Variable Surface Glycoprotein RoTat 1.2 PCR as a specific diagnostic tool for the detection of Trypanosoma evansiinfections

Abstract

BackgroundBased on the recently sequenced gene coding for the Trypanosoma evansi (T. evansi) RoTat 1.2 Variable Surface Glycoprotein (VSG), a primer pair was designed targeting the DNA region lacking homology to other known VSG genes. A total of 39 different trypanosome stocks were tested using the RoTat 1.2 based Polymerase Chain Reaction (PCR).ResultsThis PCR yielded a 205 bp product in all T. evansi and in seven out of nine T. equiperdum strains tested. This product was not detected in the DNA from T. b. brucei, T. b. gambiense, T. b. rhodesiense, T. congolense, T. vivax and T. theileri parasites. The Rotat 1.2 PCR detects as few as 10 trypanosomes per reaction with purified DNA from blood samples, i.e. 50 trypanosomes/ml.ConclusionPCR amplification of the RoTat 1.2 VSG gene is a specific marker for T. evansi strains, except T. evansi type B, and is especially useful in dyskinetoplastic strains where kDNA based markers may fail to amplify. Furthermore, our data support previous suggestions that some T. evansi stocks have been previously misclassified as T. equiperdum.

Molecular detection of Trypanosoma evansi (Kinetoplastida: Trypanosomatidae) in procyonids (Carnivora: Procyonidae) in Eastern Amazon, Brazil

“…This data was observed too on the virtual analysis by BLAST, and is consistent with the results obtained by CLAES et al (2004).…”

“…For the detection of T. evansi DNA, PCR was performed using the primers RoTat 1.2 forward (5'-GCGGGGTGTTTAAAGCAATA-3') and RoTat 1.2 reverse (5'-ATTAGTGCTGCGTGTGTTCG-3'), which amplify a 205-bp fragment of the RoTat 1.2 VSG gene, as described by CLAES et al, (2004). These primers were also used for a virtual analysis with the BLAST-Primer program, available from the National Center for Biotechnology Information (NCBI) website.…”

Development of a recombinase polymerase amplification lateral flow assay for the detection of active Trypanosoma evansi infections

“…Fifty μl of collected blood was employed to prepare purified genomic DNA for the TevPCR, whereas the remaining 50 μl of collected blood was used to obtain crude genomic DNA for the TevRPA-LF. The TevPCR was performed as described in [40] with the following modifications: the amount of purified genomic DNA as starting material (250 ng vs. 3000 ng) and the addition of 10% DMSO to the reaction mixture.…”

“…The DNA of killed trypanosomes does not remain in the blood for more than 24 to 48 hours, thus PCR-based assays are highly suitable for the detection of active infections [39]. Several genes have been investigated as targets for the PCR-based diagnosis of T. evansi; these include the RoTat1.2 VSG gene (Type A specific) [40][41][42], ribosomal DNA [43], a region from r-RNA internal transcribed spacer 1 (ITS-1) [44], the gene encoding the invariant surface glycoprotein ISG-75 [45], and the VSG JN 2118Hu gene (Type B specific) [26,28,46,47]. The drawback of PCR-based methods is that they require well-trained and experienced personnel and a laboratory environment suitable for correct protocol execution.…”

Insight into Trypanosomiasis in Animals: Various Approaches for its Diagnosis, Treatment and Control: A Review

“…Sci., 9 (5): 172-186, 2015 Davila, 2002). This PCR based detection of T. evensi is very sensitive but not validated in field (Claes et al, 2004). Comparative studies recommended that TBR primers is the most sensitive primers for detecting T. evansi (Pruvot et al, 2010) and the Phenol-Chloroform method is the most sensitive method for DNA isolation (Pruvot et al, 2013) and this can detect as less as 5-10 trypanosomes per milliliter of blood.…”