Mares-Guia Maria Angélica M M, Guterres Alexandro, Rozental Tatiana, Ferreira Michelle Dos Santos, Lemos Elba R S
Oswaldo Cruz Institute, Laboratory of Hantavirus and Rickettsioses, Fiocruz, RJ, Brazil.
Oswaldo Cruz Institute, Laboratory of Hantavirus and Rickettsioses, Fiocruz, RJ, Brazil.
Braz J Microbiol. 2018 Jan-Mar;49(1):138-143. doi: 10.1016/j.bjm.2017.04.009. Epub 2017 Aug 24.
Q fever is a worldwide zoonosis caused by Coxiella burnetii-a small obligate intracellular Gram-negative bacterium found in a variety of animals. It is transmitted to humans by inhalation of contaminated aerosols from urine, feces, milk, amniotic fluid, placenta, abortion products, wool, and rarely by ingestion of raw milk from infected animals. Nested PCR can improve the sensitivity and specificity of testing while offering a suitable amplicon size for sequencing. Serial dilutions were performed tenfold to test the limit of detection, and the result was 10× detection of C. burnetti DNA with internal nested PCR primers relative to trans-PCR. Different biological samples were tested and identified only in nested PCR. This demonstrates the efficiency and effectiveness of the primers. Of the 19 samples, which amplify the partial sequence of C. burnetii, 12 were positive by conventional PCR and nested PCR. Seven samples-five spleen tissue samples from rodents and two tick samples-were only positive in nested PCR. With these new internal primers for trans-PCR, we demonstrate that our nested PCR assay for C. burnetii can achieve better results than conventional PCR.
Q热是一种由伯氏考克斯体引起的全球性人畜共患病，伯氏考克斯体是一种小型专性细胞内革兰氏阴性细菌，存在于多种动物体内。它通过吸入来自尿液、粪便、牛奶、羊水、胎盘、流产产物、羊毛的受污染气溶胶传播给人类，很少通过摄入受感染动物的生奶传播。巢式PCR可以提高检测的灵敏度和特异性，同时提供适合测序的扩增子大小。进行了十倍系列稀释以测试检测限，结果是相对于反式PCR，使用内部巢式PCR引物对伯氏考克斯体DNA的检测灵敏度提高了10倍。对不同的生物样本进行了检测，只有在巢式PCR中才能鉴定出来。这证明了引物的效率和有效性。在19个扩增伯氏考克斯体部分序列的样本中，12个通过常规PCR和巢式PCR呈阳性。7个样本——5个来自啮齿动物的脾脏组织样本和2个蜱虫样本——仅在巢式PCR中呈阳性。使用这些用于反式PCR的新内部引物，我们证明我们的伯氏考克斯体巢式PCR检测方法比常规PCR能获得更好的结果。
