Njiru Z K, Constantine C C, Guya S, Crowther J, Kiragu J M, Thompson R C A, Dávila A M R
Division of Veterinary and Biomedical Sciences, Western Australian Biomedical Research Institute (WABRI), Murdoch University, South Street, 6150, Murdoch, Western Australia, Australia.
Parasitol Res. 2005 Feb;95(3):186-92. doi: 10.1007/s00436-004-1267-5. Epub 2004 Dec 24.
There are 11 different pathogenic trypanosomes in trypanosomiasis endemic regions of Africa. Their detection and characterisation by molecular methods relies on species-specific primers; consequently several PCR tests have to be made on each sample. Primers ITS1 CF and ITS1 BR, previously designed to amplify the internal transcribed spacer (ITS1) of rDNA, have been evaluated for use in a universal diagnostic test for all pathogenic trypanosomes. Blood was collected from 373 cattle and 185 camels. The primers gave constant PCR products with the stocks of each taxon tested. Members of subgenus Trypanozoon (T. brucei brucei, T. evansi, T. b. rhodesiense and T. b. gambiense) gave a constant product of approximately 480 bp; T. congolense, savannah 700 bp, T. congolense kilifi 620 bp and T. congolense forest 710 bp: T. simiae 400 bp, T. simiae tsavo 370 bp, T. godfreyi 300 bp and T. vivax 250 bp. The sensitivity of the test ranged from 10 pg for Trypanozoon, T. congolense clade and T. vivax to 100 pg for T. simiae and T. godfreyi. The primers detected cases of multi-taxa samples, although the sensitivity was reduced with an increase in the combinations. A better detection rate of trypanosome DNA was recorded with buffy coats than from direct blood. With the field samples, the diagnostic sensitivity was close to the sensitivity obtained using single reactions with species-specific primers for Trypanozoon 38/40 (95%) and T. congolense savannah 30/33 (90.9%) but was lower with T. vivax 25/31 (77.4%). The primers offer promise as a routine diagnostic tool through the use of a single PCR; however, further evaluation is recommended.
在非洲锥虫病流行地区存在11种不同的致病性锥虫。通过分子方法对它们进行检测和鉴定依赖于物种特异性引物；因此，每个样本都必须进行多次聚合酶链式反应（PCR）检测。先前设计用于扩增核糖体DNA内部转录间隔区（ITS1）的引物ITS1 CF和ITS1 BR，已被评估用于所有致病性锥虫的通用诊断测试。从373头牛和185头骆驼采集血液。这些引物对所测试的每个分类单元的菌株都产生了恒定的PCR产物。布氏锥虫亚属（布氏布氏锥虫、伊氏锥虫、布氏罗得西亚锥虫和布氏冈比亚锥虫）的成员产生约480 bp的恒定产物；刚果锥虫，草原型700 bp，基利菲刚果锥虫620 bp，森林刚果锥虫710 bp；西氏锥虫400 bp，察沃西氏锥虫370 bp，戈氏锥虫300 bp，活泼锥虫250 bp。该测试的灵敏度范围为：对于布氏锥虫亚属、刚果锥虫进化枝和活泼锥虫为10皮克，对于西氏锥虫和戈氏锥虫为100皮克。这些引物检测到了多分类单元样本的情况，尽管随着组合数量的增加灵敏度有所降低。与直接血液样本相比，用血沉棕黄层检测锥虫DNA的检出率更高。对于现场样本，诊断灵敏度接近使用针对布氏锥虫亚属的物种特异性引物进行单次反应时获得的灵敏度38/40（95%）和草原刚果锥虫30/33（90.9%），但活泼锥虫的灵敏度较低，为25/31（77.4%）。这些引物有望通过使用单一PCR作为常规诊断工具；然而，建议进行进一步评估。
