da Silveira Pinto A, de Lana M, Britto C, Bastrenta B, Tibayrenc M
Departamento de Microbiologia, Instituto de Ciências Biológicas, CP 486, Universidade Federal de Minas Gerais, 31270-010, MG, Belo Horizonte, Brazil.
Int J Parasitol. 2000 Jun;30(7):843-8. doi: 10.1016/s0020-7519(00)00058-8.
Monitored biclonal densities of parasites were offered to third-stage larvae of Triatoma infestans via an artificial feeding device and 30 days later, the gut contents of the insects were processed for microscopic examination and polymerase chain reaction (PCR) detection of Trypanosoma cruzi kinetoplast DNA [kDNA]). A total of 15 mixtures involving nine different stocks attributed to the 19/20, 32 and 39 major clonal genotypes of Trypanosoma cruzi were used. The presence of each T. cruzi clonal genotype after completion of the cycle through the insects was investigated by hybridising the PCR amplification products with genotype-specific minicircle kDNA probes. Sixty-five out of 90 examined insects (72.2%) were positive for parasites by microscopic examination and 85 (94.4%) were positive by PCR. The results show that almost half of the biclonal infections are not detectable after completion of the cycle, and that there are important differences in detection of such biclonal infections according to the clonal genotypes considered. Moreover, elimination of a clonal genotype by another is a frequent, but not constant, pattern in biclonal infections of T. infestans. The use of PCR and kDNA probes makes it possible to avoid the culture phase, which makes detection of mixed infections much easier in epidemiological surveys. Moreover, the fact that T. infestansdoes not transmit different T. cruzi clonal genotypes with the same efficiency has strong implications for the reliability of xenogiagnosis.
通过人工喂食装置将监测到的寄生虫双克隆密度提供给克氏锥蝽的第三期幼虫，30天后，对昆虫的肠道内容物进行处理，以便通过显微镜检查和聚合酶链反应（PCR）检测克氏锥虫动基体DNA（kDNA）。总共使用了15种混合物，涉及9种不同的虫株，这些虫株属于克氏锥虫的19/20、32和39种主要克隆基因型。通过将PCR扩增产物与基因型特异性小环kDNA探针杂交，研究了昆虫完成整个周期后每种克氏锥虫克隆基因型的存在情况。在90只接受检查的昆虫中，65只（72.2%）通过显微镜检查发现寄生虫呈阳性，85只（94.4%）通过PCR检测呈阳性。结果表明，几乎一半的双克隆感染在整个周期完成后无法检测到，而且根据所考虑的克隆基因型，此类双克隆感染的检测存在重要差异。此外，在克氏锥蝽的双克隆感染中，一种克隆基因型被另一种取代是一种常见但不固定的模式。使用PCR和kDNA探针可以避免培养阶段，这使得在流行病学调查中检测混合感染变得更加容易。此外，克氏锥蝽传播不同克氏锥虫克隆基因型的效率不同这一事实，对动物接种诊断的可靠性具有重要影响。
