000032:analysis* / Q000235:genetics*; D004587:Electrophoresis, Agar Gel; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D014345:Trypanosoma / Q000145:classification* / Q000166:cytology / Q000235:genetics*; D014346:Trypanosoma brucei brucei / Q000166:cytology* / Q000235:genetics*
D018105:DNA, Kinetoplast