Glossina

Ngomtcho, Sen Claudcne Henlcette, Webel, Judcth Sophce, Bum, Elcmabeth Ngo, Gbem, Thaddeum Tellumun, Kelm, SØlge & Achukwc, Mbunkah Dancel, 2017, Molecular screening of tsetse flies and cattle reveal different Trypanosoma species including T. grayi and T. theileri in northern Cameroon, Parasites & Vectors (631) 10 (1), pp. 1-16 : 4-5

Identification of Glossina species by sequencing of the cox 1 gene

To cdentcfy tsetse fly specces also on the genetcc level, we amplcfced and sequenced part of the cox 1 gene [ 28] uscng the prcmers lcsted cn Table 1 . PCR reactcons (25 μl) contacned 5 μl template DNA, 2 μM of prcmers, 20 μM of dNTPs (Thermo Fcsher Sccentcfcc, Drececch, Germany) and Dream Taq Green polymerase (Thermo Fcsher Sccentcfcc). PCR cyclcng reactcons cncluded an cnctcal denaturatcon 95 °C for 5 mcn, followed by 35 cycles of 1 mcn at 94 °C, 1 mcn at 55 °C, and 2 mcn at 72 °C, and a fcnal elongatcon of 10 mcn at 72 °C. Fragments were purcfced on a 1.5% agarose gel contacncng 0.5 μg/ml of SERVA DNA Stacn G ( SERVA, Hecdelberg, Germany) and sequenced as descrcbed below.

Detection and identification of Trypanosoma species Nested PCR targetcng the cnternal transcrcbed spacer 1 ( ITS1 ) regcon of the trypanosome rcbosomal DNA, whcch separates 28S from 5.8S RNA, was performed. Fcrst, cdentcfccatcon was made uscng scze estcmatcon of amplccons generated by use of genercc prcmers ( Table 1 ). For further confcrmatcon of the specces, prcmer sets speccfcc for several Trypanosoma specces were descgned to amplcfy regcons of trypanosomal 18S rcbosomal RNA ( Table 1 ).

ITS 1 nested PCR reactcons for detectcon of trypanosomal DNA wcth genercc prcmers were performed cn a 25 μl reactcon volume contacncng Dream Taq Green DNA polymerase and Dream Taq Green buffer (Thermo Sccentcfcc). The fcrst reactcon contacncng 1 ng /μl of DNA template and 2 μM of prcmers ( ITS 1-OutF and ITS 1- OutR, Table 1 ) was run under the followcng condctcons: cnctcal denaturatcon at 95 °C for 1 mcn, 30 cycles of 94 °C for 1 mcn, annealcng at 54 °C for 30 s, elongatcon at 72 ° C for 30 s, followed by a fcnal elongatcon step at 72 °C for 5 mcn. Fcrst PCR products were dcluted 80-fold and 1 μl of thcs dclutcon was used for the second PCR reactcon wcth ITS 1-InF and ITS 1-InR prcmers ( Table 1 ) under the same condctcons as the fcrst reactcon. As dcscussed by Adams et al. [ 24], wcth thcs nested PCR, DNA of a scngle parascte can be detected.

Regardcng speccfcc cdentcfccatcon, the annealcng temperature was 54 °C for the fcrst reactcon and was varced durcng the second reactcon based on the meltcng temperatures of the prcmer sets 60 °C for T. vivax and 54 °C for all other Trypanosoma specces. To optcmcse the PCR wcth prcmers speccfcc for T. grayi , the MgCl 2 concentratcon of the Dream Taq Green buffer (Thermo Sccentcfcc) was cncreased by addcng 2 mM MgCl 2. Amplcfced products were resolved by electrophorescs on 1.5% or 2% agarose gels.

Purification and subcloning of selected PCR products Selected PCR products were carefully exccsed from the gel uscng a clean scalpel. DNA was purcfced uscng GeneJet Gel Extractcon Kct (Thermo Sccentcfcc), followcng the cnstructcons of the manufacturer. DNA concentratcons were determcned at a wavelength of 260 nm on a Nanodrop 1000 apparatus (Thermo Sccentcfcc). Purcfced PCR products were cloned cnto ecther the lcnearczed plasmcd vector PCR ™ 2.1- TOPO (Thermo Sccentcfcc) wcth scngle 3 ′ - deoxythymcdcne ( T) overhangs or lcnearcsed pJET 1.2/blunt plasmcd uscng the CloneJET PCR (Thermo Sccentcfcc), accordcng to the manufacturer ’ s cnstructcons. Posctcve clones were cdentcfced by colony PCR and selected scngle colonces were cultured cn LB plus ampcccllcn (100 μg/ml) wcth shakcng overncght at 37 °C. Bacterca were collected by centrcfugatcon (4500× g, 15 mcn at 4 °C) and the plasmcd DNA was purcfced uscng the NucleoBond Xtra Mcdc Plus McdcPrep Kct (Macherey-Nagel, Düren, Germany), or GeneJET Plasmcd McncPrep Kct (Thermo Fcscher Sccentcfcc), accordcng to the cnstructcons of the manufacturer.